Cell-to-Cell Spread of HIV and Viral Pathogenesis

CHAPTER TWO

Cell-to-Cell Spread of HIV and Viral Pathogenesis K.M. Law, N. Satija, A.M. Esposito, B.K. Chen1 Immunology Institute Icahn School of Medicine at Mount Sinai, New York, NY, United States 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. T-Cell Virological Synapse 2.1 Resistance of VS to Neutralizing Antibodies 2.2 High Multiplicity of Infection by Cell-to-Cell Transmission 2.3 Drug Resistance from High MOI 2.4 Copy Number and Resistance to ART in Patients 2.5 Cell Death Mediated by Cell-Associated Virus 3. Infectious Synapses and trans-Infection 3.1 trans-Infection by Myeloid-Derived DCs 3.2 trans-Infection by Plasmacytoid DCs 3.3 trans-Infection by Monocytes and Macrophages 3.4 Macrophage Infection by T Cells 3.5 HIV Infection and Langerhans Cells 4. Role of Cell-to-Cell Infection During Sexual Transmission 4.1 Nonhuman Primate Models and Sexual Transmission 4.2 Microbreaches and Cell-Associated Virus 4.3 Hormones and Cell-Associated Virus 4.4 Atraumatic Exposure and Cell-Associated Virus 4.5 Humanized Mouse Models and Cell-to-Cell Infection 5. Conclusions References

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Abstract Human immunodeficiency virus type 1 (HIV-1) gives rise to a chronic infection that progressively depletes CD4+ T lymphocytes. CD4+ T lymphocytes play a central coordinating role in adaptive cellular and humoral immune responses, and to do so they migrate and interact within lymphoid compartments and at effector sites to mount immune responses. While cell-free virus serves as an excellent prognostic indicator for patient survival, interactions of infected T cells or virus-scavenging immune cells with uninfected T cells can greatly enhance viral spread. HIV can induce interactions between infected and uninfected T cells that are triggered by cell surface expression of viral Env, which serves as a cell adhesion molecule that interacts with CD4 on the target cell, Advances in Virus Research, Volume 95 ISSN 0065-3527 http://dx.doi.org/10.1016/bs.aivir.2016.03.001

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2016 Elsevier Inc. All rights reserved.

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before it acts as the viral membrane fusion protein. These interactions are called virological synapses and promote replication in the face of selective pressure of humoral immune responses and antiretroviral therapy. Other infection-enhancing cell–cell interactions occur between virus-concentrating antigen-presenting cells and recipient T cells, called infectious synapses. The exact roles that these cell–cell interactions play in each stage of infection, from viral acquisition, systemic dissemination, to chronic persistence are still being determined. Infection-promoting immune cell interactions are likely to contribute to viral persistence and enhance the ability of HIV-1 to evade adaptive immune responses.

ABBREVIATIONS α4β7 alpha4beta7 integrin APC antigen-presenting cell BLT bone marrow, liver, thymus CT cytoplasmic tail DC dendritic cell DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin FISH fluorescence in situ hybridization GALT gut-associated lymphatic tissue HIV-1 human immunodeficiency virus type 1 Hu-mice humanized mice Hu-PBL peripheral blood leukocyte ICAM-1 intercellular adhesion molecule 1 ICAM-3 intercellular adhesion molecule 3 IS immunological synapse LC Langerhans cell LFA-1 leukocyte function-associated antigen 1 MDDC monocyte-derived dendritic cell MDM monocyte-derived macrophage MLV murine leukemia virus MMR macrophage mannose receptor MOI multiplicity of infection NHP nonhuman primate NNRTI nonnucleoside analog reverse-transcriptase inhibitor NRTI nucleotide analog reverse-transcriptase inhibitor PBMCs peripheral blood mononuclear cells SCID severe combined immunodeficiency SIV simian immunodeficiency virus VS virological synapse

1. INTRODUCTION The human immunodeficiency virus type 1 (HIV-1) is responsible for a pandemic infection that has succeeded in spreading globally across human

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populations. The virus infects and depletes CD4+ helper T cells that coordinate adaptive immune responses (Maddon et al., 1986). In the infected individual the virus gives rise to a chronic infection that is partly controlled by a vigorous but inadequate immune response (McMichael and RowlandJones, 2001). The level of viremia that persists following acute infection is an excellent predictor of the rate of immune depletion in the absence of therapy (Mellors et al., 1996, 1997); however, the extent to which plasma virus is responsible for viral dissemination during the early or chronic stages is still unclear. Experimentally, most studies have examined infections that begin with cell-free virus (Fig. 1A), yet the interactions between infected immune cells or virus-carrying immune cells are known to allow the virus to spread more efficiently (Fig. 1B–E). As a virus that targets the immune system, HIV takes advantage of normal interactions between immune cells that promote immune surveillance and adaptive immune responses to facilitate the spread of HIV to its favored target, the activated CD4 T cell. Some physiological interactions such as those between dendritic cells (DCs) or monocytes and T cells are coopted by the virus to enhance viral spread. For example, a central cellular interaction in adaptive immune responses is the interaction between the DC and the T cell. DC–T-cell interactions have been found to greatly enhance HIV-1 infection (Cameron et al., 1992). Other interactions between immune cells appear to be specially induced by the viral glycoproteins themselves, such as the interaction between infected and uninfected T cells, which is induced by HIV Env and its interaction with CD4 ( Jolly et al., 2004). The normal disposition of T cells in lymphoid tissues is to migrate continuously and interrogate other cells for signs of danger. Within lymph nodes it is estimated that 5000 T cells can interact with a single DC within an hour (Miller et al., 2004); the efficiency of immune surveillance makes it compelling to study how these cell–cell interactions promote dissemination in vivo. In their normal functions, lymphocytes search for foreign antigens recirculating from the blood, through the lymph nodes, into the lymphatics and back into the blood again (Bromley et al., 2008). DCs sample antigens in the peripheral sites that can result in the uptake of HIV-1. This interaction of cell-free virus with DC can also promote infections, and when these cells interact with antigens in the periphery, they can return to the lymph node where they interact with lymphocytes. The continual trafficking of immune cells into and out of the lymph nodes mediates surveillance of foreign invaders but may also play important roles in the spread of HIV within the infected individual. An understanding of these interactions is critical

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A

Cell free

Cell free

CD4

LFA-1

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T cell : T cell Donor

Target

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DC : T cell

DC-SIGN

CD169 ICAM-1

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Macrophage : T cell CD4 CD169

CD169

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T cell : macrophage

Fig. 1 Routes of HIV-1 dissemination. (A) Cell-free HIV-1 infection is mediated by virus released to the extracellular milieu that binds to CD4+ T cells at a distance from the infected cell. (B) T cell-to-T-cell virological synapse occurs when an infected CD4

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to understanding the spread and pathogenesis of HIV. Here we review the mechanisms of viral spread that are facilitated by cell–cell interactions and cell migration and discuss their implications for viral transmission, pathogenesis, and prevention.

2. T-CELL VIROLOGICAL SYNAPSE The interactions between infected and uninfected T cells have been observed to be very efficient means for dissemination of HIV in culture (Dimitrov et al., 1993; Sato et al., 1992). The formation of HIV virological synapses (VSs) between T cells is initiated by the engagement of Env on the surface of the infected T cell with CD4 on the surface of an uninfected target cell (Fig. 1B; Jolly et al., 2004). VS formation is an actin-dependent process involving cell–cell adhesion, that is facilitated by cell adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1), ICAM-3, and leukocyte function-associated antigen 1 (LFA-1) which are enriched at the site of cell–cell contact ( Jolly et al., 2007a; Rudnicka et al., 2009; Vasiliver-Shamis et al., 2008). Live-imaging studies of VS formation observed that cell–cell adhesion occurred prior to virus assembly. The viral Gag protein is dynamically recruited to the site of cell–cell contact where virus is simultaneously assembled, released, and internalized by the recipient cell (Hubner et al., 2009). Long-duration imaging of cells engaged in VS revealed that newly infected cells had engaged in synapses with infected donor cells 24 h before signs of productive viral gene expression. Although the primary function of Env is to mediate membrane fusion when on the surface of the cell-free virion, when Env engages CD4 between two T cells during VS formation, cell–cell fusion is not typically observed (Chen et al., 2007; Hubner et al., 2009; Jolly et al., 2007b). The lack of T cell binds to an uninfected T cell through the engagement of HIV-1 Env on the surface of the infected cell and CD4 on the surface of the target cell. Cell adhesion occurs before virus particles are recruited to the synapse. (C) Dendritic cell-to-T-cell infectious synapse occurs via DC that has internalized virus into a sequestered compartment by binding to CD169/Siglec-1, DC-SIGN, or other virus-binding lectin. Binding of T cell does not require CD4, but does result in the enhanced infection of interacting CD4 T cells through a process of trans-infection. (D) Macrophage-to-T-cell infectious synapse can occur when macrophages internalize HIV-1 within plasma membrane invaginations through CD169/ Siglec-1. These can mediate trans-infection through an infectious synapse, which resembles that between DC and T cells. (E) T cell-to-macrophage interaction can occur when an infected CD4+ T cell is phagocytosed by a macrophage. The process of phagocytosis can result in the enhanced infection of the macrophage. (See the color plate.)

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cell–cell fusion implies that mechanisms to inhibit membrane fusion are operating. The absence of cell–cell fusion has been observed with so-called syncytium-inducing CXCR4-tropic laboratory isolates or with primary HIV-1 isolates that use CCR5 as an entry coreceptor, which are classically referred to as nonsyncytium inducing. The regulation of Env-mediated fusion may ensure that cell–cell fusion does not inhibit infection of new cells (Weng et al., 2009). The CD4–Env interaction between T cells triggers a long-lived adhesion that organizes the production of virus at the site of cell–cell contact (Chen et al., 2007; Hubner et al., 2009). Since the engagement of Env by CD4 on the virion is well appreciated as the stimulus that activates viral membrane fusion, the absence of immediate syncytia indicates that some regulation of the fusion process must occur during T-cell VS formation. Env fusion activity has been proposed to be regulated by functions encoded in its long cytoplasmic tail (CT) (Dale et al., 2011). In the context of immature virus particles, where fusion is inhibited prior to virion maturation the Env CT acts as a sensor of virion maturation that prevents premature activation of viral membrane fusion ( Jiang and Aiken, 2007; Murakami et al., 2004; Wyma et al., 2004). Maturation is triggered by the HIV protease, which itself is held in a proenzyme form within the Gagpol precursor during viral assembly. Autocatalytic processing of Gagpol liberates protease to cleave Gag and Gagpol to promote the formation of the infectious viral core. Recent studies have found that protease activity is required for entry mediated by HIV Env, and indicate that the cleavage of the Gag lattice in the immature virus particle enables Env to trigger fusion upon engagement with CD4 (Rabi et al., 2013). This may ensure that when on an immature virus particle, the Env CT engages a “safety switch” that prevents premature triggering of fusion when the viral core is not yet processed for release into the cytoplasm. The control of HIV Env fusion activity has a functional similarity to murine leukemia virus (MLV) which has a Env protein, gp70, that is controlled directly by cleavage of a C-terminal peptide—the R peptide—by the viral protease (Loving et al., 2008; Rein et al., 1994). These common mechanisms allow each virus to enable its Env protein to trigger fusion following an interaction with its receptor on the surface of the cell. In the context of cell-free virions, maturation initiates as soon as the virus is released from the cell (Konvalinka et al., 2015), and it seems likely that these same control mechanisms influence the fusogenicity of Env on the surface of the cell during cell–cell transmission (Dale et al., 2011).

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Cell surface proteins in the tetraspanin family have been found to exert an inhibitory influence on cell–cell fusion by Env and may act to prevent syncytium formation when VSs are forming. Tetraspanins CD9, CD63, and CD18 have been found to accumulate at T-cell VSs where they appear to limit cell–cell fusion (Krementsov et al., 2009; Weng et al., 2009). Additionally, Ezrin, the connector protein between actin and integral membrane proteins, is also found to have a similar inhibitory effect on cell–cell fusion (Roy et al., 2014). Cellular proteins may thus act to enhance transmission from cell to cell by limiting the formation of syncytia. The extent to which cell–cell fusion is inhibited while Env is present on the cell surface during VS formation implies that Env is maintained in a prefusogenic state. For VS formation to occur, the CD4-binding site is required to induce cell–cell adhesion; however, other factors may prevent Env from proceeding to a state where membrane fusion is activated. The exposure of key epitopes on Env on immature particles can differ from mature particles ( Joyner et al., 2011). A comprehensive study of different epitopes finds that the presence of the Env CT in Env antigens can have a dramatic effect on epitope exposure on the ectodomain of Env (Chen et al., 2015). An attractive hypothesis to consider is that interactions with the Gag lattice or interactions with proteins, eg, tetraspanins, on the cell surface may limit the exposure of key fusogenic epitopes during cell-to-cell infection. In addition to Env:CD4 interactions other adhesion-promoting molecular interactions can facilitate VS formation. LFA-1 and LFA-1-binding partners, ICAM-1 and ICAM-3, can promote VS formation ( Jolly et al., 2007a; Rudnicka et al., 2009; Vasiliver-Shamis et al., 2008). Blocking antibodies against these adhesion molecules can abrogate VS-mediated conjugate formation and reduce cell–cell transfer of HIV. In other cases, it is reported that transmission of T-cell lines and primary CD4 T cells by chronically infected T cells occurs efficiently in the absence of adhesion molecules and can proceed in the presence of blocking antibodies against ICAM-1, ICAM-3, and α and β chains of LFA-1 and, in some cases, blocking LFA-1 from binding to ICAMs enhances HIV transfer (Puigdomenech et al., 2008). It may be that under different in vitro conditions, cell adhesion molecules play an important ancillary role in promoting synapse formation. The role of ICAMs and integrins such as LFA-1 is likely to be important for both cell–cell and cell-free viral infection. ICAM-1 can be incorporated into virions and facilitate uptake kinetics that lead to enhanced productive infection (Fortin et al., 1997; Rizzuto and Sodroski, 1997; Tardif and Tremblay,

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2003). LFA-1 expression on target cells results in enhanced cell-free infection in vitro (Hioe et al., 2001). In addition to LFA-1/ICAM interactions, HIV gp120 expressed on infected donor T cells can be recognized by the activated form of alpha4beta7 integrin (α4β7) on target T cells (Arthos et al., 2008) that correlate with enhanced susceptibility to infection (Cicala et al., 2009; Ding et al., 2015; Nawaz et al., 2011). This integrin has been found to bind to a Leu-Asp-Val/Iso tripeptide motif of V2 loop of certain CCR5-tropic Env (Arthos et al., 2008; Nawaz et al., 2011). The LDI/V tripeptide motif mimics similar structure as in mucosal addressin cellular adhesion molecule and vascular cellular adhesion molecule (Hait et al., 2015). Many HIV-1 isolates do not appear to bind α4β7 which may indicate that binding is not an essential feature of HIV infection (Perez et al., 2014). Others report that HIV-1 subtype C viruses utilize α4β7 to enhance replication (Richardson et al., 2015). When HIV binds α4β7, it colocalizes with active LFA-1 and CD4 at the cell–cell interface of the VS. This gp120–α4β7 interaction triggers the activation of LFA-1 on target T cells (Hioe et al., 2011) and the active conformations of LFA-1 promote synapse formation and HIV infection. The activation of LFA-1 by gp120–α4β7 engagement increases the efficiency of infection by promoting synapse formation rather than direct capture of cell-free virus (Ding et al., 2015). This integrin is involved in migration of lymphocytes to mucosal tissues making this process relevant in memory CD4 T cell infection in the gut and vaginal mucosa that also readily express coreceptor CCR5 ( Joag et al., 2016; Mavigner et al., 2012). Targeting this interaction efficiently reduces intravaginal simian immunodeficiency virus (SIV) transmission in macaques (Byrareddy et al., 2014). An interesting hypothesis from these studies is that the virus engagement of these receptors may also influence the subsequent trafficking of infected T cells. When cell-to-cell HIV transfer occurs through VSs, the transferred virus particles are described as being internalized into an endocytic compartment (Sloan et al., 2013) where they transition from an immature to mature fusion-competent state (Dale et al., 2011). The pathway of entry may be distinct from that described for cell-free virus, which has been described as mediated by entry at the cell surface or through endocytic intermediates (Herold et al., 2014; Miyauchi et al., 2009a,b). During cell-to-cell transmission, the addition of protease inhibitors allowed endocytosed virions to be trapped in an immature state that accumulates within an endocytic

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compartment (Dale et al., 2011). These protease inhibitors do not block synapse formation, yet are potent inhibitors of virus particle fusion and promote the retention of larger numbers of virus particles. Using singleparticle imaging, fusion of these particles from within these endosomes is likely to occur within a protected compartment. Antibodies appear to have access to this compartment, as even antibodies against the membrane proximal external region in gp41 sequences that are transiently exposed during the fusion process can be inhibitory against cell-to-cell infection (Durham et al., 2012).

