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Lentivirus Vpr and Vpx accessory proteins usurp the cullin4–DDB1 (DCAF1) E3 ubiquitin ligase
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Lentivirus Vpr and Vpx accessory proteins usurp the cullin4–DDB1 (DCAF1) E3 ubiquitin ligase Bizhan Romani1 and E´ric A Cohen1,2 Myeloid cells display a differential permissivity to primate lentivirus infection that is related to their ability to encode the Vpx and to a lesser extent the Vpr accessory proteins. Vpr is encoded by all primate lentiviruses, including HIV-1 and HIV-2, while its paralog, Vpx, is unique to HIV-2 and a subset of simian lentiviruses. Both proteins usurp the CRL4A (DCAF1) E3 ligase to fulfil their functions. Vpx induces the degradation of SAMHD1, a nucleotide triphosphohydrolase that blocks lentiviral reverse transcription in myeloid cells via depletion of the intracellular pool of dNTPs. Vpr engages CRL4A (DCAF1) to degrade a yet unknown factor(s), whose proteolysis induces a G2 cell-cycle arrest in dividing cells. Although the identification of the host protein(s) targeted for degradation by Vpr will be necessary to understand its actual function, the discovery of SAMHD1 has already shed light into a new mechanism of restriction that limits infection of myeloid cells by HIV-1. Addresses 1 Institut de Recherches Cliniques de Montre´al, Montre´al, Que´bec, Canada H2W 1R7 2 Department of Microbiology and Immunology, Universite´ de Montre´al, Montre´al, Que´bec, Canada H3C 3J7 Corresponding author: Cohen, E´ric A ([email protected]) Current Opinion in Virology 2012, 2:755–763 This review comes from a themed issue on Virus replication in animals and plants Edited by Peter Nagy and Christopher Richardson For a complete overview see the Issue and the Editorial Available online 10th October 2012 1879-6257/$ – see front matter, # 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2012.09.010
Introduction Viruses have evolved ways to modulate the host cell environment in order to promote efficient viral replication and to disrupt elements of innate or acquired immunity. One strategy particularly favoured by viruses to achieve these goals is to subvert the host ubiquitin machinery in order to induce the proteasomal or lysosomal degradation of specific cellular factors that play crucial roles in inhibiting viral replication and/or inducing immune responses [1,2]. The covalent attachment of ubiquitin molecules to a target protein requires the sequential activity of three enzymes: an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase, which ultimately controls the specificity of the conjugation process since it directly binds the target protein. A www.sciencedirect.com
major class of the estimated 600–1000 E3 ligases encoded by the human genome is represented by the cullin-ring ubiquitin ligases (CRLs). CRLs are multi-subunit complexes composed of a catalytic core containing the invariant ROC/RBX ring finger protein and a substrate recognition module comprising various adaptor proteins, nucleated around a cullin scaffold protein [3] (Figure 1). When usurping the host ubiquitin machinery, viruses most often target the E3 ubiquitin ligase component. Primate lentiviruses are no exception to this principle. Human immunodeficiency virus (HIV) and several simian immunodeficiency virus (SIV) lineages encode two accessory proteins, viral infectivity factor (Vif) and viral protein U (Vpu) that hijack the host ubiquitin machinery in order to respectively degrade two potent antiviral host proteins — also called restriction factors — the cytidine deaminases APOBEC3F/3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F/ 3G) and Tetherin (BST2, CD317), which act as potent inhibitors of early (reverse transcription) and late (viral release) events of the lentiviral infection cycle [4]. In both instances, these viral proteins target specific CRLs, namely cullin1-SKP1 (bTrCP) in the case of Vpu and cullin5 (elonginB/C) for Vif [5]. Recently, we and several other investigators demonstrated that a third accessory protein encoded by all primate immunodeficiency viruses, Viral protein R (Vpr), as well as its paralog, Vpx, exclusively encoded by HIV-2 and several SIV lineages, also exert their functions by recruiting an E3 ubiquitin ligase complex composed of cullin4A (CUL4A), damaged DNA binding protein 1 (DDB1) and a member of the DCAF (DDB1–cullin4-associated-factor) family called VPRBP (viral protein R binding protein, also known as DCAF1) [6,7,8,9,10,11,12,13,14,15] (Figure 1). In this review, we highlight the latest discoveries regarding how the CUL4A–DDB1 (DCAF1) (CRL4A (DCAF1)) E3 ubiquitin ligase is exploited by these two genetically related accessory proteins to fulfil what appears to be very distinct functions during viral infection. We also discuss outstanding questions regarding SAMHD1, the recently discovered Vpx cellular target that limits lentiviral infection in myeloid cells, the identity of the yet unknown host protein(s) targeted by Vpr, and the mechanisms via which Vpr and Vpx engage the CRL4A (DCAF1) E3 ligase to degrade host proteins.
