Slipping through the door: HIV entry into the nucleus

Microbes and Infection 4 (2002) 67–73 www.elsevier.com/locate/micinf

Review

Slipping through the door: HIV entry into the nucleus Michael P. Sherman a,b,*, Warner C. Greene a,b,c a

Gladstone Institute of Virology and Immunology, P.O. Box 419100, San Francisco, CA 94141-9100, USA b Department of Medicine, University of California, San Francisco, California 94123, USA c Department of Microbiology and Immunology, University of California, San Francisco, California 94123, USA

Abstract HIV infection of non-dividing cellular targets like macrophages requires successful passage of the viral preintegration complex (PIC) across an intact nuclear envelope. Unique but redundant nuclear import signals reside within the HIV integrase, matrix, and Vpr proteins as well as the ‘DNA flap’; these signals appear to facilitate PIC transport through the limiting nuclear pores. We discuss recent studies that have advanced our understanding of this key step in the HIV life cycle. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: HIV nuclear import; Preintegration complex

1. Introduction The Retroviridae family of viruses is characterized by the ability of its members to reverse transcribe their RNA genomes into DNA prior to integration into a host cell chromosome. All retroviruses contain three major coding domains: gag, which generates the viral core, pol, which contains information for the reverse transcriptase (RT), integrase and protease enzymes, and env, which directs the production of the surface envelope. The Lentivirus genus (e.g., HIV, simian immunodeficiency virus) is distinguished by the ability to infect non-dividing cells [1]. Conversely, the gamma-retroviruses (or oncoretroviruses), like Moloney murine leukemia virus, require nuclear membrane dissolution or at least the passage of cells into mitosis for the viral integration machinery to access the host cell DNA. In the case of HIV-1, both non-dividing macrophages and resting T cells serve as important targets for viral infection. In addition to the shared products of the gag, pol, and env genes, the lentiviruses encode additional proteins like viral protein R (Vpr) that contribute to productive infection of such non-dividing cellular hosts (Figs 1 and 2A). The HIV preintegration complex (PIC), which contains the viral RNA/DNA and the machinery to facilitate import and integration, must traverse the nuclear pore complex

* Corresponding author. Tel.: +1-415-695-3840; fax: +1-415-826-1817. E-mail address: [email protected] (M.P. Sherman). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 1 2 8 6 - 4 5 7 9 ( 0 1 ) 0 1 5 1 1 - 8

(NPC) studding the double lipid bilayer of the nuclear membrane in order to gain access to the nucleoplasm (Fig. 2B). Of note, the HIV PIC displays a Stokes diameter of 56 nm that greatly exceeds the 25-nm central channel of the nuclear pore. How HIV successfully enters the nucleus and what viral proteins mediate this feat of molecular gymnastics have been the focus of many recent studies. As is often the case in HIV biology, the virus employs redundancy as a solution to this problem. Recent studies have identified nuclear import signals in three different viral proteins (integrase, matrix (MA), and Vpr), as well as a ‘DNA flap’ produced during reverse transcription, that appear to contribute to successful nuclear targeting of the HIV PIC. We discuss in the following sections our current understanding of each of these karyophiles. 2. Nucleocytoplasmic transport Eukaryotic cells possess a nuclear envelope that is studded with multiple nuclear pores. These pores serve as the conduits for bi-directional transport of macromolecules that are critically required for maintenance of normal cellular physiology [2]. The NPC corresponds to a 125MDa macromolecular assembly of 50–100 polypeptides (reviewed in [3]). The NPC spans the nuclear membrane and creates an aqueous channel with a passive diffusion pore size of 9 nm in diameter, allowing for the theoretical passage of a globular protein up to approximately 60 kDa in size. During active transport, however, the central channel

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Fig. 1. The integrated provirus of a retrovirus is flanked by terminal repeats (U3, R, U5) that contain the promoter for transcription. The gag, pol and env genes are shared by all Retroviridae, but HIV-1 also contains two regulatory genes (tat, rev) and four accessory genes (vpr, vpu, nef, vif) that contribute to viral replication and host pathogenesis. Highlighted are the genes that contribute, along with the ‘central DNA flap’ formed within integrase, to nuclear import of HIV. MA (matrix), CA (capsid), NC (nulceocapsid), RT (reverse transcriptase).