2.1 Resistance of VS to Neutralizing Antibodies When cell-free infection is compared to cell-to-cell infection, neutralizing antibodies are generally less efficient at blocking cell-to-cell infection (Abela et al., 2012; Chen et al., 2007; Durham et al., 2012; Martin et al., 2010; Zhong et al., 2013). Interestingly, the magnitude of the resistance to neutralizing antibodies conferred by cell-to-cell infection is highly dependent upon the antibody epitopes targeted (Abela et al., 2012; Durham et al., 2012). Differences between neutralization of cell-associated virus and cell-free virus were less dramatic when chronically infected cells were used as the donor cells (Martin et al., 2010), which may be attributable to the presence of cell surface-bound cell-free virus particles on these cells (Schiffner et al., 2013). Different viral strains exhibit distinct inhibitory sensitivities when a panel of potent broadly neutralizing antibodies are tested in cell-to-cell transmission (Reh et al., 2015). This also occurred during cellto-cell transmission between infected monocytes and uninfected CD4 T cells (Duncan et al., 2014). These studies indicate that the resistance of cell-associated HIV to neutralizing antibodies is highly dependent upon the accessibility or presence of different epitopes on the surface of the infected cell. An interesting clue to the mechanisms behind neutralization resistance of cell-associated HIV is that deletion of the CT of Env does diminish the magnitude of resistance, ie, becomes easier to neutralize (Durham et al., 2012). Since the Env CT plays an important role in controlling fusion during the particle maturation process, this function of the CT may control the epitopes exposed on the surface of the cell. The CT truncation mutation removes 144 amino-terminal amino acids from the gp41 domain and results in higher levels of gp160 on the surface of infected cells. Despite the higher expression

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of △CT Env on the surface of infected cells, the cell-to-cell infection from these cells is increased in its sensitivity to neutralizing antibodies. It is hypothesized that the structures that are targeted for neutralization are frequently unavailable on the surface of cells and that neutralizing epitopes may be revealed during the maturation process that occurs after the virus particle has been transferred into the target cell endosome.

2.2 High Multiplicity of Infection by Cell-to-Cell Transmission VS between infected and uninfected cells can drive the direct cell-to-cell transfer of large amounts of virus and viral antigens per infection event or high multiplicity of infection (MOI) (Blanco et al., 2004; Chen et al., 2007). An early study by Estes and colleagues observed that large amounts of viral p24 were transferred by cell–cell contact between chronically infected cells and uninfected CD4 T cells through a mechanism that does not require coreceptor interactions. These cell–cell interactions were not reported to lead to productive infection. Instead, the acceptor T cells released significant amounts of infectious virus when separated from the chronically infected cells. This study supported a model by which uninfected T cells can sequester virus and release it at a later time, to promote viral spread. The importance of T cell-to-T-cell contact for the spread of HIV within a cell culture has been demonstrated with a culture system where infected cells were prevented from interacting with one another by constant agitation, or allowed to settle to the bottom of the culture dish (Sourisseau et al., 2007). The study found that stable cell contact was critical for viral spread in these T-cell cultures (Sourisseau et al., 2007). A comparison of the amount of virus transferred from infected to uninfected cells revealed that cell–cell contacts are much more efficient than exposure to cell-free virus at mediating transfer of virus particles to recipient cells (Chen et al., 2007). T cells exposed to cell-associated virus exhibited nearly 100-fold higher viral uptake than cells exposed to high concentrations of cell-free virus produced by transient transfection of highly productive 293 T cells. The differences in viral uptake are even further exaggerated when it is considered that the cell-free viral stocks employed experimentally have much higher titers of virus than supernatants released by infected T cells. This increase in viral uptake by cell–cell contact is found to directly result in enhanced infection as revealed by experiments that sorted cells that internalized large amounts of virus and found that these give rise to newly infected

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cells (Del Portillo et al., 2011). Live-imaging studies have also directly illustrated that cells that synapse are the cells that become infected (Hubner et al., 2009). The increased uptake of viral particles that occurs in cell-to-cell transmission results in a greater frequency of target cells becoming infected with more than one copy of HIV (Del Portillo et al., 2011). To ascertain the frequency with which more than one virus was cotransmitted from one T cell to another T cell, investigators have examined the cotransmission of two fluorescent reporter viruses when donor cells expressed more than one genotype per cell. When comparing cell-to-cell vs cell-free transmission, authors have found that the frequency of dually infected cells was significantly greater than that which was found in cell-free infection, which follows a random Poisson distribution (Del Portillo et al., 2011). When titrating cell-associated inoculums in vitro, the fraction of infected cells that were coinfected stayed constant at low inocula. This indicates that the minimal titratable unit of transmission is a cell that cotransmits multiple viruses to new cells at a fairly constant frequency. From these titration experiments the authors concluded that infections through cell-associated inocula are intrinsically multiploid. The study also measured the copy number of genomes per cell by fluorescence in situ hybridization (FISH) and found a mean proviral copy number of 3.7 in cells infected by cell-to-cell transmission compared to a mean of 1.1 in cells infected by cell-free virus. Multiploid inheritance of HIV may enhance the ability HIV to support genetic diversity by complementing spontaneous mutations as they arise. The high MOI afforded by cell-to-cell transmission has also been found to help the virus to overcome restriction factors such as TRIM5α (Richardson et al., 2008). In the case for TRIM5, rhesus TRIM5α can restrict incoming viruses by binding to the viral capsid and preventing successful reverse transcription and entering the nucleus. This restriction of HIV-1 in simian cells can be saturated by exposure to an excess of cell-free virus. However, the high level of virus transferred across a VS is also sufficient to saturate the restriction activity of TRIM5α. At the cell surface viral restriction mechanisms mediated by the host factor tetherin appear to preferentially impact cell-free viral infection over cell-to-cell infections. Tetherin is an interferon-induced cell surface protein that inhibits the release of cell-free HIV by tethering it to the cell surface (Neil et al., 2008; Van Damme et al., 2008). While tetherin’s effects on the release of cell-free virus are clear, its ability to impair cell-to-cell spread

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of HIV is not consistent between different studies (Casartelli et al., 2010; Jolly et al., 2010; Kuhl et al., 2010; Zhong et al., 2013). Recent reports indicate that the tethering of virus to the surface by tetherin may serve other important roles by activating antibody-dependent clearance of infected cells by natural killer cells through antibody-dependent cellular cytotoxicity (Alvarez et al., 2014; Arias et al., 2014).

2.3 Drug Resistance from High MOI The higher MOI of cell-to-cell HIV infection compared to that of cell-free infection is believed to have other advantages in addition to the increased genetic diversity it gives to viruses. It has been shown that viral infection under conditions of cell-to-cell spread is resistant to the nucleotide analog reverse-transcriptase inhibitor (NRTI), tenofovir, compared to cell-free infection (Sigal et al., 2011). In peripheral blood mononuclear cells (PBMCs) there was almost complete inhibition of infection in cell-free conditions, while cocultured cells showed significantly less inhibition. This correlated with higher MOI in cell-to-cell transmission. When a very high MOI of cell-free virus was used, it recapitulated the tenofovir resistance seen in cell-to-cell transfer of virus. This observation of reduced drug sensitivity during cell-to-cell transmission further extended to other types of antiretroviral compounds (Agosto et al., 2014). A study by Mothes and colleagues examined six NRTIs, four nonnucleoside analog reverse-transcriptase inhibitors (NNRTIs), four entry inhibitors, and four protease inhibitors and observed that some of these compounds have similar levels of inhibition in both cell-to-cell and cell-free inhibition assays. Notable exceptions were NRTI such as tenofovir, AZT, and stavudine that inhibited cell-to-cell transmission significantly less efficiently than cell free. Interestingly it was found that combinations of these antiretrovirals that were individually less effective at inhibiting cell-to-cell transmission could increase their efficiency against cell-to-cell transmission when used in combination. This study also found that the differences observed in inhibition between cell-to-cell and cell-free modes of transmission were due to the higher MOI of cell-to-cell transmission in agreement with Sigal et al. Reduced drug sensitivity during cell-to-cell transmission may apply to other synapses besides the T cell-to-T-cell synapse. A similar phenomenon was observed in another study that examined antiretroviral efficacy on cellto-cell transmission in a macrophage to CD4+ T-cell system. This study used

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monocyte-derived macrophages (MDMs) and CD4+ T, cells isolated from PBMCs with a luciferase-based reporter virus to characterize the potency of AZT, nevirapine and raltegravir on cell-to-cell vs cell-free transmission of HIV in the context of macrophage transmission (Duncan et al., 2013). Viral transmission was controlled by coculturing donor and target cells in a static culture to support cell-to-cell transmission or by gently shaking the culture to allow only cell-free transmission. This study further validated the finding that the antiretrovirals tested were significantly less efficient at inhibiting cell-to-cell transmission compared to cell-free transmission. This difference between modes of transmission could be negated by increasing the MOI of cell-free infection to that of cell-to-cell transmission, further supporting the idea that the higher MOI of cell-to-cell transmission is responsible for the higher resistance to antiretrovirals observed in this mode of transmission. These findings indicate that cell-to-cell transmission may be relevant to macrophage T-cell interactions. Although the predominant mode of drug resistance in cell-to-cell transmission appears to be due to higher MOI, there are other properties of cellto-cell transmission, which may allow a virus to overcome fitness barriers that are driven by drug pressures. A study examining the integrase inhibitor dolutegravir (DTG) found that viruses with specific integrase mutations that developed in patients undergoing DTG treatment exhibited impaired replication (Bastarache et al., 2014). When cultured in vitro, the cell-to-cell transmission capacity of these viruses was comparable to WT virus. The cell-free infection capacity of these mutants was found to be decreased. Furthermore, these mutants were found to be capable of establishing latency and reactivating from latency. It was hypothesized that the higher copy number of genomes transmitted during cell-to-cell transmission may promote replication in the presence of certain antiretrovirals and provide a selective advantage for establishing latency. While cell-to-cell transmission may bolster resistance to particular antiretroviral therapies, some studies have found specific antiretroviral therapies to be equally efficient against both cell-to-cell and cell-free transmission. Protease inhibitors and reverse-transcriptase inhibitors exhibited varying abilities to differentiate cell-to-cell vs cell-free transmission (Titanji et al., 2013). The reverse-transcription inhibitors tested (nevirapine, tenofovir, and zidovudine) were significantly less effective at inhibiting cell-to-cell transmission of HIV compared to cell-free transmission, in agreement with previous findings by other groups. In contrast, the protease inhibitors tested (lopinavir and darunavir) were equally efficient at inhibiting cell-to-cell

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transmission, as they were at cell free (Titanji et al., 2013). Because of the ability of protease inhibitors to block multiple steps in the replication cycle, these may be particularly potent antagonists against both cell-free and cell– cell infection (Rabi et al., 2013). These results suggest that while some methods of inhibition of HIV replication may target pathways which are different in cell-to-cell transmission compared to cell free, it is possible to develop inhibitors which target common pathways utilized by both modes of transmission, hopefully leading to greater inhibition efficiency in vivo.