The CUL4A–DDB1 (DCAF1) E3 ubiquitin ligase The evolutionary conserved CUL4 E3 ubiquitin ligase (CRL4) family, in concert with its large b-propeller Current Opinion in Virology 2012, 2:755–763
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Figure 1
NEDD8 Cul4A DDB1 H box
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? DCAF1
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Proposed model for the mode of induction of G2 arrest by HIV-1 Vpr. The cullin4A–DDB1 (DCAF1) E3 ubiquitin ligase is a multisubunit complex that comprises a catalytic core and a substrate recognition module nucleated around a cullin4A (Cul4A) scaffold protein (pink). The catalytic core consists of a small RING protein (ROC, purple) that binds and activates and E2 enzyme (dark brown), which transfers ubiquitin (Ub, red) to a substrate. The substrate recognition module comprises a WD40-containing DCAF1 substrate specificity receptor (yellow) bound to the three-b-propeller protein, DDB1 (dark blue) via its WD40 domain and a putative structural H box element, as indicated. Vpr (orange) is proposed to bind the E3 ubiquitin ligase complex through DCAF1 to recruit a yet unknown protein target(s) (blue) whose ubiquitination and proteaosomal degradation results in activation of ATR and ultimately in induction of a G2 cell cycle arrest. Covalent conjugation of the ubiquitinlike modifier NEDD8 (light brown) activates cullin4A.
DDB1 adaptor, regulates a diverse set of cellular processes including development, transcription, replication and DNA repair [16]. Mass spectrometric analyses of the CUL4A–DDB1 complex have revealed physical interactions with a set of more than 50 WD40-containing proteins, referred to as DCAFs that act as substrate specificity receptors [17]. At present, more than twodozen substrates have been shown to be regulated by these complexes, a large fraction of those being chromatin-associated proteins [16]. In addition to controlling basic cellular processes, early studies revealed that CRL4A ligases are hijacked by several pathogenic viruses, including members of paramyxovirus and hepadnavirus families, to ubiquitinate host proteins, which are not normally targeted by this E3 ligase family. Hence, proteins V and X encoded by paramyxovirus simian virus Current Opinion in Virology 2012, 2:755–763
5 (SV5) and hepatitis B virus (HBV), respectively, interact directly with DDB1 to redirect the CUL4A–DDB1 E3 ubiquitin ligase towards new substrates [18]. While the biological implications of HBV X engagement to CRL4A remain poorly understood, SV5 V protein hijacking of the CRL4A E3 ligase complex was shown to promote the ubiquitination and subsequent proteasomal degradation of STAT1, so that cellular antiviral responses are impeded via inhibition of interferon (IFN) production [19]. Importantly, recent structural studies have provided key insights into how these viral proteins interact with DDB1 despite their sequence divergence. They reveal that binding to DDB1 is mediated through an alpha-helical motif, also called H box, which is indeed shared by the various cellular DCAFs. Thus, these studies have identified a common structural element that is crucial for anchoring these viral hijackers as well as cellular substrate-recruiting adapters to CRL4A E3 ubiquitin ligase complexes [20]. The DCAF1/VprBP WD40-containing substrate receptor that is part of the CRL4A recruited by Vpr and Vpx was identified less than two decades ago as an HIV-1 Vprinteracting protein [21,22]. However, the cellular target(s) normally recruited by DCAF1 remained elusive until recently. Huang and Chen reported that CRL4A (DCAF1) induces the rapid degradation of the tumour suppressor Merlin (NF2, neurofibromin 2) following serum starvation [23]. More recently, DCAF1 was also found to act as the substrate recognition subunit of the CRL4A complex that mediates the stress-induced proteolysis of Mcm10, a replication factor that plays an essential role in initiation and elongation of DNA replication [24]. Interestingly, mutations in Mcm10 have been shown to lead to stalled replication and cell cycle arrest, while depletion of the protein resulted in activation of the G2 checkpoint pathway [25]. However, depletion of DCAF1 was also found to reduce the rate of DNA replication and to impede cellular proliferation suggesting a crucial role for DCAF1 in regulating factors involved in DNA replication and the cell cycle [11,26].