of the NPC allows bi-directional transport of macromolecules up to 25 nm in diameter. Translocation across the NPC and into the nucleoplasm and, alternatively, into the cytoplasm are governed by a class of proteins known as importins and exportins, respectively. Both are members of the karyopherin family of proteins (reviewed in [3] and [4]). The importins and exportins engage the appropriate import or export signals within the cargo proteins and mediate their transport. While the precise factors governing directionality of transport remain poorly understood, it is thought that the steep gradient of RanGTP/RanGDP (high ratio in the nucleus and low ratio in the cytoplasm) plays a key role. This gradient reflects the fact that RCC1, the nucleotide exchange factor for Ran, is abundantly expressed in the nucleus while the RanGTPase, RanGAP, is present in the cytoplasm [5]. The hypothesis that the gradient of RanGTP controls the destination of targeted proteins is supported by recent work where the directionality of nucleocytoplasmic transport was inverted by increasing the concentration of the RanGTP in the cytoplasm [6]. For proteins in the cytoplasm to be directly transported to the nucleus they must contain a nuclear localization signal (NLS). A classic NLS comprises either a short stretch of basic lysine residues, such as in the SV 40 large T antigen (PKKKRKV), or a bipartite basic NLS with two interdependent basic amino acid clusters with an intervening spacer as found in nucleoplasmin (KRPAATKKAGQAKKKK). These nuclear import signals are recognized by a cellular protein, importin α, that in turn binds to a second protein, importin β. This trimeric complex then engages the nucleoporin components of NPCs and progressively moves across the pore in a series of sequential binding and release steps that require energy. Release of the bound protein cargo occurs when RanGTP binds to importin β in the nucleus (reviewed in [2] and [7]). Alternatively, a growing class of proteins bypass the requirement for importin α binding and instead directly associate with importin β. The nuclear import signals in these proteins are generally rich in arginine rather than lysine [8,9]. Such signals have been identified in HIV-1 Tat and Rev, human T-cell leukemia virus Rex, cyclin B1 and

hTAP. The Tat, Rev and Rex proteins in fact compete with importin for a binding site present on importin β. Conversely, cyclin B1 appears to bind to a unique site on importin β [10]. Of note, all of these proteins also contain a nuclear export signal (NES) and are able to shuttle into and out of the nucleus. The first recognized NES was described in the HIV shuttling protein Rev [11,12]. The NES is recognized by the chromosome maintenance region 1 protein (CRM1) or exportin-1 [13,14]. In the presence of RanGTP, CRM1/exportin-1 binds to a leucine-rich signal distinct from the NLS and mediates export through the NPC. This process is interrupted by addition of leptomycin B, a compound that covalently modifies and inactivates CRM1-dependent nuclear export. A second nuclear import signal, termed M9, has been identified in the hnRNP A1 protein [15]. Although similarly dependent on the RanGTP gradient, this signal exhibits no sequence homology to the classic NLS. Indeed, the M9 sequence is rich in aromatic amino acids and is recognized by transportin, a different karyopherin family member displaying 25% homology with importin β. The M9 region binds to transportin, and is also the signal recognized by an unidentified nuclear carrier process that targets hnRNP A1 for export from the nucleus into the cytoplasm. Thus, the M9 sequence corresponds to a nucleocytoplasmic shuttling signal [16]. Based on knowledge gained from study of yeast transporters, there are likely to be several importin β-like proteins and numerous cargo that bind to unique sites with as yet uncharacterized import and export signals [17]. In addition, there are now a number of proteins, including the transportins themselves, which are able to enter and exit the nucleus in an energy- and RanGTP-independent manner [18]. Thus, while significant progress has been made in understanding select components of the nuclear import and export machinery, much remains unknown.

3. Are all cells created equally? Early studies identified non-dividing macrophages as permissive targets for HIV infection in vitro [19]. Clinically,

M.P. Sherman, W.C. Greene / Microbes and Infection 4 (2002) 67–73 Fig. 2. A) The mature virion is fully assembled after budding. B) The viral preintegration complex (PIC) measures more than twice the size of the central channel within the nuclear pore complex yet is able to successfully negotiate into the nucleus. C) The HIV life cycle. HIV fuses with the CD4 and cognate coreceptor, initiating fusion and uncoating (represented here as a single step). The subsequent complex (sometimes called a reverse transcriptase complex) facilitates conversion of viral RNA into cDNA and sheds several proteins while it traverses the cytoplasm along microtubules towards the nucleus. The PIC, or at least the components required for retroviral integration, somehow enter through the limiting NPC to gain access to host chromosomal DNA. After integration, genomic viral RNA is exported along with the immature viral particle components which assemble and bud together out of the cytoplasm through lipid rafts.