2.4 Copy Number and Resistance to ART in Patients Given the studies that find reduced efficacy of some antiretroviral drugs in in vitro settings, it becomes critical to assess the importance of cell-to-cell transmission in vivo as this may impact resistance to drugs in different anatomical sites. Studies of patient splenocytes have supported that viral replication spreads locally within lymphoid tissues and infects cells with a high MOI (Delassus et al., 1992; Gratton et al., 2000). Through examination of viruses isolated from individual germinal centers from splenic tissue of infected patients, the viral sequences have been found to be organized in clusters, indicating that viral spread within a tissue compartment is strongly influenced by local cell–cell interactions, and not the diffusion of cell-free virus or immune complexes over large areas (Cheynier et al., 1994; Delassus et al., 1992; Gratton et al., 2000). Viruses isolated from individual clusters were genetically distinct from neighboring clusters. It has been found that splenocytes from HIV-infected patients harbored genetically distinct viruses which were the result of single cells being infected with multiple copies of HIV-1 ( Jung et al., 2002). Splenocytes isolated from two HIVinfected patients and sequence divergence were quantified using FISH. They found that 75% of cells had more than 2 proviruses with the mean number of proviruses per cell being 3.5. The authors suggested that this higher MOI allows for a greater amount of recombination events leading to a more diverse population of viruses. These studies generally favor that the replication within lymphoid tissues occurs within local clusters rather than as a result of the broad diffusion of a viral swarm. Because of the association of cell–cell transmission with multicopy infection, it is interesting to study infected patients to see if the number of cells infected with multiple copies of HIV is greater than one might expect given random infection by cell-free virus. Recent studies have used single-cell PCR approaches to assess the number of distinct HIV DNA molecules

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per cell in the lymphoid tissues and peripheral blood of five HIV-infected patients ( Josefsson et al., 2013). In this case it was found that while a majority of infected cells may be detected as singly infected cells, a significant number of infected cells in each sample were multiply infected (>20% in many cases). These studies indicate that the occurrence of multicopy infections in vivo is not rare, a phenomenon which occurs at a much greater frequency with direct cell-to-cell transmission of HIV and indicates that infection mechanisms that promote multicopy infection are operative in vivo. It is also important to consider that in patients, cells that are infected with a single proviral copy may be less susceptible to cell death and/or more likely to produce a latent infection which would enhance the survival of these cells (Dixit and Perelson, 2005; Temin, 1988). It is therefore possible that the pool of cells that support active replication may contain a higher frequency of multiply infected cells. The lower potency of some antiretroviral drugs against cell–cell transmission may be particularly relevant when considering viral spread within lymphoid tissues where most infection is likely to occur. Recent studies have indicated that the concentrations of antiretroviral drugs achieved in lymphoid tissues can also be less well penetrated by certain antiretrovirals. One study, examining 12 HIV-infected individuals undergoing ART, found that although the antiretrovirals concentrations were at expected levels in the peripheral blood, they were significantly lower in tissue biopsies taken from lymph nodes (Fletcher et al., 2014). The lower drug penetration correlated with increased viral replication, as indicated by larger inhibition of virus in plasma than in lymphoid tissue where drug concentrations were lower. Another study examining rectal, vaginal, and cervical tissue samples from 15 healthy uninfected individuals given a single dose of the preexposure prophylaxis cocktail Truvada (a combination of emtricitabine and tenofovir) found that the concentrations of the antiretrovirals in Truvada varied from tissue to tissue (Patterson et al., 2011). For example, tenofovir was found to be 100-fold lower in vaginal tissue compared to rectal, while emtricitabine was higher in vaginal tissue. The lower penetration of antiretrovirals into tissues that may support higher levels of cell-to-cell spread may promote viral persistence in patients on ART. Recent deep sequencing data of viruses from lymphoid tissue from HIVinfected patients support the model that replicating HIV reservoirs exist in lymphoid tissues where antiretroviral drug concentrations are lower (Lorenzo-Redondo et al., 2016). This study developed a model through

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phylogenetic analysis that drug-sensitive strains were able to propagate deep within lymphoid tissue where drug concentrations are lower. Presumably because drug concentrations are lower in these tissues, these allow for replication of a pool of drug-sensitive strains, with a higher level of fitness under conditions of no drugs. These are predicted to propagate in lymphoid tissue even when plasma drug levels are highly inhibitory.

2.5 Cell Death Mediated by Cell-Associated Virus As previously discussed, cell-mediated HIV transmission facilitates infections in which a large number of virions that are transferred to the target T cell. This process can also contribute to viral pathogenesis through the depletion of CD4 T cells that are susceptible or even resistant to infection. Multiple innate sensing pathways may be triggered following cell-to-cell transmission that can lead to cell death. A cell death mechanism mediated by the recognition of HIV occurs in bystander CD4 T cells that are not productively infected (Doitsh et al., 2010). This bystander cell death is observed during infection of lymphoid-derived cells and is found to be dependent on cytosolic accumulation of incomplete HIV reverse transcripts mediated by cell-to-cell HIV transmission but not by cell-free transmission (Doitsh et al., 2010; Galloway et al., 2015). In CD4+ T cells, these incomplete HIV transcripts are sensed by IFI16, a DNA sensor that detects viral DNAs to active innate inflammatory responses through the inflammasome activation pathway (Doitsh et al., 2014; Monroe et al., 2014). The inflammasome is an innate immune signaling complex that activates caspase-1 which initiates the cleavage-mediated activation of proinflammatory cytokines, prointerleukin-1β (IL-1β) and IL-18, into their bioactive form and causes pyroptosis, a highly inflammatory form of programmed cell death (Guo et al., 2015). Cells undergoing pyroptosis exhibit plasma membrane pore formation and leakage of cytoplasmic contents leading to an inflammatory response. Disruption of VSs between infected T cells and tonsilar T cells by coculturing cells on different sides of a semipermeable barrier can inhibit bystander pyroptosis of tonsilar T cells (Galloway et al., 2015). In addition, LFA-1–ICAM-1 interaction is necessary to facilitate this program cell death pathway as shown with antibody blockade experiment that inhibits the formation of the VS (Galloway et al., 2015). Bystander cell death is abrogated in the presence of efavirenz, an NNRTI that allosterically inhibits HIV reverse transcriptase, and by AMD3100, an entry inhibitor that blocks gp120

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engagement of the CXCR4 coreceptor. Bystander cell death, however, is unchanged in the presence of nucleoside analog reverse-transcriptase inhibitors (NRTIs) which presumably allow some short reverse-transcriptase products to be formed (Doitsh et al., 2010). Pyroptosis involves oncosis, plasma membrane rupture, and release of cytoplasmic contents into the surrounding tissue environment, which may attribute to further pathophysiological damage at the site of HIV infection. In addition to resting cells derived from tonsil tissues, CD4 T cells derived from spleen and gut-associated lymphatic tissue (GALT) are also susceptible to this form of cell death (Doitsh et al., 2014; Steele et al., 2014). IL-1β causes breakdown of the gut epithelium that provides a major barrier to protect the body against pathogens. Chronic immune activation may result from pathogenic events in the GALT during infection leading to massive killing of lamina propria CD4 T cells and microbial translocation leading to systemic immune activation (Brenchley et al., 2004; Mehandru et al., 2004). Cell-to-cell transmission may be relevant during infection of GALT tissue as cell–cell synapses have been observed by three-dimensional ultrastructural examination using transmission electron microscopy (Ladinsky et al., 2014). Evidence for pyroptotic cell death has also been observed during SIV infection. Members of this pathway, IFI16, caspase-1, and IL-1β are significantly upregulated in expression in CD4 T cells in draining lymph nodes (Lu et al., 2015). However, not all tissues are equally susceptible to pyroptosis. Resting peripheral blood CD4 T cells are resistant to pyroptosis during abortive infection, which may be due to different levels of IFI16, different resting states, or distinct cytokine milieux (Trinite et al., 2015). Coculture of peripheral blood CD4+ T cells with lymphoid tissue lymphocytes is sufficient to alter the resistance to pyroptosis (Mun˜oz-Arias et al., 2015). It has been reported that 95% of dying cells undergo death by pyroptosis as bystander cells or resting cells that are not permissive for infection, while 5% of CD4 T-cell death is attributable to another form of cell death, apoptosis, in productively infected cells (Doitsh et al., 2010). Apoptosis has been proposed as a key mechanism in productively infected cells and is mediated through DNA-dependent protein kinase (DNA-PK), a central integrator of the DNA damage response (Steele et al., 2014). Following viral integration events, this kinase is activated causing phosphorylation of p53 and histone H2AX (Cooper et al., 2013). It has yet to be shown directly whether the high multiplicity of cell–cell transmission mediates greater DNA-PKmediated cell death.

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3. INFECTIOUS SYNAPSES AND trans-INFECTION Enhancement of HIV infection by immune cells that do not become infected is often referred to as trans-infection because the donor cell passes the virus in an infectious form to a recipient target cell. When this mode of infection occurs by cells that also can participate in immunological synapse (IS), it can be referred to as an infectious synapse. These synapses form between an uninfected antigen-presenting cell (APC) that scavenges cellfree HIV into an internal compartment and present it to an uninfected target T cell through a durable cell–cell adhesion (Cameron et al., 1992). transInfection is distinct from cis-infection in which the donor cell itself is not infected and spread the infection via CD4–Env interaction as well as additional adhesion pathways that normally promote APC/T-cell interactions. Numerous adhesion molecules and lectins have been implicated in this process, and while these are thought of as occurring independently of cognate antigen interactions, it is likely that cognate APC–T-cell interactions influence the frequency of these interactions (Rodriguez-Plata et al., 2013). These virus-binding molecules do not function as receptors for viral entry into APCs but rather facilitate efficient virus uptake and trans-infection of T cells. Additionally, as these cells can migrate from tissue sites to draining lymph nodes, they may transport of virus to secondary lymphoid organs rich in T cells to enhance dissemination through cell–cell contacts. This section will review some of the important molecules and cell types involved in this trans-infection process.

3.1 trans-Infection by Myeloid-Derived DCs The most well-studied example of the infectious synapses involves DCs as the donor APC (Fig. 1C). The major role of DCs is to scavenge for pathogens, migrate from their resident tissues to lymph nodes, and process and present antigen to T cells that initiate adaptive immunity (Bromley et al., 2008). HIV has been described as infecting cells through a Trojan horse mechanism as the virus coopts the multistep process of antigen presentation to gain access to a special compartment in APCs for dissemination to T cells. In this process, DCs first capture and bind HIV. Second, HIV traffics within these DCs to a protected and nondegradative compartment. Third, HIV is transferred to CD4+ T cells by exocytosis of intact virus. Direct HIV infection of DCs is much less efficient in comparison to infection of CD4 T cells

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in vivo (Wu and KewalRamani, 2006). Low infection rates have been attributed to low levels of expression of CD4 and coreceptor, degradation of HIV, and abundance of restriction factors (Laguette et al., 2011). The ability of DCs to greatly enhance transmission of cell-free virus to target T cells was first described by Steinman and colleagues (Cameron et al., 1992), and this was shown to be an independent pathway than that of productive infection (Blauvelt et al., 1997). The mechanism by which DCs enhance infection of T cells was further described through live confocal imaging studies by McDonald and Hope who found that the virus captured by DCs is rapidly relocalized to the site of cell–cell interaction when the DC encounters an uninfected T cell. CD4, CCR5, and CXCR4 on the T cell are recruited to the interface, while the monocyte-derived DCs (MDDCs) concentrated HIV to the same region of an infectious synapse (McDonald et al., 2003). At the point of cell–cell contact, T-cell filopodia have been observed to sample the virus-rich deep, membrane invaginations on the DC (Do et al., 2014). This process has been readily seen in a number of DC subsets (Wu and KewalRamani, 2006). Immature DCs survey mucosal tissues for unwanted potential pathogens for internalization, lysosomal degradation, and antigen presentation. HIV uses this process to mediate efficient infection into T cells in part by a C-type lectin receptor, dendritic cell-specific intercellular adhesion molecule-3-frabbing nonintegrin (DC-SIGN), a C-type lectin receptor expressed by mucosal DCs, and a subset of macrophages (Geijtenbeek et al., 2000; Kwon et al., 2002). DC-SIGN expressed on immature DCs mediates rapid internalization of HIV for trans-infection. DC-SIGN interacts with HIV gp120 with an affinity five times greater than for the cognate receptor CD4, and this binding facilitates enhanced exposure of the CD4binding site increasing the affinity of Env to enhance infection (IzquierdoUseros et al., 2014). Mature DCs that are frequently found within lymphoid tissues can effectively transfer HIV to T cells, but DC-SIGN may be dispensable for trans-infection (Wang et al., 2007). DCs present in mucosal tissue can capture HIV viral particles through other viral receptors that promote trans-infection. Siglec-1 (CD169) is a cell surface receptor that mediates trans-enhancement of HIV infection through recognition of sialic acid moieties of glycosphingolipids in virus membrane (Izquierdo-Useros et al., 2012). Host cell-derived glycosphingolipids, like GM3, in the viral membrane are important for efficient capture in a manner similar to DC-SIGN (Akiyama et al., 2014; Puryear et al., 2012). However,

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DC-SIGN recognizes the viral envelope glycoprotein and GM3-CD169 binding is gp120 independent for sequestration and infection (Yu et al., 2014). Upon virus assembly, these glycosphingolipids are incorporated into the virus membrane by matrix domain of Gag (Akiyama et al., 2014). During capture, CD169 mediates compartmentalization of virus into deep plasma membrane invaginations within the DC that reduces the efficiency of anti-gp120 antibodies from neutralizing trans-infection (Akiyama et al., 2015). This localization has been found to be important for efficient trans-infection by mature DCs. Capture of HIV mediated by CD169, as well as the subsequent trans-infection to T cells, can be further enhanced in response to IFNα (Pino et al., 2015). LFA-1–ICAM-1 interaction is known to be an integral component of the IS. HIV takes advantage of this adhesive interaction to facilitate, binding to target cells, formation of the IS and optimal cell-mediated spread (Vasiliver-Shamis et al., 2008). Both LFA-1 and ICAM-1 molecules are upregulated on activated mature DC (Sanders et al., 2002). It has been reported that ICAM-1 and LFA-1 interaction is important for stabilization of the IS (Grakoui et al., 1999; Lee et al., 2002; Monks et al., 1998). DC-mediated viral transmission is inhibited when ICAM-1 on DCs or LFA-1 on CD4 T cells is blocked by antibodies or knocked down by interfering RNAs. However, blockade of ICAM-1 on target cells does not significantly inhibit DC-mediated HIV transmission. Overexpression and antibody blockade studies have demonstrated that DC-mediated HIV transmission to CD4 T cells is independent of ICAM-2 and ICAM-3 (Wang et al., 2009). Transfer of virus from DCs to CD4+ T cells might occur by several distinct mechanisms that include viral transfer via infectious synapses, de novo viral production, or by exosome-associated viruses. In this later process, virus is rapidly internalized by mature MDDCs into endosomal, multivesicular bodies, which are endocytic bodies that are enriched in tetraspanins (Garcia et al., 2008). These include CD9, CD63, CD81, and CD82, which are not found on immature DCs. HIV is able to circumvent immune destruction after capture by DCs. Virus particles are rerouted to this tetraspanin-rich, low-pH compartment following virus capture (Garcia et al., 2005). Some tetraspanins, particularly CD63, are incorporated into the viral membrane upon budding from the DC ( Jolly and Sattentau, 2007). Exosomes captured by mDCs can be efficiently transmitted to T lymphocytes in an envelope gp120-independent manner (IzquierdoUseros et al., 2009).

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3.2 trans-Infection by Plasmacytoid DCs Plasmacytoid DCs (pDCs) are a subtype of circulating DCs that are derived from a lymphoid lineage and are particularly effective at initiating early responses to viral infections. Productive infection within pDC is less efficient than myeloid DC, even though both subsets express CD4 and coreceptors (Smed-Sorensen et al., 2005). pDCs are able to sense HIV-1infected cells and react by producing proinflammatory cytokines such as type-I IFN (Lepelley et al., 2011). The interaction of pDC by cell-associated virus is a more potently activating of these cells than cell-free virus. This immune activation in turn leads to the recruitment of DCs and other immune cells to the site of infection, which may facilitate the transfer of virus from infected lymphocytes to pDCs (Li et al., 2009). This innate immune sensing of virus by virus in pDC is impaired by bNAbs that also reduce lymphocyte-to-lymphocyte viral spread (Malbec et al., 2013). In addition, HIV gp120 has been shown to interfere with Toll-like receptor signaling in pDCs, reducing the secretion of antiviral and inflammatory cytokines for the development of effective immune responses (Martinelli et al., 2007).