Viral protein R, an enigmatic HIV accessory protein The genome of primate lentiviruses encodes a group of proteins comprising Vif, Vpr, Vpx, Vpu, and Nef — the so-called accessory proteins — whose functions are to modify the host cell environment to facilitate viral replication and to evade innate and adaptive antiviral immune responses [5]. Of these, Vpr remains one of the least understood accessory proteins in terms of its role in viral replication and pathogenesis. Vpr is a small phosphorylated nuclear protein that is capable of shuttling between the nucleus and the cytoplasm [27]. This protein, which is conserved across all primate lentiviruses, including HIV-1 and HIV-2, is packaged into progeny virions through an interaction with the Gag precursor protein, thus pointing to an early role during the viral life cycle. Most of the www.sciencedirect.com
Lentivirus Vpr and Vpx accessory proteins Romani and Cohen 757
information regarding the roles of Vpr in virus replication has been derived from the study of HIV-1 Vpr. While many functions have been attributed to Vpr, the most widely accepted are induction of an arrest at the G2 phase of the cell cycle in dividing cells and enhancing infection in terminally differentiated myeloid cells, such as monocyte-derived macrophages (MDM) [28,29]. The functional relevance of Vpr-mediated G2 arrest remains unclear since efficient HIV-1 replication can be observed during ex vivo infection of dividing CD4+ T cells even in the absence of Vpr. However, one key advance in the field was the finding that the mechanism underlying Vprmediated G2 arrest involved the engagement of the CRL4A (DCAF1) E3 ubiquitin ligase by Vpr [6,7,8,9,10,11,12] (Figure 1). This recruitment, which was dependent on a physical interaction with the substrate-recruiting receptor DCAF1, was required to establish an intracellular environment that mimicked a DNA damage response initiated by the Ataxia Telangectasia and Rad3-related (ATR) protein kinase, ultimately resulting in the activation of a G2 checkpoint pathway [28]. While the architecture and the composition of the E3 ligase complexed with Vpr are still not entirely defined, mutagenic studies of Vpr have highlighted the existence of at least two functional domains (Figure 2). The first domain is involved in engaging DCAF1 and is dependent on conserved residues located within the third alpha helix of the protein and perhaps a conserved motif (Wx4Fx2Fx3AFxH; where x are residues that are highly variable and F corresponds to residues with bulky hydrophobic side chains) within the first helix [6,7,8,9,11,30]. The second one encompasses the C-terminal region of the protein, a domain of Vpr that is dispensable for DCAF1 binding, yet is essential for the induction of a G2 arrest [7,8,9,31]. The simplest model to explain these findings is that Vpr would recruit the E3 ligase complex through DCAF1 to induce the ubiquitination of a yet unknown cellular substrate(s), whose proteasomal degradation would lead to ATR
activation and G2 arrest (Figure 1). Indeed, results reported by our group revealed that Vpr could induce the lysine (K)48-linked poly-ubiquitination and proteasomal degradation of as-yet-unknown cellular proteins. Furthermore, Vpr-mediated K48-polyubiquitination and proteasomal degradation of these putative substrates were found necessary for the activation of the ATR pathway [32]. While the identity of the host protein(s) targeted by Vpr remains unknown, indirect evidence suggests that it might be chromatin-associated. Specifically, Vpr was found to associate with chromatin through its C-terminus and form mobile nuclear structures, designated as Vpr nuclear foci, where indeed DCAF1 and DNA repair foci components such as g-histone 2AX, 53BP1 and RPA32 co-localized [31]. Interestingly, inhibition of ATR kinase activity or depletion of DCAF1, two processes that prevent Vpr-mediated G2 arrest, did not inhibit the formation of Vpr nuclei foci, suggesting that formation of these Vpr-containing nuclear structures may represent a crucial early event in the process of Vprmediated ATR activation and G2 arrest. It is still unclear whether Vpr binds its