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macrophages likely play an important role as a viral reservoir leading to the production of the macrophage-tropic strains of HIV utilizing the CCR5 HIV coreceptor that are preferentially involved in horizontal and vertical transmission [20]. Several investigations have now shown that resting and/or naïve CD4+ T cells are infected within the peripheral blood mononuclear cells (PBMCs) and lymphoid tissue compartments of patients and provide an important reservoir for HIV production during the course of AIDS [21–23]. These findings stand in sharp contrast to in vitro studies where this resting sub-population of T cells appeared quite resistant to de novo infection without at least some degree of activation [24]. This raised the question of whether resting naïve T cells are truly infected de novo or instead are infected during a period of activation and proliferation and only later return to the resting state. Recent studies employing ex vivo lymphoid histoculture and human tonsil and spleen tissue for infection with HIV in the absence of added growth factors have revealed that truly resting, naïve T cells can be productively infected with HIV [25]. The resting state of these cells has been demonstrated by their failure to incorporate BrdU (non-dividing) and by the presence of low RNA levels (quiescent). Together, these findings suggest that the earlier failure to detect de novo infection of resting T cells in peripheral blood samples likely reflects differences in the culture conditions that were employed. These tissues, which contain a heterogeneous population of cells, may provide factors in trans to the resting naïve T cells that allow HIV to complete its life cycle [26]. In contrast to the CCR5 tropism displayed by viruses productively infecting non-dividing macrophages, CXCR4-tropic viruses predominate in the infection of resting T cells. This finding is consistent with the more prevalent expression of the CXCR4 coreceptor on these resting, naïve T cells. The ability of HIV and other primate lentiviruses to infect non-dividing cells has permitted the generation of a valuable set of gene transfer vectors. For example, lentiviral vectors have been successfully used to introduce foreign genes into a variety of non-dividing cellular hosts, including terminally differentiated neurons, myocytes, retinal cells, and hepatocytes [27]. Of note, the overall levels of integration and infection are reduced in these non-dividing host cells compared with their dividing counterparts, suggesting that lentiviruses may prefer the actively replicating subpopulation of cells but nevertheless have preserved the ability to enter quiescent cells. However, in terms of the work on hepatocytes, a note of caution must be added. Recent studies suggest that the process of introducing the lentiviral vector may stimulate liver cells to divide, promoting a more permissive state for gene transfer [28]. However, since the efficiency of lentiviral infection of truly nondividing cells is clearly reduced, it may have been difficult to discern in these studies whether the fully differentiated non-dividing hepatocytes were also infected, albeit at a lower level.

4. What factors govern PIC entry into the nucleus? 4.1. Viral life cycle HIV-1 binds to target cells initially through high-affinity interactions of its gp120 envelope glycoprotein with surface CD4 receptors. This interaction triggers conformational changes in gp120 which promote engagement of the HIV coreceptor (principally CCR5 or CXCR4). These events in turn activate the gp41 envelope protein to mediate fusion of the viral and cellular membranes (Fig. 2C). Fusion leads to ‘microinjection’ of the HIV-1 capsid component of the virion. Once inside the cell, the HIV capsid undergoes ‘uncoating’. Uncoating is followed by reverse transcription of the viral RNA genome which culminates in formation of the PIC [29]. Various core viral proteins are lost during formation of the PIC. Components of the PIC include the double-stranded DNA version of the viral genome as well as the reverse transcriptase, matrix, integrase, and Vpr proteins (Fig. 2B). Select host proteins are incorporated into the PIC including barrier to auto-integration factor and HMG I(Y). Prior to integration, the viral PIC must traverse from the plasma membrane to the nuclear envelope, a distance which may approach 20 µm. Dr Tom Hope and colleagues (University of Illinois, Chicago) have assembled preliminary evidence indicating that this transit involves the dynamic association of the HIV PIC with microtubules. Real-time fluorescence microscopy has revealed that the sub-viral particle slides down these microtubules in an energydependent fashion delivering the PIC to the vicinity of the nuclear envelope. We will now discuss each of the viral components which have been implicated in PIC import across the nuclear envelope. 4.2. Integrase While integrase is clearly essential for the introduction of retroviral cDNA into the host chromosome, this viral protein also appears to play a role in nuclear uptake of the PIC. Early work on PIC import showed that removing the C-terminal half of the gag p55 precursor protein during infection of growth-arrested MT-4 cells did not eliminate the production of two-LTR circles, a dead-end viral DNA product that is formed by host factors only in the nucleus of target cells [30]. More recent studies have revealed a non-classical nuclear import signal buried within the catalytic domain of integrase that plays a key role in the nuclear accumulation of the viral PIC [31]. Mutation of this nuclear import signal revealed that the import function of integrase could be selectively disrupted while preserving overall catalytic activity, and also that integrase import is required whether the cells are dividing or are in a resting state. This latter observation may change the dogma that oncoretroviruses require nuclear membrane breakdown to bypass the