3.3 trans-Infection by Monocytes and Macrophages Like DCs, HIV infection in monocytes and macrophages is inefficient due to their low expression of viral entry receptors and high expression of restriction factors. However, infected macrophages have the capacity to transfer HIV at a high MOI to CD4+ T cells in a similar manner to the T cell:T-cell synapse, which reduces viral sensitivity to reverse-transcriptase inhibitors and some bNAbs (Duncan et al., 2014). In addition, monocytes and macrophages may all mediate trans-infection similar to DCs, using conserved machinery expressed on both cell types (Fig. 1D). Infectious virions persist within these cells and extend their infectivity for long periods of time for up to 6 weeks (Sharova et al., 2005). Monocytes and macrophages normally do not express DC-SIGN in vitro, but can be induced by Th2 cytokine, Il-13; however, DC-SIGN-positive macrophages do play important immunological roles in vivo (Conde et al., 2015). Sampling by CD169 has been proposed to be an early step toward the initiation of host immune responses against incoming viruses, but similar to DCs can also be utilized by HIV to efficiently infect the host. CD169 expression is increased on CD14+ monocytes and macrophages during HIV infection (Rempel et al., 2008; van der Kuyl et al., 2007; Zou et al., 2011). The efficient capture of cell-free virus in lymphoid tissues is mediated

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by CD169 on macrophages located within the subcapsular sinus of lymph nodes from humanized mice (Hu-mice) (Sewald et al., 2015). Sewald and colleagues demonstrated that CD169, expressed on the surface of macrophages, captures both HIV and MLV cell-free particles and mediates their transfer to proliferating lymphocytes in vivo in a manner similar to DC: T-cell infectious synapses (Sewald et al., 2015). In vitro studies indicate that MDDCs have the highest capacity for CD169-mediated trans-infection, followed by monocytes, with the weakest trans-infection capacity demonstrated by macrophages, which may be due to faster viral degradation in this cell type. In macrophages that lack DC-SIGN, macrophage mannose receptor (MMR) is an alternative receptor that mediates trans-infection to T cells (Nguyen and Hildreth, 2003). MMR-mediated infection is blocked up to 80% using inhibitors of MMR binding, such as mannan, D-mannose, EDTA, and soluble mannose-binding lectin.

3.4 Macrophage Infection by T Cells A recent study by Sattentau and colleagues revealed a novel cell-mediated mechanism that enhances the infection of macrophages. In this study, it was found that MDMs can selectively engulf autologous HIV-infected T cells in a manner that facilitates efficient macrophage infection (Fig. 1E). In this process, both healthy, living and dead or dying infected primary CD4+ T cells are preferentially taken up by macrophages. This process is independent of Env expression on the T cell because infected cells expressing a nonfunctional Env remain preferential for engulfment by MDMs. This infection is restricted to T cells chronically infected with CCR5 macrophagetropic strains (Bal, YU2, or transmitted founder Env-CH077, H040, REJO, WITO), but not X4-tropic HIV (MN, IIIB) (Baxter et al., 2014). Surprisingly, these T cells may reside within the internal compartment of the macrophages for hours to even days after engulfment. This mode of spread is highly efficient compared to cell-free transmission, which could be explained by a high MOI similar to VS- and IS-mediated spread. This process is blocked by inhibiting phagocytosis with the actin–antagonist jasplakinolide. It remains unknown which pattern recognition marker expressed on dead and dying infected cells is preferential for this mode of infection.

3.5 HIV Infection and Langerhans Cells Also from the myeloid lineage, Langerhans cells (LCs) act as sentinels of epidermal and mucosal epithelia but are largely confined to local sites of

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infection within the epithelial layer where they are involved in innate and adaptive immune responses. These anatomic, phenotypic, and functional properties may allow for frequent interactions with HIV within genital mucosal epithelium during sexual transmission. LCs do express CD4, CXCR4, and CCR5 (Zaitseva et al., 1997). However, the maturation states of LCs may determine their susceptibility to infection as immature resident LC expresses surface CCR5, but not surface CXCR4, whereas mature LC expresses less CCR5 and higher levels of CXCR4. Following intravaginal exposure, these cells comprise a major cell subset that can be infected by SIV in rhesus macaques (Hu et al., 2000; Spira et al., 1996). Similar to other myeloid lineages, LCs can efficiently capture and transfer HIV to CD4+ T cells as observed in ex vivo explant models (Peressin et al., 2014; Reece et al., 1998; Sugaya et al., 2004). LCs may have multiple processes to bind and capture HIV-1. Following endocytosis of HIV-1, virions may remain intact in the LC cytoplasm for several days (Hladik et al., 2007). LCs express additional adhesive molecules, such as C-type lectins that recognize HIV, in particular DC-SIGN and LC-specific C-type lectin, Langerin. Unlike DC-SIGN, Langerin reduces HIV transmission mediated by LCs as Langerin inhibition allowed for further trans-infection into T cells as well as direct infection of LCs themselves (de Witte et al., 2007). This demonstrates that, at the initial sites of mucosal acquisition, the role of LCs to either facilitate HIV transmission or serve a protective function is determined by the virus-interacting lectins expressed on the cell surface.

4. ROLE OF CELL-TO-CELL INFECTION DURING SEXUAL TRANSMISSION Sexual transmission of HIV requires that the virus inoculum crosses mucosal surfaces that protect the host and provide barriers to transmission. The initial sites of replication are believed to be local within target cells of mucosal tissue (Hu et al., 2000; Miller et al., 2005; Zhang et al., 1999). During mucosal transmission, there is a complex interplay between the virus and immune cells at the site of infection that have a role in exacerbating or regulating the infection process (Haase, 2005; Zhang et al., 1999, 2004). In situ characterization of mucosal SIV infection revealed that resting and activated CD4 T cells are recruited to the site of infection, along with macrophages and DCs (Haase, 2005; Li et al., 2009; Pope and Haase, 2003). Following local expansion of incoming virus, HIV further disseminates to the draining lymph node, and subsequently through the lymphatics to the bloodstream

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for establishment of infection in secondary lymphoid organs (Haase, 2011; Zhang et al., 1999). During mucosal infection, DCs and CD4+ T lymphocytes are the initial target cells for HIV along with monocytes and macrophages although less abundant (Spira et al., 1996). These cells are relevant for establishing early HIV reservoirs and contributing to HIV persistence. Many of these cells are thought to be vehicles for HIV that mediate systemic dissemination in vivo (Murooka et al., 2012; Sewald et al., 2015). During the acute infection through mucosal routes, it is likely that the movement of cells into and out of the mucosal site may be important in facilitating the spread of HIV systemically. A long-standing and critical question regarding the sexual transmission of HIV is what form of virus within genital secretions gives rise to the initial infection. Semen represents the major source of virus in sexual transmission and is a complex mix of plasma and cells that contains both cell-free virus and HIV-infected cells (Houzet et al., 2014). The cellular portion is composed of spermatozoa, leukocytes, and epithelial cells, while the plasma contains thousands of different proteins (Pilch and Mann, 2006). In infected individuals, HIV can be found in the semen during acute, chronic stages of infection, during AIDS, or in patients receiving HAART (Bernard-Stoecklin et al., 2014; Houzet et al., 2014). An estimated 21–75% of seminal leukocytes contain HIV proviral DNA in HIV-infected men (Anderson et al., 2010; Bernard-Stoecklin et al., 2014). Replication-competent HIV can be isolated from macrophages and T cells isolated from semen by coculture with PBMCs in vitro (Quayle et al., 1997). While both cell-free and cellassociated virus are infectious, the relative contributions of each have been challenging to assess yielding different answers with different models. This distinction may be particularly critical as we identify more differences in the susceptibility of cell-associated viruses to neutralization by antibodies or inhibition by antiretroviral drugs in tissues. In the following sections we review the older literature and examine new studies that may provide a clearer view of the question.

4.1 Nonhuman Primate Models and Sexual Transmission Chimpanzees are the only nonhuman primate (NHP) that can be readily infected with HIV-1. No longer employed as an experimental model, chimpanzees were the focus of early studies examining what viral inocula could be infectious when presented through a vaginal route. A study in 1998 examined a chimpanzee model for mucosal HIV transmission and found that

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persistent HIV infection could be established in adult chimpanzees upon intravaginal exposure with high titers of cell-free or cell-associated HIV (LAI-1). They reported that for productive infection to take place more than one exposure is required, which was considered similar to human sexual transmission (Girard et al., 1998). Currently, infection of rhesus macaques with SIV is the most extensively studied experimental animal model for immunodeficiency virus transmission. SIV infection in macaques can be established with an acute early viremia, subsequently causing lymphadenopathy and a specific drop in CD4+ PBMCs (Daniel et al., 1985; Letvin et al., 1985). While early chimp studies examined both cell-free and cell-associated SIV inocula, most macaque studies have employed high-dose cell-free SIV inoculums (Miller et al., 1989, 1994; Sodora et al., 1998). These studies provide insights into mucosal transmission and help to generate working models for the events that occur during acute infection. Studies that use infected cells as a source of infection are very few specifically in context of mucosal infection. Although SIV-infected cells efficiently infect macaques when delivered intravenously (Almond et al., 1995), the vaginal mucosa of rhesus macaques presents a barrier to both infected cells and virus during mucosal challenge (Almond et al., 1995; Miller et al., 1989). Vaginal mucosal barrier of macaques is relatively refractory to transmission when cell-associated SIV inoculum is administered through this route. A study by Sodora et al. demonstrated that cell-free SIV could transmit SIV more efficiently in comparison to cell-associated HIV when inoculated vaginally (Sodora et al., 1998). As few as 2 SIVinfected PBMCs could efficiently transmit the virus through intravenous route, whereas 10,000 SIV-infected PBMCs from the same stock failed to infect macaques through vaginal route. Although the cell-free infections are attractive because of their reproducibility, the high doses involved in the challenges generally represent more viral copies than are present in most seminal fluids (Mayer et al., 1999). The low efficiency of cell-associated inoculums in SIV vaginal transmission studies has been speculated to be in part due to the limited viability of the viral stock used or an inadequate dosing (Anderson, 2010). The early SIV studies (Miller et al., 1989, 1994; Sodora et al., 1998) discouraged the use of cell-associated virus for mucosal challenge of rhesus macaques. More recent studies have reexamined cell-associated SIV transmission via vaginal route in the presence of microbreaches with the rationale that these occur commonly during sexual intercourse (Kaizu et al., 2006; Weiler et al., 2008).

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4.2 Microbreaches and Cell-Associated Virus In humans, mucosal exposure to HIV occurs during vaginal and/or anal sex between infected and uninfected individuals. Intercourse causes microbreaches in the mucosal surface of vagina, penis, and anus. These breaches can be exacerbated by repeated sexual intercourse and set the stage for greater access to target cells for HIV. A disrupted mucosal surface and local inflammatory response are features of genital coinfections such as HSV-2, which are strongly associated with increased risk of transmission of HIV (Van de Perre et al., 2008; Wald and Link, 2002). These result in an environment that makes the target cells of HIV more accessible to the virus and infected cells. To study transmission of virus in the presence of microbreaches, Kaizu et al. used a model where genital ulcers were induced in the female reproductive tract. In the setting of ulcerative genital process they reported intravaginal administration of SIVmac239-infected PBMCs yielded persistent infection of mature female cynomolgus macaques (Kaizu et al., 2006). A follow-up report from the same group dissecting the earliest events in vaginal dissemination found a preferential association of vRNA with induced ulcers during the first few days of infection (Weiler et al., 2008). In this model, the allogenic cells were found to migrate rapidly from the vaginal site of the inoculum to the draining lymph nodes. In 2002, another group examined the events that take place when virusinfected cells cross the epithelial barrier (Ignatius et al., 2002). The study used immature DCs and T cells infected with SIV ex vivo as a viral source that was administered through a subcutaneous route. They found both cellfree and cell-associated virus to be infectious. However, in the acute phase of infection, the draining lymph nodes of cell-free inoculated monkeys were predominated by virus-producing CD4+ T cells, whereas in case of cellassociated inoculum both macrophages and T cells were positive for SIV. The different patterns of infection may indicate that the modes of infection can influence which host cells are initially targeted.

4.3 Hormones and Cell-Associated Virus In addition to sexual intercourse-related microtrauma, the hormonal state of the individual can have a bearing on the occurrence of infection, particularly in the female genital tract. Progestin treatment of female macaques can enable systemic and persistent infection following intravaginal exposure to cell-associated SIV (Marx et al., 1996). Progesterone treatment mimics the luteal phase of menstrual cycle and facilitates infection. Progestins block

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estrogen production from ovaries and induce thinning of the vaginal epithelium. The infected cells were tracked in tissues and blood after vaginal application using CFSE labeling. Studies using cell-associated cervico vaginal inocula found that infected cells could penetrate the mucosa and contribute to the spread to distal sites. Frozen stocks of macaque splenocytes at the peak of viremia (SIVmac251) were used as the source of inoculum intravaginally in medroxyprogesterone acetate (Depo-Provera)-treated macaques (Salle et al., 2010). They detected labeled cells in draining lymph nodes and peripheral blood as early as 21 h postinfection. SIV-infected cells in lamina propria of vaginal mucosa were identified by in situ hybridization at 21 h postexposure, as well as in T-cell areas of distal lymph nodes at 21 and 45 h postexposure, indicating inoculated SIV disseminated quickly to distant tissues such as axillary lymph nodes as early as 45 h.

4.4 Atraumatic Exposure and Cell-Associated Virus In the absence of microtrauma or hormonal factors, infected cells can potentially infiltrate mucosal layers to initiate infection. CD4+ T cells may actively migrate through mucosal tissue (Ibata et al., 1997; Khanna et al., 2002; Salle et al., 2010) with potential to transmit to resident cells either by contacting them directly or by releasing free virus. Also macrophages and DCs can contribute to virus spread by trans-infection as reviewed in Section 3. In 2013, a report by Kolodkin-Gal et al. examined the relative efficiency at which HIV- and SIV-infected donor mononuclear cells vs cell-free virus initiates mucosal infection under atraumatic conditions (Kolodkin-Gal et al., 2013). A three-dimensional sealed human colonic mucosa explant system was developed to study early events in infection. In this system, PBMCs infected with a replication-competent R5-tropic HIV-1, GFP-expressing strain, and NL4-3-BaL-GFP were observed to cross the epithelium and initiate infection of host target cell, whereas cell-free could not. This observation was also demonstrated using cell-associated and cell-free SIVmac251 virus in simian colonic tissue explants. In order to validate these results in vivo, cell-associated and cell-free SIVmac251 was administered intrarectally in macaques in four successive challenges. Cell-associated virus could initiate infection following two challenges, whereas cell-free virus did not indicating a higher efficiency of cell-associated virus in transmission. The viral titer of the inoculum (24,000 viral DNA copies/inoculum) employed is comparable to that found in semen of HIV-infected males physiologically (up to 80,000 viral DNA copies/mL) (Politch et al., 2009; Salle et al., 2010;

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Wolff and Anderson, 1988). When considering high-dose intravaginal challenge in rhesus macaques, these generally exceed levels in genital secretions reported in HIV-infected patients (Barouch et al., 2012; Keele et al., 2009; Liu et al., 2010; Stekler et al., 2008). Given the differences in neutralization and sensitivity to antiretrovirals of cell-associated HIV, further studies of transmission using cell-associated virus are needed to assess the efficacy of antibodies, drugs, or microbicides to prevent mucosal transmission.