substrate(s) directly on chromatin or alternatively via a potential cellular co-factor(s) that would be in proximity or even physically interacting with the substrate as it is the case for the SV5 V protein, which targets STAT1 by binding STAT2 [19]. The latter scenario would indeed account for the presence of Vpr nuclear foci in G2, when in all probability the putative Vpr cellular target(s) is degraded. Importantly, whether Vpr acts as an adaptor to recruit a new substrate(s) to the CRL4A (DCAF1) E3 ligase for ubiquitination and proteasomal degradation, a model analogous to that used by other HIV accessory proteins such as Vif or Vpx (see below), or whether Vpr forcibly enhances the ubiquitination of a natural substrate(s) targeted by DCAF1 remains unresolved. In that regard, it is interesting to note that uracil DNA glycosylases, UNG2 and SMUG1, two proteins previously shown to be targeted by HIV-1 Vpr for degradation but not linked to Vpr-mediated G2 arrest [33,34], might be novel natural substrates of CRL4A (DCAF1). In that particular context, Vpr was
Figure 2
α helix I
SAMHD1-binding domain
α helix II
α helix III
Predicted Vpr targetbinding domain
Elements involved in DCAF1 binding Current Opinion in Virology
Amino acid sequence alignment of Vpr and Vpx proteins encoded by HIV-1/HIV-2 and mapping of the functional regions of the proteins. Amino acid residues that are conserved in all the three sequences are highlighted in green while those conserved in two of the sequences are shown in red. Sequences were aligned using ClustalX (1.83) and highlighted using GeneDoc (2.7). Amino acids that constitute or predicted to form a-helices are indicated in purples boxes. The SAMHD1 binding site of HIV-2 Vpx at the N-terminus and the predicted target-binding site of HIV-1 Vpr at the Cterminus are also indicated. Position of HIV-1/2 Vpr a-helices were indicated as previously reported [40], while those of HIV-2 Vpx were shown as predicted by Mahnke et al. [41]. Gaps are indicated by (–). GeneBank accession numbers for HIV-1 pNL4.3 and HIV-2 ROD are U26942.1 and M15390.1, respectively. www.sciencedirect.com
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found to promote constitutive DCAF1-dependent UNG2 turnover by augmenting rather than permitting the interaction between UNG2 and the E3 ligase [35]. The functional relevance of Vpr-mediated G2 arrest in HIV-1 replication and pathogenesis will require the identification of the host protein(s) targeted by Vpr. While induction of a G2/M checkpoint in dividing cells could indeed be the function that Vpr was evolved to fulfil, it is also possible that it could be due to another function orchestrated by Vpr. However, the fact that transcription of viral genes is modestly upregulated when cells are in G2 suggests that Vpr-mediated G2 arrest could provide an optimal cellular environment for virus production. Such an effect would probably confer the virus with a selective advantage given the very short half-life of HIV-1-infected T cells [36]. More recent studies have also pointed towards an immuno-modulatory role of Vpr-mediated activation of DNA damage response. Indeed, our group and others have shown that Vpr-mediated activation of ATR leads to an upregulation of ligands of the activating natural killer (NK) cell receptor, NKG2D, in particular ULBP1 and ULBP2, at the surface of HIV-1-infected T cells, an effect that results in increased susceptibility to NK-cell cytolytic activity [37,38]. Although the purpose of upregulating NKG2D ligands remains ambiguous at this time, it is possible that it could further contribute to T cell depletion by rendering infected and Vpr-transducedbystander cells targets for NK-cell mediated killing. Additionally, upregulation of NKG2D ligands could be a contributing factor in the NK-cell dysfunction observed during chronic HIV infection through sustained effector activation, as previously observed in cancer models [39]. That being said, by analogy to the function of other viral accessory proteins, it is quite possible that the function of Vpr is to degrade a host factor that is unfavourable for viral replication or persistence during