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NPC and implies that all retroviruses may enter via active import via the NPC, but perhaps at different times of the cell cycle. 4.3. DNA flap Additional factors contribute to nuclear import of the viral PIC, including a central DNA flap. This element corresponds to a triple-stranded intermediate created during reverse transcription. HIV reverse transcription begins with tRNA priming to generate a minus-strand, strong-stop DNA that contains the U5 and R sequences (viral RNA spans from 5' R-U5 to U3-R on the 3' side, see Fig. 1). This nascent viral DNA then forms a primer on the 3' end of the viral genome and continues to generate minus-strand DNA while the RNA is degraded by the RNase H activity of the RT. When the minus-strand DNA reaches the center of the viral genome, within the integrase open reading frame, it engages a region termed the polypurine tract (PPT, named for its base composition). The PPTs are very conserved within retroviruses, but are not recognized by RTs between viruses. A pause in reverse transcription at the PPT due to a relative resistance to RNase H activity allows for its specific cleavage and generation of an RNA primer (derived from the genomic RNA–[minus-strand] DNA hybrid) used to initiate plus-strand DNA synthesis. All retroviruses generate a specific plus-strand primer at the PPT, but HIV also generates an upstream plus-strand primer resulting in discontinuous DNA production as two half-genomic fragments. The upstream genome is transcribed until it reaches the PPT and a region 99 nucleotides downstream termed the central termination sequence. At the latter, the RT is ejected leaving behind a linear complementary cDNA and this 99-nucleotide plus-strand overlap. Together, these elements form a triple-stranded intermediate termed the central DNA flap. Despite having an upstream template for DNA synthesis, sequence maintenance of the PPT is critical for virus replication [32]. However, early studies suggested that mutations in the central PPT interfered with HIV replication at a step after reverse transcription [33]. Subsequent studies have revealed that the central DNA flap acts as an import signal for the PIC [34]. Zennou and colleagues engineered an HIV genome that contained mutations in the PPT where pyrimidines were exchanged for purines while otherwise maintaining reverse transcription and the integrity of the integrase gene (the PPT overlaps with the integrase coding region). This DNA flap mutant of HIV was impaired in single-round infection assays. Further more, replication was reduced in both dividing and growtharrested cells, a situation reminiscent of mutation of the integrase NLS. To follow on their observation, the investigators assayed HIV-infected cells for the presence of integrated viral DNA and found the PPT mutant integration efficiency was reduced, placing the block to replication at the level of nuclear entry. These mutant HIV-1 genomes accumulated at or just inside the nuclear envelope. Finally,

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the investigators showed that by inserting a central cisacting DNA flap into an HIV-based vector lacking a PPT, they could greatly enhance the infection of growth-arrested cells. The mechanism by which a nucleic acid structure alters transport through the NPC remains unclear; however, there is precedence for such control in the case of RNA export [35]. The fact that the import defect is observed in both dividing and non-dividing cells implies that all retroviruses enter the nucleus through the NPC and that the requirement for mitosis may not be for nuclear membrane breakdown. 4.4. Matrix The earliest work on HIV PIC import in growth-arrested cells used the formation of two-LTR circles as a surrogate for nuclear import. The formation of these circles is confined to the nucleus. These studies revealed that HIV PIC import was an energy-dependent process that occurred independently of the cell cycle [30]. The search for the key viral nuclear proteins mediating this response ensued. HIV-1 MA, an abundant PIC protein, was found to display nucleophilic properties and to contain a classical nuclear import signal. When this signal was mutated, HIV replication in growth-arrested cells was sharply inhibited [36]. In contrast, these NLS mutations did not impair virus growth in replicating cells [37]. Other studies supported a role for MA in the nuclear accumulation of the HIV PIC in cultured monocyte-derived macrophages (MDMs) since small NLS peptide competitors blocked infection of these cells in a MA-dependent fashion. Of note, subsequent studies by these same investigators revealed that mutations in both the MA NLS and Vpr produced only partial and dose-dependent inhibition of HIV nuclear import, a finding that launched the search for additional import factors [38]. However, more recent work has challenged the notion that MA plays any role in delivery of the PIC across the NPC, as the entire globular head of this protein can be removed without significant inhibitory effects in single infection assays in MDM [39,40]. Most recently an export signal within the p55gag precursor protein has been identified. MA relies on both the NLS and an NES to localize viral RNA to the correct cellular compartment for packaging [41]. Thus, MA nuclear localization is involved in producing competent virions, but its precise role in directing nuclear import of the HIV PIC remains unresolved. 4.5. Vpr Perhaps the most controversial protein with respect to its role in PIC import is Vpr. This HIV protein is a 96-aminoacid polypeptide that is packaged into progeny virions through its interaction with the C-terminal p6Gag domain of the Pr55Gag precursor protein. Although several hundred molecules of Vpr were initially detected in each virion, more recent studies involving Vpr expression in cis rather