4.5 Humanized Mouse Models and Cell-to-Cell Infection NHP models have been the dominant model for studying transmission and dissemination following mucosal challenge. While the similarities between SIV and HIV infection are clear, there is no substitute for testing vaccines or preventive strategies to studying HIV (Deruaz and Luster, 2013). This is especially true in case of testing candidate inhibitors against HIV that may not have efficacy against SIV. The development of Hu-mice has been an important advance for studying HIV biology in vivo which has allowed researchers to overcome numerous blocks to replication of HIV in mice (Aldrovandi et al., 1993; Mosier et al., 1991; Namikawa et al., 1988). Hu-mice are generated by xenografting human cells and tissues into immunocompromised mice to reconstitute a functional human immune system for many months. The presence of human CD4 T cells in Hu-mice enables the study of HIV infection of human cells in an in vivo context. The humanized peripheral blood leukocyte (Hu-PBL) model employs severe combined immunodeficient (SCID) mice and transplanted them with human peripheral mononuclear cells to study mucosal transmission of HIV. Two early studies used this model to demonstrate effective vaginal transmission of cell-associated HIV. Khanna et al. demonstrated that human PBMCs infected with HIVBa-L (CCR5 tropic) are transmitted when applied intravaginally into Hu-PBL mice (Khanna et al., 2002). The mice were pretreated with the progestin, Depo-Provera, to increase the susceptibility of vaginal tissue to infection. They found that R5-tropic virus could be transmitted more efficiently in comparison to X4-tropic virus, a situation closely resembling the one in human beings where founder viruses that are detected early on in infection are mostly R5-tropic. In addition, their work provides a model for mucosal transmission of cell-associated HIV-1. The paucity of human cells engraftment of the reproductive tract of the Hu-PBL mice directly controls for any infection resulting from cell-free virus released in vaginal tissue and makes a case for transmission by migrating

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infected cells. Using the same model another group used a fluorescently labeled cell-associated HIV to demonstrate vaginal transmission resulting in virus replication and systemic dissemination (Di Fabio et al., 2001). Subsequently the Hu-PBL has been used to evaluate the efficacy of several microbicidal agents by various groups (Di Fabio et al., 2001; Khanna et al., 2002; Olmsted et al., 2005). The Hu-PBL models are predominantly comprised of xenoreactive T cells and lack other lineages that may be involved in transmission such as myeloid and DCs. The engraftment of human stem cells and stromal tissue to promote their development is exploited in the bone marrow, liver, thymus (BLT)humanized mouse model. The use of human fetal liver and thymus tissue is cotransplanted under the kidney capsule of NOD/SCID mice and followed by engraftment with autologous CD34+ hematopoietic stem cells. The BLT model supports the development of all major lineages of human immune system with the presence of engrafted cells in lymph nodes, gastrointestinal tract, and genital tract (Akkina, 2013; Denton and Garcia, 2011, 2012). Transmission of HIV after vaginal (Stoddart et al., 2011) and rectal (Sun et al., 2007) exposure by cell-free virus has been successfully demonstrated in the BLT mouse model under atraumatic conditions. Cellassociated HIV has also been found to mediate vaginal transmission in the BLT model (Purcell et al., 2012). When testing microbicide strategies, Purcell et al. reported a lower efficacy of topical application of tenofovir against cell-associated virus in comparison to cell-free virus. Both NHPs and small animals have served as models for vaginal transmission of HIV using both cell-free as well as cell-associated inoculums. Clarity regarding the relative contribution of each requires careful consideration of the relative titers of the two sources of virus in physiological sources, which is not frequently assessed. Nonetheless, two conclusions can be drawn from the data generated from both the model systems: the first is that infected cells can give rise to HIV infection, and the second is that infection can be disseminated systemically when infected cells are the source of initial infection. A related question is whether cell-to-cell transfer of HIV occurs in vivo for spread within or between lymphoid tissue sites. Cell culture data have readily shown that in vitro, formation of VSs between infected and uninfected T cells when they are in contact leads to more efficient viral transfer. To examine the role of cell–cell transmission in in vivo context, Murooka et al. investigated cell–cell transmission mechanisms during the spread of virus locally and systemically using the BLT model

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(Murooka et al., 2012). They employed a GFP-encoding R5-tropic virus and multiphoton intravital microscopy to study the dynamic behavior of infected cells in the lymph nodes of infected mice. HIV–GFP-infected T central memory cells were injected into the footpad of BLT mice, and the draining lymph nodes were observed for reduction in motility that may be indicative of putative VSs. They observed reduced motility of these cells and elongated infected cells in a subset of these cells. The reduced motility of infected cells correlated with a tethering phenotype whereby the infected cells appeared to be tethered in place within the draining lymph node. A key experiment in this study was the treatment of infected mice with an inhibitor of sphingosine monophosphate receptor, which can block egress of T cells from lymph nodes. When administered infected T-cell egress from the draining lymph nodes to efferent lymph vessels was blocked and this prevented systemic viral dissemination. This study provides evidence that lymphocyte migration can be essential for spread of HIV from a local draining lymph node to become a systemic infection. The humanized mouse models have also been exploited to understand the contribution of trans-infection for virus spread in vivo (Sewald et al., 2015). By injecting HIV into footpads of Hu-mice and monitoring their arrival at the draining lymph nodes, they found that trans-infection by sinus-lining macrophages CD169/Siglec-1 was involved acquisition of cell-free HIV-1. The capture by CD169 was required to initiate transinfection of T cells in this model, indicating that cell-free virus can depend upon cells to concentrate the virus and pass it on to susceptible T cells. This study provided compelling evidence for the importance of trans-infection in vivo.

5. CONCLUSIONS HIV exploits the interactions between immune cells to facilitate its spread and to evade innate and adaptive immune responses. We are becoming increasingly aware that cell–cell interactions may drive much of the immunopathogenesis of HIV infection, so blocking these interactions specifically may have the potential to alter the natural history of HIV infections. Because cell-to-cell infections can differ in their sensitivity to antibodies, drugs, and other restriction factors, the role that various cell–cell interactions play during transmission and chronic infection should be established with greater clarity. Understanding the mechanisms of antibody evasion will be important for vaccine design. Consideration of cell-to-cell transmission

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and local tissue penetration when choosing drug regimens may help to address the possibility that low levels of cell–cell replication may continue even during treatment with HAART. Lastly, the role that cell-associated virus plays during transmission and dissemination should help to inform us about what antibodies or cellular immune responses may be effective when developing preventive vaccines.

REFERENCES Abela, I.A., Berlinger, L., Schanz, M., Reynell, L., Gunthard, H.F., Rusert, P., Trkola, A., 2012. Cell-cell transmission enables HIV-1 to evade inhibition by potent CD4bs directed antibodies. PLoS Pathog. 8, e1002634. Agosto, L.M., Zhong, P., Munro, J., Mothes, W., 2014. Highly active antiretroviral therapies are effective against HIV-1 cell-to-cell transmission. PLoS Pathog. 10, e1003982. Akiyama, H., Miller, C., Patel, H.V., Hatch, S.C., Archer, J., Ramirez, N.G., Gummuluru, S., 2014. Virus particle release from glycosphingolipid-enriched microdomains is essential for dendritic cell-mediated capture and transfer of HIV-1 and henipavirus. J. Virol. 88, 8813. Akiyama, H., Ramirez, N.G., Gudheti, M.V., Gummuluru, S., 2015. CD169-mediated trafficking of HIV to plasma membrane invaginations in dendritic cells attenuates efficacy of anti-gp120 broadly neutralizing antibodies. PLoS Pathog. 11, e1004751. Akkina, R., 2013. New generation humanized mice for virus research: comparative aspects and future prospects. Virology 435, 14. Aldrovandi, G.M., Feuer, G., Gao, L., Jamieson, B., Kristeva, M., Chen, I.S., Zack, J.A., 1993. The SCID-hu mouse as a model for HIV-1 infection. Nature 363, 732. Almond, N., Kent, K., Cranage, M., Rud, E., Clarke, B., Stott, E.J., 1995. Protection by attenuated simian immunodeficiency virus in macaques against challenge with virusinfected cells. Lancet 345, 1342. Alvarez, R.A., Hamlin, R.E., Monroe, A., Moldt, B., Hotta, M.T., Rodriguez Caprio, G., Fierer, D.S., Simon, V., Chen, B.K., 2014. HIV-1 Vpu antagonism of tetherin inhibits antibody-dependent cellular cytotoxic responses by natural killer cells. J. Virol. 88, 6031. Anderson, D.J., 2010. Finally, a macaque model for cell-associated SIV/HIV vaginal transmission. J. Infect. Dis. 202, 333. Anderson, D.J., Politch, J.A., Nadolski, A.M., Blaskewicz, C.D., Pudney, J., Mayer, K.H., 2010. Targeting Trojan horse leukocytes for HIV prevention. AIDS 24, 163. Arias, J.F., Heyer, L.N., von Bredow, B., Weisgrau, K.L., Moldt, B., Burton, D.R., Rakasz, E.G., Evans, D.T., 2014. Tetherin antagonism by Vpu protects HIV-infected cells from antibody-dependent cell-mediated cytotoxicity. Proc. Natl. Acad. Sci. U.S.A. 111, 6425. Arthos, J., Cicala, C., Martinelli, E., Macleod, K., Van Ryk, D., Wei, D., Xiao, Z., Veenstra, T.D., Conrad, T.P., Lempicki, R.A., McLaughlin, S., Pascuccio, M., Gopaul, R., McNally, J., Cruz, C.C., Censoplano, N., Chung, E., Reitano, K.N., Kottilil, S., Goode, D.J., Fauci, A.S., 2008. HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nat. Immunol. 9, 301. Barouch, D.H., Liu, J., Li, H., Maxfield, L.F., Abbink, P., Lynch, D.M., Iampietro, M.J., SanMiguel, A., Seaman, M.S., Ferrari, G., Forthal, D.N., Ourmanov, I., Hirsch, V.M., Carville, A., Mansfield, K.G., Stablein, D., Pau, M.G., Schuitemaker, H., Sadoff, J.C., Billings, E.A., Rao, M., Robb, M.L., Kim, J.H., Marovich, M.A., Goudsmit, J.,

74

K.M. Law et al.

Michael, N.L., 2012. Vaccine protection against acquisition of neutralization-resistant SIV challenges in rhesus monkeys. Nature 482, 89. Bastarache, S.M., Mesplede, T., Donahue, D.A., Sloan, R.D., Wainberg, M.A., 2014. Fitness impaired drug resistant HIV-1 is not compromised in cell-to-cell transmission or establishment of and reactivation from latency. Viruses 6, 3487. Baxter, A.E., Russell, R.A., Duncan, C.J., Moore, M.D., Willberg, C.B., Pablos, J.L., Finzi, A., Kaufmann, D.E., Ochsenbauer, C., Kappes, J.C., Groot, F., Sattentau, Q.J., 2014. Macrophage infection via selective capture of HIV-1-infected CD4 + T cells. Cell Host Microbe 16, 711. Bernard-Stoecklin, S., Gommet, C., Cavarelli, M., Le Grand, R., 2014. Nonhuman primate models for cell-associated simian immunodeficiency virus transmission: the need to better understand the complexity of HIV mucosal transmission. J. Infect. Dis. 210 (Suppl. 3), S660. Blanco, J., Bosch, B., Fernandez-Figueras, M.T., Barretina, J., Clotet, B., Este, J.A., 2004. High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells. J. Biol. Chem. 279, 51305. Blauvelt, A., Asada, H., Saville, M.W., Klaus-Kovtun, V., Altman, D.J., Yarchoan, R., Katz, S.I., 1997. Productive infection of dendritic cells by HIV-1 and their ability to capture virus are mediated through separate pathways. J. Clin. Invest. 100, 2043. Brenchley, J.M., Schacker, T.W., Ruff, L.E., Price, D.A., Taylor, J.H., Beilman, G.J., Nguyen, P.L., Khoruts, A., Larson, M., Haase, A.T., Douek, D.C., 2004. CD4 + T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J. Exp. Med. 200, 749. Bromley, S.K., Mempel, T.R., Luster, A.D., 2008. Orchestrating the orchestrators: chemokines in control of T cell traffic. Nat. Immunol. 9, 970. Byrareddy, S.N., Kallam, B., Arthos, J., Cicala, C., Nawaz, F., Hiatt, J., Kersh, E.N., McNicholl, J.M., Hanson, D., Reimann, K.A., Brameier, M., Walter, L., Rogers, K., Mayne, A.E., Dunbar, P., Villinger, T., Little, D., Parslow, T.G., Santangelo, P.J., Villinger, F., Fauci, A.S., Ansari, A.A., 2014. Targeting alpha4beta7 integrin reduces mucosal transmission of simian immunodeficiency virus and protects gut-associated lymphoid tissue from infection. Nat. Med. 20, 1397. Cameron, P.U., Freudenthal, P.S., Barker, J.M., Gezelter, S., Inaba, K., Steinman, R.M., 1992. Dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to CD4+ T cells. Science 257, 383. Casartelli, N., Sourisseau, M., Feldmann, J., Guivel-Benhassine, F., Mallet, A., Marcelin, A.G., Guatelli, J., Schwartz, O., 2010. Tetherin restricts productive HIV-1 cell-to-cell transmission. PLoS Pathog. 6, e1000955. Chen, P., Hubner, W., Spinelli, M.A., Chen, B.K., 2007. Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Envdependent neutralization-resistant virological synapses. J. Virol. 81, 12582. Chen, J., Kovacs, J.M., Peng, H., Rits-Volloch, S., Lu, J., Park, D., Zablowsky, E., Seaman, M.S., Chen, B., 2015. HIV-1 ENVELOPE. Effect of the cytoplasmic domain on antigenic characteristics of HIV-1 envelope glycoprotein. Science 349, 191. Cheynier, R., Henrichwark, S., Hadida, F., Pelletier, E., Oksenhendler, E., Autran, B., Wain-Hobson, S., 1994. HIV and T cell expansion in splenic white pulps is accompanied by infiltration of HIV-specific cytotoxic T lymphocytes. Cell 78, 373. Cicala, C., Martinelli, E., McNally, J.P., Goode, D.J., Gopaul, R., Hiatt, J., Jelicic, K., Kottilil, S., Macleod, K., O’Shea, A., Patel, N., Van Ryk, D., Wei, D., Pascuccio, M., Yi, L., McKinnon, L., Izulla, P., Kimani, J., Kaul, R., Fauci, A.S., Arthos, J., 2009. The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1. Proc. Natl. Acad. Sci. U.S.A. 106, 20877.