natural infection in vivo or in a specific cell type. Whether this host factor is targeted by the Vpr/CRL4A ligase complex to enhance HIV-1 infection of macrophages remains unknown. One could envision that degradation of a host factor(s) by Vpr in dividing cells would lead to induction of a G2 checkpoint, while Vpr-mediated degradation of the same cellular target in non-dividing macrophages would enhance HIV-1 replication or/and promote viral persistence. Identification of the host protein (s) targeted for degradation by the Vpr/CRL4A (DCAF1) complex will clearly provide important insights into the actual function of Vpr. Furthermore, the crystal structure of the DDB1DCAF1-Vpr substrate-recruiting module will be necessary to fully understand the architecture of the complex and the role of Vpr in either redirecting the specificity of the E3 ubiquitin ligase or in modulating its activity towards a natural substrate(s). Current Opinion in Virology 2012, 2:755–763
Vpx, an antagonist of SAMHD1 Primate lentiviruses in the phylogenetic group, which comprises HIV-2, its SIV precursor that naturally infects sooty mangabey (SIVsm) and SIVsm-derived rhesus macaques SIV(SIVmac), encode both Vpr, an orthologue of the HIV-1 Vpr protein, and Vpx, a related protein that displays sequence and predicted structural similarities [40,41] (Figure 2). As Vpr, Vpx is packaged into the core of progeny viral particles, is localized in the nucleus of infected cells and engages the CRL4A (DCAF1) E3 ubiquitin ligase through binding to the DCAF1 substrate specificity receptor [13,14,42]. Despite these similarities, the functional outcome of engaging the same E3 ubiquitin ligase is very different for Vpr and Vpx. Vpx expression does not induce a G2 cell cycle arrest. Rather, the protein was found to be crucial for the ability to efficiently infect myeloid cells, such as monocytes, dendritic cells (DC) and MDMs. In contrast, HIV-1 is inefficient at infecting DCs and although HIV-1 Vpr enhances HIV-1 replication in MDM, its effect is quite modest compared to that of Vpx during HIV-2 and SIVsm/mac infections [29]. Interestingly, the fact that both MDMs and DCs preloaded with Vpx-containing virus-like particles (VLPs) became extremely sensitive to HIV-1, suggested that not only could Vpx counteract a restriction against cognate viruses but it also enabled HIV-1 to infect DCs and to more efficiently infect MDMs [14,15,43]. Consistent with the notion that Vpx could overcome a dominant restriction factor in myeloid cells, heterokaryons generated between permissive COS cells and non-permissive/ restrictive MDMs, supported infection by wt SIV but not Vpx-deficient SIV [15]. Studies in myeloid cells designed to determine which step of the virus life cycle is affected by Vpx, revealed that the viral protein targeted the process of reverse transcription [13,14,44,45]. Importantly, the recruitment of the CRL4A (DCAF1) E3 ligase by Vpx was found to be required for enhancement of viral infectivity in myeloid cells. Furthermore, treatment of monocytes or DCs with the proteasome inhibitor MG132 restored partially the infectivity of Vpx-deficient SIVmac particles [45]. Taken together, these findings suggest a model in which Vpx redirects the activity of the CRL4A (DCAF1) to eliminate a host factor that is detrimental to efficient reverse transcription in MDMs and DCs. This assumption has now been readily demonstrated. Using immunoprecipitation combined with mass spectrometry, two groups have recently identified sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) as the cellular restriction factor targeted by Vpx [46,47]. While expression of SAMHD1 was associated with the restrictive or permissive phenotype of various cell types, the correlation was far from perfect, suggesting that additional requirements may be at play. Notably, Vpx was shown to relieve the SAMHD1mediated block of reverse transcription in myeloid cells by diverting the CRL4A (DCAF1) E3 ligase towards the www.sciencedirect.com
Lentivirus Vpr and Vpx accessory proteins Romani and Cohen 759
restriction factor, leading to its rapid and efficient proteasomal degradation (Figure 3).