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than in trans suggest roughly 14 Vpr molecules/virion. Despite the fact that Vpr is dispensable for HIV replication in cultured PBMCs, it is highly conserved in vivo and in the other primate lentiviruses, HIV-2 and simian immunodeficiency virus, which also encode Vpr2 as well as a Vprrelated gene product termed Vpx, also believed to participate in PIC import. Vpr is principally expressed in the nucleus because of at least two distinct and novel NLS [42,43]. Further, Vpr, like MA, displays nucleocytoplasmic shuttling properties reflecting the presence of an exportin1-dependent NES that likely guarantees its incorporation into the PIC, albeit in small amounts [43]. The nucleophilic properties of Vpr contribute to the ability of HIV to infect non-dividing target cells such as macrophages [44,45]. However, the phenotype in MDM requires low input of virus and weeks of culture. This could be due to the fact that MDM incorporate BrdU and hence mask the relatively inefficient HIV replication in nondividing cells or that rather Vpr is only a modifier of PIC import. While Vpr itself does not possess a canonical NLS, it is thought to bind to the soluble import factors and possibly stabilize import by MA for example. In the experimental context of lymphoid histocultures, Dr Mark Goldsmith’s laboratory and we have observed that Vpr plays an important role in HIV replication in tissue macrophages. Conversely Vpr does not appear to play a significant role in HIV growth in cycling T cells, a finding that is consistent with prior studies performed with PBMCs. Of note, we have found that Vpr is not required for HIV replication in a newly recognized target cell population, quiescent, naïve T cells [46]. Recent studies by BouyacBertoia et al. have shown that Vpr is not essential for HIV PIC import in growth-arrested indicator cell lines [31]. Thus, it is unclear at this time whether Vpr enhances import of the PIC into tissue macrophages present in the histoculture, or instead alters the intracellular millieu in these cells in a specific manner that enhances virus growth. It is likely that the G2 cell-cycle-arresting property of Vpr that is observed in cycling T cells is a marker for a phenotype of increased virus production in the relevant macrophage cell target. Finally, in recent studies, we have shown that Vpr alters the structure of the nuclear lamina in a manner that leads to the formation of nuclear herniations that intermittently rupture [47]. These ruptures in the nuclear envelope may provide a freely accessible portal for uptake of the large HIV PIC uptake in select situations. Notwithstanding, HIV clearly contains other means for entry into the nucleus of non-dividing cells, since HIV-based gene transfer vectors lacking Vpr effectively transduce such cells as neurons.

5. Conclusion The initial observation that HIV effectively infects terminally differentiated macrophages in vivo and various

growth-arrested cells in vitro contrasted sharply with the notion that oncogenic retroviruses require cell division and attendant nuclear membrane breakdown to establish productive infection. This difference has spawned a broad series of studies that have highlighted the karyophylic properties of the HIV integrase, MA, and Vpr proteins as well as the potential nuclear targeting function of the DNA flap in nuclear import of the HIV PIC. The ability of HIV to establish productive infections in non-dividing cells has also led to exploitation of HIV as a gene transfer vehicle permitting stable expression of various target genes in non-replicating cells of various tissues. Of note, the overall level of infection of such non-dividing cells is consistently less than that observed in proliferating permissive cellular targets. Taken together, the currently available data suggest that the HIV integrase plays a leading role in nuclear import of the PIC, with the DNA flap, MA, and possibly Vpr proteins serving as supporting cast for this process. It will be important to determine how the integrase protein mediates nuclear uptake with an eye toward the potential for disrupting this pathway in a virus-specific manner. Such an approach could provide an attractive alternative to blocking the HIV replicative life cycle at a point prior to proviral integration into the host chromosome.

Acknowledgements Our own work in this area is supported by the National Institutes of Health (R01 AI45324 and K08 AI01866). We also acknowledge the UCSF-GIVI Center for AIDS Research (NIH #P30 MH59037). We thank John Carroll for graphics illustration and Sue Cammack and Robin Givens for manuscript preparation.

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