HIV-1 Cell-to-Cell Transmission

75

Conde, P., Rodriguez, M., van der Touw, W., Jimenez, A., Burns, M., Miller, J., Brahmachary, M., Chen, H.M., Boros, P., Rausell-Palamos, F., Yun, T.J., Riquelme, P., Rastrojo, A., Aguado, B., Stein-Streilein, J., Tanaka, M., Zhou, L., Zhang, J., Lowary, T.L., Ginhoux, F., Park, C.G., Cheong, C., Brody, J., Turley, S.J., Lira, S.A., Bronte, V., Gordon, S., Heeger, P.S., Merad, M., Hutchinson, J., Chen, S.H., Ochando, J., 2015. DC-SIGN(+) macrophages control the induction of transplantation tolerance. Immunity 42, 1143. Cooper, A., Garcı´a, M., Petrovas, C., Yamamoto, T., Koup, R.A., Nabel, G.J., 2013. HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration. Nature 498, 376. Dale, B.M., McNerney, G.P., Thompson, D.L., Hubner, W., de Los Reyes, K., Chuang, F.Y., Huser, T., Chen, B.K., 2011. Cell-to-cell transfer of HIV-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion. Cell Host Microbe 10, 551. Daniel, M.D., Letvin, N.L., King, N.W., Kannagi, M., Sehgal, P.K., Hunt, R.D., Kanki, P.J., Essex, M., Desrosiers, R.C., 1985. Isolation of T-cell tropic HTLV-III-like retrovirus from macaques. Science 228, 1201. Del Portillo, A., Tripodi, J., Najfeld, V., Wodarz, D., Levy, D.N., Chen, B.K., 2011. Multiploid inheritance of HIV-1 during cell-to-cell infection. J. Virol. 85, 7169. Delassus, S., Cheynier, R., Wain-Hobson, S., 1992. Nonhomogeneous distribution of human immunodeficiency virus type 1 proviruses in the spleen. J. Virol. 66, 5642. Denton, P.W., Garcia, J.V., 2011. Humanized mouse models of HIV infection. AIDS Rev. 13, 135. Denton, P.W., Garcia, J.V., 2012. Mucosal HIV-1 transmission and prevention strategies in BLT humanized mice. Trends Microbiol. 20, 268. Deruaz, M., Luster, A.D., 2013. BLT humanized mice as model to study HIV vaginal transmission. J. Infect. Dis. 208 (Suppl. 2), S131. de Witte, L., Nabatov, A., Pion, M., Fluitsma, D., de Jong, M.A., de Gruijl, T., Piguet, V., van Kooyk, Y., Geijtenbeek, T.B., 2007. Langerin is a natural barrier to HIV-1 transmission by Langerhans cells. Nat. Med. 13, 367. Di Fabio, S., Giannini, G., Lapenta, C., Spada, M., Binelli, A., Germinario, E., Sestili, P., Belardelli, F., Proietti, E., Vella, S., 2001. Vaginal transmission of HIV-1 in hu-SCID mice: a new model for the evaluation of vaginal microbicides. AIDS 15, 2231. Dimitrov, D.S., Willey, R.L., Sato, H., Chang, L.J., Blumenthal, R., Martin, M.A., 1993. Quantitation of human immunodeficiency virus type 1 infection kinetics. J. Virol. 67, 2182. Ding, J., Tasker, C., Lespinasse, P., Dai, J., Fitzgerald-Bocarsly, P., Lu, W., Heller, D., Chang, T.L., 2015. Integrin alpha4beta7 expression increases HIV susceptibility in activated cervical CD4+ T cells by an HIV attachment-independent mechanism. J. Acquir. Immune Defic. Syndr. 69, 509. Dixit, N.M., Perelson, A.S., 2005. HIV dynamics with multiple infections of target cells. Proc. Natl. Acad. Sci. U.S.A. 102, 8198. Do, T., Murphy, G., Earl, L.A., Del Prete, G.Q., Grandinetti, G., Li, G.H., Estes, J.D., Rao, P., Trubey, C.M., Thomas, J., Spector, J., Bliss, D., Nath, A., Lifson, J.D., Subramaniam, S., 2014. Three-dimensional imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission. J. Virol. 88, 10327. Doitsh, G., Cavrois, M., Lassen, K.G., Zepeda, O., Yang, Z., Santiago, M.L., Hebbeler, A.M., Greene, W.C., 2010. Abortive HIV infection mediates CD4 T cell depletion and inflammation in human lymphoid tissue. Cell 143, 789–801. http://dx.doi.org/10.1016/ j.cell.2010.11.001. Doitsh, G., Galloway, N.L., Geng, X., Yang, Z., Monroe, K.M., Zepeda, O., Hunt, P.W., Hatano, H., Sowinski, S., Mun˜oz-Arias, I., Greene, W.C., 2014. Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection. Nature 505, 509.

76

K.M. Law et al.

Duncan, C.J., Russell, R.A., Sattentau, Q.J., 2013. High multiplicity HIV-1 cell-to-cell transmission from macrophages to CD4 + T cells limits antiretroviral efficacy. AIDS 27, 2201. Duncan, C.J., Williams, J.P., Schiffner, T., Gartner, K., Ochsenbauer, C., Kappes, J., Russell, R.A., Frater, J., Sattentau, Q.J., 2014. High-multiplicity HIV-1 infection and neutralizing antibody evasion mediated by the macrophage-T cell virological synapse. J. Virol. 88, 2025. Durham, N.D., Yewdall, A.W., Chen, P., Lee, R., Zony, C., Robinson, J.E., Chen, B.K., 2012. Neutralization resistance of virological synapse-mediated HIV-1 infection is regulated by the gp41 cytoplasmic tail. J. Virol. 86, 7484. Fletcher, C.V., Staskus, K., Wietgrefe, S.W., Rothenberger, M., Reilly, C., Chipman, J.G., Beilman, G.J., Khoruts, A., Thorkelson, A., Schmidt, T.E., Anderson, J., Perkey, K., Stevenson, M., Perelson, A.S., Douek, D.C., Haase, A.T., Schacker, T.W., 2014. Persistent HIV-1 replication is associated with lower antiretroviral drug concentrations in lymphatic tissues. Proc. Natl. Acad. Sci. U.S.A. 111, 2307. Fortin, J.F., Cantin, R., Lamontagne, G., Tremblay, M., 1997. Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity. J. Virol. 71, 3588. Galloway, N.L., Doitsh, G., Monroe, K.M., Yang, Z., Mun˜oz-Arias, I., Levy, D.N., Greene, W.C., 2015. Cell-to-cell transmission of HIV-1 is required to trigger pyroptotic death of lymphoid-tissue-derived CD4 T cells. Cell Rep. 12, 1555. Garcia, E., Pion, M., Pelchen-Matthews, A., Collinson, L., Arrighi, J.F., Blot, G., Leuba, F., Escola, J.M., Demaurex, N., Marsh, M., Piguet, V., 2005. HIV-1 trafficking to the dendritic cell-T-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse. Traffic 6, 488. Garcia, E., Nikolic, D.S., Piguet, V., 2008. HIV-1 replication in dendritic cells occurs through a tetraspanin-containing compartment enriched in AP-3. Traffic 9, 200. Geijtenbeek, T.B., Kwon, D.S., Torensma, R., van Vliet, S.J., van Duijnhoven, G.C., Middel, J., Cornelissen, I.L., Nottet, H.S., KewalRamani, V.N., Littman, D.R., Figdor, C.G., van Kooyk, Y., 2000. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100, 587. Girard, M., Mahoney, J., Wei, Q., van der Ryst, E., Muchmore, E., Barre-Sinoussi, F., Fultz, P.N., 1998. Genital infection of female chimpanzees with human immunodeficiency virus type 1. AIDS Res. Hum. Retroviruses 14, 1357. Grakoui, A., Bromley, S.K., Sumen, C., Davis, M.M., Shaw, A.S., Allen, P.M., Dustin, M.L., 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221. Gratton, S., Cheynier, R., Dumaurier, M.J., Oksenhendler, E., Wain-Hobson, S., 2000. Highly restricted spread of HIV-1 and multiply infected cells within splenic germinal centers. Proc. Natl. Acad. Sci. U.S.A. 97, 14566. Guo, H., Callaway, J.B., Ting, J.P., 2015. Inflammasomes: mechanism of action, role in disease, and therapeutics. Nat. Med. 21, 677. Haase, A.T., 2005. Perils at mucosal front lines for HIV and SIV and their hosts. Nat. Rev. Immunol. 5, 783. Haase, A.T., 2011. Early events in sexual transmission of HIV and SIV and opportunities for interventions. Annu. Rev. Med. 62, 127. Hait, S.H., Soares, E.A., Sprinz, E., Arthos, J., Machado, E.S., Soares, M.A., 2015. Worldwide genetic features of HIV-1 Env alpha4beta7 binding motif: the local dissemination impact of the LDI tripeptide. J. Acquir. Immune Defic. Syndr. 70, 463. Herold, N., Anders-Osswein, M., Glass, B., Eckhardt, M., Muller, B., Krausslich, H.G., 2014. HIV-1 entry in SupT1-R5, CEM-ss, and primary CD4 + T cells occurs at the plasma membrane and does not require endocytosis. J. Virol. 88, 13956.

HIV-1 Cell-to-Cell Transmission

77

Hioe, C.E., Chien Jr., P.C., Lu, C., Springer, T.A., Wang, X.H., Bandres, J., Tuen, M., 2001. LFA-1 expression on target cells promotes human immunodeficiency virus type 1 infection and transmission. J. Virol. 75, 1077. Hioe, C.E., Tuen, M., Vasiliver-Shamis, G., Alvarez, Y., Prins, K.C., Banerjee, S., Nadas, A., Cho, M.W., Dustin, M.L., Kachlany, S.C., 2011. HIV envelope gp120 activates LFA-1 on CD4 T-lymphocytes and increases cell susceptibility to LFA-1-targeting leukotoxin (LtxA). PLoS One 6, e23202. Hladik, F., Sakchalathorn, P., Ballweber, L., Lentz, G., Fialkow, M., Eschenbach, D., McElrath, M.J., 2007. Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1. Immunity 26, 257. Houzet, L., Matusali, G., Dejucq-Rainsford, N., 2014. Origins of HIV-infected leukocytes and virions in semen. J. Infect. Dis. 210 (Suppl. 3), S622. Hu, J., Gardner, M.B., Miller, C.J., 2000. Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells. J. Virol. 74, 6087. Hubner, W., McNerney, G.P., Chen, P., Dale, B.M., Gordon, R.E., Chuang, F.Y., Li, X.D., Asmuth, D.M., Huser, T., Chen, B.K., 2009. Quantitative 3D video microscopy of HIV transfer across T cell virological synapses. Science 323, 1743. Ibata, B., Parr, E.L., King, N.J., Parr, M.B., 1997. Migration of foreign lymphocytes from the mouse vagina into the cervicovaginal mucosa and to the iliac lymph nodes. Biol. Reprod. 56, 537. Ignatius, R., Tenner-Racz, K., Messmer, D., Gettie, A., Blanchard, J., Luckay, A., Russo, C., Smith, S., Marx, P.A., Steinman, R.M., Racz, P., Pope, M., 2002. Increased macrophage infection upon subcutaneous inoculation of rhesus macaques with simian immunodeficiency virus-loaded dendritic cells or T cells but not with cell-free virus. J. Virol. 76, 9787. Izquierdo-Useros, N., Naranjo-Gomez, M., Archer, J., Hatch, S.C., Erkizia, I., Blanco, J., Borras, F.E., Puertas, M.C., Connor, J.H., Fernandez-Figueras, M.T., Moore, L., Clotet, B., Gummuluru, S., Martinez-Picado, J., 2009. Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway. Blood 113, 2732. Izquierdo-Useros, N., Lorizate, M., Puertas, M.C., Rodriguez-Plata, M.T., Zangger, N., Erikson, E., Pino, M., Erkizia, I., Glass, B., Clotet, B., Keppler, O.T., Telenti, A., Krausslich, H.G., Martinez-Picado, J., 2012. Siglec-1 is a novel dendritic cell receptor that mediates HIV-1 trans-infection through recognition of viral membrane gangliosides. PLoS Biol. 10, e1001448. Izquierdo-Useros, N., Lorizate, M., McLaren, P.J., Telenti, A., Kra¨usslich, H.-G.G., Martinez-Picado, J., 2014. HIV-1 capture and transmission by dendritic cells: the role of viral glycolipids and the cellular receptor Siglec-1. PLoS Pathog. 10, e1004146. Jiang, J., Aiken, C., 2007. Maturation-dependent human immunodeficiency virus type 1 particle fusion requires a carboxyl-terminal region of the gp41 cytoplasmic tail. J. Virol. 81, 9999. Joag, V.R., McKinnon, L.R., Liu, J., Kidane, S.T., Yudin, M.H., Nyanga, B., Kimwaki, S., Besel, K.E., Obila, J.O., Huibner, S., Oyugi, J.O., Arthos, J., Anzala, O., Kimani, J., Ostrowski, M.A., Toronto, H.I.V.R.G., Kaul, R., 2016. Identification of preferential CD4(+) T-cell targets for HIV infection in the cervix. Mucosal Immunol. 9, 1. Jolly, C., Sattentau, Q.J., 2007. Human immunodeficiency virus type 1 assembly, budding, and cell-cell spread in T cells take place in tetraspanin-enriched plasma membrane domains. J. Virol. 81, 7873. Jolly, C., Kashefi, K., Hollinshead, M., Sattentau, Q.J., 2004. HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse. J. Exp. Med. 199, 283.

78

K.M. Law et al.