which is induced by interferon-g and in response to viral infection [48]. Mutations in SAMHD1 cause Aicardi-Goutie`res syndrome (AGS), a rare and heterogenous genetic disease associated with inflammatory encephalopathy and characterized by inappropriate triggering of innate
The gene encoding human SAMHD1 was initially identified in DCs as a homologue of the mouse MG11 gene, Figure 3
(a)
HIV-1 ΔVpx HIV-2/SIVsm
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dNTP depletion Nuclear pore complex
dNTP depletion SAMHD1 dNTP
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(b) Virion incorporated Vpx
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SAMHD1 dNTP pools are increased
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Vpx interacts with the CRL4A (DCAF1) E3 ubiquitin ligase to induce the degradation of SAMHD1, a restriction factor that blocks reverse transcription in myeloid cells by depleting the pool of dNTPs. (a) In non-permissive myeloid cells, SAMHD1 blocks reverse transcription of HIV-1 (which, does not encode Vpx) or Vpx-deficient HIV-2/SIVsm by depleting the intracellular pools of deoxynucleoside triphosphates (dNTP) through its dNTP triphosphohydrolase activity. (b) HIV-2 and its precursor, SIVsm, express Vpx to counteract the SAMHD1-mediated restriction. Vpx diverts the activity of the CRL4A (DCAF1) E3 ligase to degrade SAMHD1 via the proteasome. This degradation increases the intracellular pool of dNTPs to levels that are required for efficient synthesis of the viral DNA by reverse transcription. dN, deoxynucleosides; PPP, triphosphates. www.sciencedirect.com
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immune responses, including overproduction of IFNa [49]. Like TREX1, another gene associated with AGS, SAMHD1 has been proposed to act as a negative regulator of the innate response by preventing the accumulation of reverse transcription products of virus or transposable elements that might otherwise be sensed by the cellular intrinsic type 1 IFN response [50,51]. SAMHD1 contains a SAM domain involved in protein–protein and protein– RNA interactions and a central HD domain, which is predicted to have nucleotidase and phosphodiesterase activities, raising the possibility that SAMHD1 may have a role in nucleotide metabolism. Indeed, structural and biochemical studies of the HD domain revealed that SAMHD1 is a potent dGTP-regulated triphosphohydrolase that converts deoxynucleoside triphosphates (dNTPs) into deoxynucleoside and triphosphate [52,53] (Figure 3a). Importantly, site-specific mutation of the crucial catalytic amino-acids of the HD domain responsible for the triphosphohydrolase activity of SAMHD1 was found to alleviate the restriction of HIV-1 infection in non-permissive cells [46], indicating that the antiviral activity of SAMHD1 would be mediated by an effect on dNTP metabolism. Indeed, in an elegant series of experiments, Lahouassa and colleagues recently provided evidence that SAMHD1 regulates the pool of dNTPs in myeloid cells, thus exerting a block on HIV-1 reverse transcription by reducing intracellular levels of dNTPs to below those required for the synthesis of viral DNA [54]. Degradation of SAMHD1 in non-permissive cells via transduction of Vpx-containing VLPs, resulted in increased dNTP levels and rescued HIV-1 restriction. Consistently, incubation of purified MDMs with extracellular deoxynucleosides to increase the intracellular pool of dNTPs enhanced the ability of DVpx SIVmac to infect cells, indicating that depletion of the dNTP pool is probably the mechanism underlying SAMHD1-mediated restriction of primate lentivirus infection of myeloid cells. This mechanism provides a rationale as to why SAMHD1 restriction functions only in terminally differentiated and non-dividing cells. DCs and MDMs are non-dividing cells that do not have to keep high concentrations of dNTPs. Furthermore, it explains the apparent paradox between the localization of SAMHD1, which is nuclear, and the site where it exerts its restriction on reverse transcription, which is cytosolic (Figure 3a). In fact, recent evidence suggests that Vpx-mediated degradation of SAMHD1 would be initiated in the nucleus. Indeed, while cytoplasmic variants of SAMHD1 were found to potently block lentiviral infection, they were resistant to degradation induced by the nuclear Vpx protein [55] (Figure 3b). Overall, these findings suggest that SAMHD1 is distinct from other host restriction factors identified up to now, such as APOBEC3F/3G, Tetherin or TRIM5, in that it does not appear to exert its antiviral activity by directly targeting a viral component of lentiviruses. Instead, Current Opinion in Virology 2012, 2:755–763