Jolly, C., Mitar, I., Sattentau, Q.J., 2007a. Adhesion molecule interactions facilitate human immunodeficiency virus type 1-induced virological synapse formation between T cells. J. Virol. 81, 13916. Jolly, C., Mitar, I., Sattentau, Q.J., 2007b. Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1. J. Virol. 81, 5547. Jolly, C., Booth, N.J., Neil, S.J., 2010. Cell-cell spread of human immunodeficiency virus type 1 overcomes tetherin/BST-2-mediated restriction in T cells. J. Virol. 84, 12185. Josefsson, L., Palmer, S., Faria, N.R., Lemey, P., Casazza, J., Ambrozak, D., Kearney, M., Shao, W., Kottilil, S., Sneller, M., Mellors, J., Coffin, J.M., Maldarelli, F., 2013. Single cell analysis of lymph node tissue from HIV-1 infected patients reveals that the majority of CD4 + T-cells contain one HIV-1 DNA molecule. PLoS Pathog. 9, e1003432. Joyner, A.S., Willis, J.R., Crowe Jr., J.E., Aiken, C., 2011. Maturation-induced cloaking of neutralization epitopes on HIV-1 particles. PLoS Pathog. 7, e1002234. Jung, A., Maier, R., Vartanian, J.P., Bocharov, G., Jung, V., Fischer, U., Meese, E., WainHobson, S., Meyerhans, A., 2002. Recombination: multiply infected spleen cells in HIV patients. Nature 418, 144. Kaizu, M., Weiler, A.M., Weisgrau, K.L., Vielhuber, K.A., May, G., Piaskowski, S.M., Furlott, J., Maness, N.J., Friedrich, T.C., Loffredo, J.T., Usborne, A., Rakasz, E.G., 2006. Repeated intravaginal inoculation with cell-associated simian immunodeficiency virus results in persistent infection of nonhuman primates. J. Infect. Dis. 194, 912. Keele, B.F., Li, H., Learn, G.H., Hraber, P., Giorgi, E.E., Grayson, T., Sun, C., Chen, Y., Yeh, W.W., Letvin, N.L., Mascola, J.R., Nabel, G.J., Haynes, B.F., Bhattacharya, T., Perelson, A.S., Korber, B.T., Hahn, B.H., Shaw, G.M., 2009. Low-dose rectal inoculation of rhesus macaques by SIVsmE660 or SIVmac251 recapitulates human mucosal infection by HIV-1. J. Exp. Med. 206, 1117. Khanna, K.V., Whaley, K.J., Zeitlin, L., Moench, T.R., Mehrazar, K., Cone, R.A., Liao, Z., Hildreth, J.E., Hoen, T.E., Shultz, L., Markham, R.B., 2002. Vaginal transmission of cell-associated HIV-1 in the mouse is blocked by a topical, membrane-modifying agent. J. Clin. Invest. 109, 205. Kolodkin-Gal, D., Hulot, S.L., Korioth-Schmitz, B., Gombos, R.B., Zheng, Y., Owuor, J., Lifton, M.A., Ayeni, C., Najarian, R.M., Yeh, W.W., Asmal, M., Zamir, G., Letvin, N.L., 2013. Efficiency of cell-free and cell-associated virus in mucosal transmission of human immunodeficiency virus type 1 and simian immunodeficiency virus. J. Virol. 87, 13589. Konvalinka, J., Krausslich, H.G., Muller, B., 2015. Retroviral proteases and their roles in virion maturation. Virology 479–480, 403. Krementsov, D.N., Weng, J., Lambele, M., Roy, N.H., Thali, M., 2009. Tetraspanins regulate cell-to-cell transmission of HIV-1. Retrovirology 6, 64. Kuhl, B.D., Sloan, R.D., Donahue, D.A., Bar-Magen, T., Liang, C., Wainberg, M.A., 2010. Tetherin restricts direct cell-to-cell infection of HIV-1. Retrovirology 7, 115. Kwon, D.S., Gregorio, G., Bitton, N., Hendrickson, W.A., Littman, D.R., 2002. DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection. Immunity 16, 135. Ladinsky, M.S., Kieffer, C., Olson, G., Deruaz, M., Vrbanac, V., Tager, A.M., Kwon, D.S., Bjorkman, P.J., 2014. Electron tomography of HIV-1 infection in gut-associated lymphoid tissue. PLoS Pathog. 10, e1003899. Laguette, N., Sobhian, B., Casartelli, N., Ringeard, M., Chable-Bessia, C., Segeral, E., Yatim, A., Emiliani, S., Schwartz, O., Benkirane, M., 2011. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474, 654. Lee, K.H., Holdorf, A.D., Dustin, M.L., Chan, A.C., Allen, P.M., Shaw, A.S., 2002. T cell receptor signaling precedes immunological synapse formation. Science 295, 1539.

HIV-1 Cell-to-Cell Transmission

79

Lepelley, A., Louis, S., Sourisseau, M., Law, H.K., Pothlichet, J., Schilte, C., Chaperot, L., Plumas, J., Randall, R.E., Si-Tahar, M., Mammano, F., Albert, M.L., Schwartz, O., 2011. Innate sensing of HIV-infected cells. PLoS Pathog. 7, e1001284. Letvin, N.L., Daniel, M.D., Sehgal, P.K., Desrosiers, R.C., Hunt, R.D., Waldron, L.M., MacKey, J.J., Schmidt, D.K., Chalifoux, L.V., King, N.W., 1985. Induction of AIDS-like disease in macaque monkeys with T-cell tropic retrovirus STLV-III. Science 230, 71. Li, Q., Estes, J.D., Schlievert, P.M., Duan, L., Brosnahan, A.J., Southern, P.J., Reilly, C.S., Peterson, M.L., Schultz-Darken, N., Brunner, K.G., Nephew, K.R., Pambuccian, S., Lifson, J.D., Carlis, J.V., Haase, A.T., 2009. Glycerol monolaurate prevents mucosal SIV transmission. Nature 458, 1034. Liu, J., Keele, B.F., Li, H., Keating, S., Norris, P.J., Carville, A., Mansfield, K.G., Tomaras, G.D., Haynes, B.F., Kolodkin-Gal, D., Letvin, N.L., Hahn, B.H., Shaw, G.M., Barouch, D.H., 2010. Low-dose mucosal simian immunodeficiency virus infection restricts early replication kinetics and transmitted virus variants in rhesus monkeys. J. Virol. 84, 10406. Lorenzo-Redondo, R., Fryer, H.R., Bedford, T., Kim, E.Y., Archer, J., Kosakovsky Pond, S.L., Chung, Y.S., Penugonda, S., Chipman, J.G., Fletcher, C.V., Schacker, T.W., Malim, M.H., Rambaut, A., Haase, A.T., McLean, A.R., Wolinsky, S.M., 2016. Persistent HIV-1 replication maintains the tissue reservoir during therapy. Nature 530, 51. Loving, R., Li, K., Wallin, M., Sjoberg, M., Garoff, H., 2008. R-Peptide cleavage potentiates fusion-controlling isomerization of the intersubunit disulfide in Moloney murine leukemia virus Env. J. Virol. 82, 2594. Lu, W., Demers, A.J., Ma, F., Kang, G., Yuan, Z., Wan, Y., Li, Y., Xu, J., Lewis, M., Li, Q., 2015. Next-generation mRNA sequencing reveals pyroptosis-induced CD4 + T cell death in early simian immunodeficiency virus-infected lymphoid tissues. J. Virol. 90, 1080. Maddon, P.J., Dalgleish, A.G., McDougal, J.S., Clapham, P.R., Weiss, R.A., Axel, R., 1986. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47, 333. Malbec, M., Porrot, F., Rua, R., Horwitz, J., Klein, F., Halper-Stromberg, A., Scheid, J.F., Eden, C., Mouquet, H., Nussenzweig, M.C., Schwartz, O., 2013. Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission. J. Exp. Med. 210, 2813. Martin, N., Welsch, S., Jolly, C., Briggs, J.A., Vaux, D., Sattentau, Q.J., 2010. Virological synapse-mediated spread of human immunodeficiency virus type 1 between T cells is sensitive to entry inhibition. J. Virol. 84, 3516. Martinelli, E., Cicala, C., Van Ryk, D., Goode, D.J., Macleod, K., Arthos, J., Fauci, A.S., 2007. HIV-1 gp120 inhibits TLR9-mediated activation and IFN-{alpha} secretion in plasmacytoid dendritic cells. Proc. Natl. Acad. Sci. U.S.A. 104, 3396. Marx, P.A., Spira, A.I., Gettie, A., Dailey, P.J., Veazey, R.S., Lackner, A.A., Mahoney, C.J., Miller, C.J., Claypool, L.E., Ho, D.D., Alexander, N.J., 1996. Progesterone implants enhance SIV vaginal transmission and early virus load. Nat. Med. 2, 1084. Mavigner, M., Cazabat, M., Dubois, M., L’Faqihi, F.E., Requena, M., Pasquier, C., Klopp, P., Amar, J., Alric, L., Barange, K., Vinel, J.P., Marchou, B., Massip, P., Izopet, J., Delobel, P., 2012. Altered CD4 + T cell homing to the gut impairs mucosal immune reconstitution in treated HIV-infected individuals. J. Clin. Invest. 122, 62. Mayer, K.H., Boswell, S., Goldstein, R., Lo, W., Xu, C., Tucker, L., DePasquale, M.P., D’Aquila, R., Anderson, D.J., 1999. Persistence of human immunodeficiency virus in semen after adding indinavir to combination antiretroviral therapy. Clin. Infect. Dis. 28, 1252.

80

K.M. Law et al.

McDonald, D., Wu, L., Bohks, S.M., KewalRamani, V.N., Unutmaz, D., Hope, T.J., 2003. Recruitment of HIV and its receptors to dendritic cell-T cell junctions. Science 300, 1295. McMichael, A.J., Rowland-Jones, S.L., 2001. Cellular immune responses to HIV. Nature 410, 980. Mehandru, S., Poles, M.A., Tenner-Racz, K., Horowitz, A., Hurley, A., Hogan, C., Boden, D., Racz, P., Markowitz, M., 2004. Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J. Exp. Med. 200, 761. Mellors, J.W., Rinaldo Jr., C.R., Gupta, P., White, R.M., Todd, J.A., Kingsley, L.A., 1996. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 272, 1167. Mellors, J.W., Munoz, A., Giorgi, J.V., Margolick, J.B., Tassoni, C.J., Gupta, P., Kingsley, L.A., Todd, J.A., Saah, A.J., Detels, R., Phair, J.P., Rinaldo Jr., C.R., 1997. Plasma viral load and CD4 + lymphocytes as prognostic markers of HIV-1 infection. Ann. Intern. Med. 126, 946. Miller, C.J., Alexander, N.J., Sutjipto, S., Lackner, A.A., Gettie, A., Hendrickx, A.G., Lowenstine, L.J., Jennings, M., Marx, P.A., 1989. Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus. J. Virol. 63, 4277. Miller, C.J., Marthas, M., Torten, J., Alexander, N.J., Moore, J.P., Doncel, G.F., Hendrickx, A.G., 1994. Intravaginal inoculation of rhesus macaques with cell-free simian immunodeficiency virus results in persistent or transient viremia. J. Virol. 68, 6391. Miller, M.J., Hejazi, A.S., Wei, S.H., Cahalan, M.D., Parker, I., 2004. T cell repertoire scanning is promoted by dynamic dendritic cell behavior and random T cell motility in the lymph node. Proc. Natl. Acad. Sci. U.S.A. 101, 998. Miller, C.J., Li, Q., Abel, K., Kim, E.Y., Ma, Z.M., Wietgrefe, S., La Franco-Scheuch, L., Compton, L., Duan, L., Shore, M.D., Zupancic, M., Busch, M., Carlis, J., Wolinsky, S., Haase, A.T., 2005. Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus. J. Virol. 79, 9217. Miyauchi, K., Kim, Y., Latinovic, O., Morozov, V., Melikyan, G.B., 2009a. HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes. Cell 137, 433. Miyauchi, K., Kozlov, M.M., Melikyan, G.B., 2009b. Early steps of HIV-1 fusion define the sensitivity to inhibitory peptides that block 6-helix bundle formation. PLoS Pathog. 5, e1000585. Monks, C.R., Freiberg, B.A., Kupfer, H., Sciaky, N., Kupfer, A., 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82. Monroe, K.M., Yang, Z., Johnson, J.R., Geng, X., Doitsh, G., Krogan, N.J., Greene, W.C., 2014. IFI16 DNA sensor is required for death of lymphoid CD4 T cells abortively infected with HIV. Science 343, 428. Mosier, D.E., Gulizia, R.J., Baird, S.M., Wilson, D.B., Spector, D.H., Spector, S.A., 1991. Human immunodeficiency virus infection of human-PBL-SCID mice. Science 251, 791. Mun˜oz-Arias, I., Doitsh, G., Yang, Z., Sowinski, S., Ruelas, D., Greene, W.C., 2015. Blood-derived CD4 T cells naturally resist pyroptosis during abortive HIV-1 infection. Cell Host Microbe 18, 463. Murakami, T., Ablan, S., Freed, E.O., Tanaka, Y., 2004. Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity. J. Virol. 78, 1026. Murooka, T.T., Deruaz, M., Marangoni, F., Vrbanac, V.D., Seung, E., von Andrian, U.H., Tager, A.M., Luster, A.D., Mempel, T.R., 2012. HIV-infected T cells are migratory vehicles for viral dissemination. Nature 490, 283.

HIV-1 Cell-to-Cell Transmission

81

Namikawa, R., Kaneshima, H., Lieberman, M., Weissman, I.L., McCune, J.M., 1988. Infection of the SCID-hu mouse by HIV-1. Science 242, 1684. Nawaz, F., Cicala, C., Van Ryk, D., Block, K.E., Jelicic, K., McNally, J.P., Ogundare, O., Pascuccio, M., Patel, N., Wei, D., Fauci, A.S., Arthos, J., 2011. The genotype of earlytransmitting HIV gp120s promotes alpha (4) beta(7)-reactivity, revealing alpha (4) beta(7) +/CD4+ T cells as key targets in mucosal transmission. PLoS Pathog. 7, e1001301. Neil, S.J., Zang, T., Bieniasz, P.D., 2008. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451, 425. Nguyen, D.G., Hildreth, J.E., 2003. Involvement of macrophage mannose receptor in the binding and transmission of HIV by macrophages. Eur. J. Immunol. 33, 483. Olmsted, S.S., Khanna, K.V., Ng, E.M., Whitten, S.T., Johnson 3rd, O.N., Markham, R.B., Cone, R.A., Moench, T.R., 2005. Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model. BMC Infect. Dis. 5, 79. Patterson, K.B., Prince, H.A., Kraft, E., Jenkins, A.J., Shaheen, N.J., Rooney, J.F., Cohen, M.S., Kashuba, A.D., 2011. Penetration of tenofovir and emtricitabine in mucosal tissues: implications for prevention of HIV-1 transmission. Sci. Transl. Med. 3, 112re4. Peressin, M., Proust, A., Schmidt, S., Su, B., Lambotin, M., Biedma, M.E., Laumond, G., Decoville, T., Holl, V., Moog, C., 2014. Efficient transfer of HIV-1 in trans and in cis from Langerhans dendritic cells and macrophages to autologous T lymphocytes. AIDS 28, 667. Perez, L.G., Chen, H., Liao, H.X., Montefiori, D.C., 2014. Envelope glycoprotein binding to the integrin alpha4beta7 is not a general property of most HIV-1 strains. J. Virol. 88, 10767. Pilch, B., Mann, M., 2006. Large-scale and high-confidence proteomic analysis of human seminal plasma. Genome Biol. 7, R40. Pino, M., Erkizia, I., Benet, S., Erikson, E., Fernandez-Figueras, M.T., Guerrero, D., Dalmau, J., Ouchi, D., Rausell, A., Ciuffi, A., Keppler, O.T., Telenti, A., Krausslich, H.G., Martinez-Picado, J., Izquierdo-Useros, N., 2015. HIV-1 immune activation induces Siglec-1 expression and enhances viral trans-infection in blood and tissue myeloid cells. Retrovirology 12, 37. Politch, J.A., Mayer, K.H., Anderson, D.J., 2009. Depletion of CD4+ T cells in semen during HIV infection and their restoration following antiretroviral therapy. J. Acquir. Immune Defic. Syndr. 50, 283. Pope, M., Haase, A.T., 2003. Transmission, acute HIV-1 infection and the quest for strategies to prevent infection. Nat. Med. 9, 847. Puigdomenech, I., Massanella, M., Izquierdo-Useros, N., Ruiz-Hernandez, R., Curriu, M., Bofill, M., Martinez-Picado, J., Juan, M., Clotet, B., Blanco, J., 2008. HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4. Retrovirology 5, 32. Purcell, D., Cunningham, A., Turville, S., Tachedjian, G., Landay, A., 2012. Biology of mucosally transmitted sexual infection-translating the basic science into novel HIV intervention: a workshop summary. AIDS Res. Hum. Retroviruses 28, 1389. Puryear, W.B., Yu, X., Ramirez, N.P., Reinhard, B.M., Gummuluru, S., 2012. HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells. Proc. Natl. Acad. Sci. U.S.A. 109, 7475. Quayle, A.J., Xu, C., Mayer, K.H., Anderson, D.J., 1997. T lymphocytes and macrophages, but not motile spermatozoa, are a significant source of human immunodeficiency virus in semen. J. Infect. Dis. 176, 960.

82

K.M. Law et al.

Rabi, S.A., Laird, G.M., Durand, C.M., Laskey, S., Shan, L., Bailey, J.R., Chioma, S., Moore, R.D., Siliciano, R.F., 2013. Multi-step inhibition explains HIV-1 protease inhibitor pharmacodynamics and resistance. J. Clin. Invest. 123, 3848. Reece, J.C., Handley, A.J., Anstee, E.J., Morrison, W.A., Crowe, S.M., Cameron, P.U., 1998. HIV-1 selection by epidermal dendritic cells during transmission across human skin. J. Exp. Med. 187, 1623. Reh, L., Magnus, C., Schanz, M., Weber, J., Uhr, T., Rusert, P., Trkola, A., 2015. Capacity of broadly neutralizing antibodies to inhibit HIV-1 cell-cell transmission is strain- and epitope-dependent. PLoS Pathog. 11, e1004966. Rein, A., Mirro, J., Haynes, J.G., Ernst, S.M., Nagashima, K., 1994. Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J. Virol. 68, 1773. Rempel, H., Calosing, C., Sun, B., Pulliam, L., 2008. Sialoadhesin expressed on IFNinduced monocytes binds HIV-1 and enhances infectivity. PLoS One 3, e1967. Richardson, M.W., Carroll, R.G., Stremlau, M., Korokhov, N., Humeau, L.M., Silvestri, G., Sodroski, J., Riley, J.L., 2008. Mode of transmission affects the sensitivity of human immunodeficiency virus type 1 to restriction by rhesus TRIM5alpha. J. Virol. 82, 11117. Richardson, S.I., Gray, E.S., Mkhize, N.N., Sheward, D.J., Lambson, B.E., Wibmer, C.K., Masson, L., Werner, L., Garrett, N., Passmore, J.A., Karim, Q.A., Karim, S.S., Williamson, C., Moore, P.L., Morris, L., 2015. South African HIV-1 subtype C transmitted variants with a specific V2 motif show higher dependence on alpha4beta7 for replication. Retrovirology 12, 54. Rizzuto, C.D., Sodroski, J.G., 1997. Contribution of virion ICAM-1 to human immunodeficiency virus infectivity and sensitivity to neutralization. J. Virol. 71, 4847. Rodriguez-Plata, M.T., Puigdomenech, I., Izquierdo-Useros, N., Puertas, M.C., Carrillo, J., Erkizia, I., Clotet, B., Blanco, J., Martinez-Picado, J., 2013. The infectious synapse formed between mature dendritic cells and CD4(+) T cells is independent of the presence of the HIV-1 envelope glycoprotein. Retrovirology 10, 42. Roy, N.H., Lambele, M., Chan, J., Symeonides, M., Thali, M., 2014. Ezrin is a component of the HIV-1 virological presynapse and contributes to the inhibition of cell-cell fusion. J. Virol. 88, 7645. Rudnicka, D., Feldmann, J., Porrot, F., Wietgrefe, S., Guadagnini, S., Prevost, M.C., Estaquier, J., Haase, A.T., Sol-Foulon, N., Schwartz, O., 2009. Simultaneous cell-tocell transmission of human immunodeficiency virus to multiple targets through polysynapses. J. Virol. 83, 6234. Salle, B., Brochard, P., Bourry, O., Mannioui, A., Andrieu, T., Prevot, S., DejucqRainsford, N., Dereuddre-Bosquet, N., Le Grand, R., 2010. Infection of macaques after vaginal exposure to cell-associated simian immunodeficiency virus. J. Infect. Dis. 202, 337. Sanders, R.W., de Jong, E.C., Baldwin, C.E., Schuitemaker, J.H., Kapsenberg, M.L., Berkhout, B., 2002. Differential transmission of human immunodeficiency virus type 1 by distinct subsets of effector dendritic cells. J. Virol. 76, 7812. Sato, H., Orenstein, J., Dimitrov, D., Martin, M., 1992. Cell-to-cell spread of HIV-1 occurs within minutes and may not involve the participation of virus particles. Virology 186, 712. Schiffner, T., Sattentau, Q.J., Duncan, C.J., 2013. Cell-to-cell spread of HIV-1 and evasion of neutralizing antibodies. Vaccine 31, 5789. Sewald, X., Ladinsky, M.S., Uchil, P.D., Beloor, J., Pi, R., Herrmann, C., Motamedi, N., Murooka, T.T., Brehm, M.A., Greiner, D.L., Shultz, L.D., Mempel, T.R., Bjorkman, P.J., Kumar, P., Mothes, W., 2015. Retroviruses use CD169-mediated trans-infection of permissive lymphocytes to establish infection. Science 350, 563.

HIV-1 Cell-to-Cell Transmission

83

Sharova, N., Swingler, C., Sharkey, M., 2005. Macrophages archive HIV-1 virions for dissemination in trans. EMBO J. 24, 2481. Sigal, A., Kim, J.T., Balazs, A.B., Dekel, E., Mayo, A., Milo, R., Baltimore, D., 2011. Cellto-cell spread of HIV permits ongoing replication despite antiretroviral therapy. Nature 477, 95. Sloan, R.D., Kuhl, B.D., Mesplede, T., Munch, J., Donahue, D.A., Wainberg, M.A., 2013. Productive entry of HIV-1 during cell-to-cell transmission via dynamin-dependent endocytosis. J. Virol. 87, 8110. Smed-Sorensen, A., Lore, K., Vasudevan, J., Louder, M.K., Andersson, J., Mascola, J.R., Spetz, A.L., Koup, R.A., 2005. Differential susceptibility to human immunodeficiency virus type 1 infection of myeloid and plasmacytoid dendritic cells. J. Virol. 79, 8861. Sodora, D.L., Lee, F., Dailey, P.J., Marx, P.A., 1998. A genetic and viral load analysis of the simian immunodeficiency virus during the acute phase in macaques inoculated by the vaginal route. AIDS Res. Hum. Retroviruses 14, 171. Sourisseau, M., Sol-Foulon, N., Porrot, F., Blanchet, F., Schwartz, O., 2007. Inefficient human immunodeficiency virus replication in mobile lymphocytes. J. Virol. 81, 1000. Spira, A.I., Marx, P.A., Patterson, B.K., Mahoney, J., Koup, R.A., Wolinsky, S.M., Ho, D.D., 1996. Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J. Exp. Med. 183, 215. Steele, A.K., Lee, E.J., Manuzak, J.A., Dillon, S.M., Beckham, J.D., McCarter, M.D., Santiago, M.L., Wilson, C.C., 2014. Microbial exposure alters HIV-1-induced mucosal CD4+ T cell death pathways ex vivo. Retrovirology 11, 14. Stekler, J., Sycks, B.J., Holte, S., Maenza, J., Stevens, C.E., Dragavon, J., Collier, A.C., Coombs, R.W., 2008. HIV dynamics in seminal plasma during primary HIV infection. AIDS Res. Hum. Retroviruses 24, 1269. Stoddart, C.A., Maidji, E., Galkina, S.A., Kosikova, G., Rivera, J.M., Moreno, M.E., Sloan, B., Joshi, P., Long, B.R., 2011. Superior human leukocyte reconstitution and susceptibility to vaginal HIV transmission in humanized NOD-scid IL-2Rgamma(-/-) (NSG) BLT mice. Virology 417, 154. Sugaya, M., Lore, K., Koup, R.A., Douek, D.C., Blauvelt, A., 2004. HIV-infected Langerhans cells preferentially transmit virus to proliferating autologous CD4 + memory T cells located within Langerhans cell-T cell clusters. J. Immunol. 172, 2219. Sun, Z., Denton, P.W., Estes, J.D., Othieno, F.A., Wei, B.L., Wege, A.K., Melkus, M.W., Padgett-Thomas, A., Zupancic, M., Haase, A.T., Garcia, J.V., 2007. Intrarectal transmission, systemic infection, and CD4 + T cell depletion in humanized mice infected with HIV-1. J. Exp. Med. 204, 705. Tardif, M.R., Tremblay, M.J., 2003. Presence of host ICAM-1 in human immunodeficiency virus type 1 virions increases productive infection of CD4+ T lymphocytes by favoring cytosolic delivery of viral material. J. Virol. 77, 12299. Temin, H.M., 1988. Mechanisms of cell killing/cytopathic effects by nonhuman retroviruses. Rev. Infect. Dis. 10, 399. Titanji, B.K., Aasa-Chapman, M., Pillay, D., Jolly, C., 2013. Protease inhibitors effectively block cell-to-cell spread of HIV-1 between T cells. Retrovirology 10, 161. Trinite, B., Chan, C.N., Lee, C.S., Levy, D.N., 2015. HIV-1 Vpr- and reverse transcriptioninduced apoptosis in resting peripheral blood CD4 T cells and protection by common gamma-chain cytokines. J. Virol. 90, 904. Van Damme, N., Goff, D., Katsura, C., Jorgenson, R.L., Mitchell, R., Johnson, M.C., Stephens, E.B., Guatelli, J., 2008. The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell Host Microbe 3, 245.

84

K.M. Law et al.

Van de Perre, P., Segondy, M., Foulongne, V., Ouedraogo, A., Konate, I., Huraux, J.M., Mayaud, P., Nagot, N., 2008. Herpes simplex virus and HIV-1: deciphering viral synergy. Lancet Infect. Dis. 8, 490. van der Kuyl, A.C., van den Burg, R., Zorgdrager, F., Groot, F., Berkhout, B., Cornelissen, M., 2007. Sialoadhesin (CD169) expression in CD14 + cells is upregulated early after HIV-1 infection and increases during disease progression. PLoS One 2, e257. Vasiliver-Shamis, G., Tuen, M., Wu, T.W., Starr, T., Cameron, T.O., Thomson, R., Kaur, G., Liu, J., Visciano, M.L., Li, H., Kumar, R., Ansari, R., Han, D.P., Cho, M.W., Dustin, M.L., Hioe, C.E., 2008. Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells. J. Virol. 82, 9445. Wald, A., Link, K., 2002. Risk of human immunodeficiency virus infection in herpes simplex virus type 2-seropositive persons: a meta-analysis. J. Infect. Dis. 185, 45. Wang, J.H., Janas, A.M., Olson, W.J., Wu, L., 2007. Functionally distinct transmission of human immunodeficiency virus type 1 mediated by immature and mature dendritic cells. J. Virol. 81, 8933. Wang, J.-H.H., Kwas, C., Wu, L., 2009. Intercellular adhesion molecule 1 (ICAM-1), but not ICAM-2 and -3, is important for dendritic cell-mediated human immunodeficiency virus type 1 transmission. J. Virol. 83, 4195. Weiler, A.M., Li, Q., Duan, L., Kaizu, M., Weisgrau, K.L., Friedrich, T.C., Reynolds, M.R., Haase, A.T., Rakasz, E.G., 2008. Genital ulcers facilitate rapid viral entry and dissemination following intravaginal inoculation with cell-associated simian immunodeficiency virus SIVmac239. J. Virol. 82, 4154. Weng, J., Krementsov, D.N., Khurana, S., Roy, N.H., Thali, M., 2009. Formation of syncytia is repressed by tetraspanins in human immunodeficiency virus type 1-producing cells. J. Virol. 83, 7467. Wolff, H., Anderson, D.J., 1988. Immunohistologic characterization and quantitation of leukocyte subpopulations in human semen. Fertil. Steril. 49, 497. Wu, L., KewalRamani, V.N., 2006. Dendritic-cell interactions with HIV: infection and viral dissemination. Nat. Rev. Immunol. 6, 859. Wyma, D.J., Jiang, J., Shi, J., Zhou, J., Lineberger, J.E., Miller, M.D., Aiken, C., 2004. Coupling of human immunodeficiency virus type 1 fusion to virion maturation: a novel role of the gp41 cytoplasmic tail. J. Virol. 78, 3429. Yu, X., Feizpour, A., Ramirez, N.G., Wu, L., Akiyama, H., Xu, F., Gummuluru, S., Reinhard, B.M., 2014. Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells. Nat. Commun. 5, 4136. Zaitseva, M., Blauvelt, A., Lee, S., Lapham, C.K., Klaus-Kovtun, V., Mostowski, H., Manischewitz, J., Golding, H., 1997. Expression and function of CCR5 and CXCR4 on human Langerhans cells and macrophages: implications for HIV primary infection. Nat. Med. 3, 1369. Zhang, Z., Schuler, T., Zupancic, M., Wietgrefe, S., Staskus, K.A., Reimann, K.A., Reinhart, T.A., Rogan, M., Cavert, W., Miller, C.J., Veazey, R.S., Notermans, D., Little, S., Danner, S.A., Richman, D.D., Havlir, D., Wong, J., Jordan, H.L., Schacker, T.W., Racz, P., Tenner-Racz, K., Letvin, N.L., Wolinsky, S., Haase, A.T., 1999. Sexual transmission and propagation of SIV and HIV in resting and activated CD4 + T cells. Science 286, 1353. Zhang, Z.Q., Wietgrefe, S.W., Li, Q., Shore, M.D., Duan, L., Reilly, C., Lifson, J.D., Haase, A.T., 2004. Roles of substrate availability and infection of resting and activated CD4 + T cells in transmission and acute simian immunodeficiency virus infection. Proc. Natl. Acad. Sci. U.S.A. 101, 5640.

HIV-1 Cell-to-Cell Transmission

85

Zhong, P., Agosto, L.M., Ilinskaya, A., Dorjbal, B., Truong, R., Derse, D., Uchil, P.D., Heidecker, G., Mothes, W., 2013. Cell-to-cell transmission can overcome multiple donor and target cell barriers imposed on cell-free HIV. PLoS One 8, e53138. Zou, Z., Chastain, A., Moir, S., Ford, J., Trandem, K., Martinelli, E., Cicala, C., Crocker, P., Arthos, J., Sun, P.D., 2011. Siglecs facilitate HIV-1 infection of macrophages through adhesion with viral sialic acids. PLoS One 6, e24559.