SAMHD1 renders specific target cells non-permissive to infection by depleting the building blocks that are central to lentivirus replication. Whether SAMHD1 restriction activity is limited to cells of the myeloid lineage or also affects other non-dividing cells with low pools of dNTPs, remains to be determined. The molecular mechanism for Vpx-mediated recruitment of SAMHD1 to the CRL4A (DCAF1) E3 ligase complex and the domains on the respective molecules that are involved have provided insights into the architecture of the Vpx–E3 ligase complex. Vpx binding with DCAF1 involves a conserved central helical domain in the Vpx protein, which indeed displays homology with the corresponding putative DCAF1-binding domain of HIV-1/ HIV-2 Vpr [13,14] (Figure 2). Recent genetic and biochemical evidence suggest that in complex with DCAF1, the structure of Vpx may be modulated in such way that the N-terminal domain of the protein forms a new interface to recruit SAMHD1 to the CRL4A (DCAF1) E3 ligase for ubiquitination [42]. This region of Vpx, which was previously suggested to bind a cellular restriction factor, is distinct from the region of HIV/SIV Vpr that is believed to bind its cellular target, which is located in its C-terminal region [28,56] (Figure 2). Whether DCAF1 also participates by binding to SAMHD1 directly or indirectly remains to be demonstrated. Nevertheless, the observation that no stable association was detected with DCAF1 alone discards the possibility that SAMHD1 is a natural substrate of the ligase and that Vpx’s role is to modulate its activity [42]. Rather, it argues that the mechanism used by Vpx to antagonize SAMHD1 is based on diverting the activity the CRL4A (DCAF1) E3 ligase towards the restriction factor to induce its ubiquitination and degradation (Figure 3b). Crystallographic studies will be required to reveal the detailed structural basis of the interactions occurring between DCAF-1, Vpx and SAMHD1. Recent studies have analysed the restriction activity of SAMHD1 from divergent primate species and their potential sensitivities to degradation in presence of Vpx protein from cognate SIVs [57,58]. Although primate SAMHD1 restriction activity towards HIV-1 was maintained through evolution, the sensitivity of primate SAMHD1 to Vpx-mediated degradation was species-specific. Indeed, SIVmac Vpx recruitment of human SAMHD1 to the E3 ligase complex was found to be dependent on an interaction with the C-terminal region of SAMHD1 [42,57]. This region of the protein, which appears to contain an autonomous sequence element recognized by SIVmac Vpx, at least in vitro [42], contains a cluster of amino acid residues that display strong positive selection during primate evolution. This provides a rationale for the species-specific targeting of human SAMHD1 by different SIV Vpx alleles [57]. However, although mutational analysis of the sites under www.sciencedirect.com
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positive selection in the C-terminus of human SAMHD1 revealed their potential importance for interaction with Vpx, the conservation of identical amino acid residues at a crucial position (V626) in owl monkey and gray mouse lemur SAMHD1 was not predictive of their sensitivities to SIVmac Vpx [57]. This finding raises the possibility that other sites may be involved in SAMHD1 recognition. Indeed, Lim and colleagues have also shown that positively selected residues within the N-terminal domain of SAMHD1 could determine both binding and sensitivity to Vpx-mediated degradation [58]. One unanswered question is why Vpx-encoding HIV-2 and SIVs have evolved mechanisms to degrade SAMHD1 and overcome restriction in DCs and MDMs, while HIV1, which is highly sensitive to SAMHD1, has not? One clue to this important question may lie in a recent observation made by Manel and colleagues. When DCs are rendered permissive to HIV-1 infection through expression of Vpx, these cells sense the infection via the presence of a cryptic sensor that detects newly synthesized viral proteins and, as a result produce IFNs [43]. Thus, the lack of Vpx may have allowed HIV-1 to avoid infecting myeloid cells so that, the activation of cytoplasmic sensors and the induction of inflammatory cytokines can be limited. In contrast, the ability of HIV-2 and some SIVs to establish infection of myeloid cells may have contributed to a better overall control of the invading virus by the immune system of the host. Whether these genetic and functional differences between HIV-1 and HIV-2 account at least in part for the different virulence properties of these two closely related viruses is an open question. Clearly, further investigation will be needed to provide additional insights into the role of SAMHD1 antagonism by Vpx in mediating the innate immune response and in influencing viral pathogenesis.
Concluding remarks Investigations of the evolutionary relationship of SAMHD1 orthologues in different primate species reveal that SAMHD1 antagonism may have resulted from a new function acquired by an ancestral Vpr protein during viral evolution [58]. Indeed, evolutionary analysis suggests that this new function appears to have preceded the acquisition of a Vpx gene in some primate lentiviruses. In fact, present day SIV lineages, such as SIV from African green monkeys (agm) and sykes (syk) monkeys, were found to encode Vpr proteins that display both SAMHD1 antagonism and the capacity to induce a G2 arrest. The new ability of this ancestral Vpr to degrade SAMHD1 is believed to have initiated an evolutionary arm race with SAMHD1, subjecting the protein to high levels of diverse selection during primate evolution. These novel and exciting notions led Lim and colleagues [58] to propose that this co-evolution led to the acquisition of Vpx through a gene duplication or recombination events, as previously proposed [59]. It may have become too costly for Vpr to www.sciencedirect.com
compete at the same time in two separate arm races with different primate host proteins. The selective advantage and functional implications of keeping solely Vpr and its associated G2 arrest activity, as for HIV-1, or encoding both Vpr and Vpx, as in the case of HIV-2, will require further investigation. Clearly, the identification and functional details of the host protein(s) targeted by Vpr will help address these important questions. However, it is reasonable to predict that Vpr and Vpx through their ability to usurp the CRL4A (DCAF1) E3 ligase and target host proteins for degradation, like SAMHD1, could have further effects on lentivirus immune evasion and pathogenesis.
Acknowledgements The authors thank Vibhuti Dave for helpful discussions and apologize to the authors of many interesting studies that could not be cited due to space limitations. EAC is recipient of the Canada Research Chair in Human Retrovirology. This work was supported by grants from the Canadian Institute of Health Research (HET 85519 and MOP 12381) to EAC.
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Le Rouzic E, Belaidouni N, Estrabaud E, Morel M, Rain JC, Transy C, Margottin-Goguet F: HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the CUL4–DDB1 ubiquitin ligase. Cell Cycle 2007, 6:182-188. This paper reported for the first time that HIV-1 Vpr engages the CRL4A (DCAF1) E3 ligase, via an interaction with DCAF1, to induce a G2 cell cycle arrest.
7.
Belzile JP, Duisit G, Rougeau N, Mercier J, Finzi A, Cohen EA: HIV1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase. PLoS Pathog 2007, 3:e85. See annotation to Ref. [6].
8.
Tan L, Ehrlich E, Yu XF: DDB1 and Cul4A are required for human immunodeficiency virus type 1 Vpr-induced G2 arrest. J Virol 2007, 81:10822-10830. See annotation to Ref. [6].
9.
DeHart JL, Zimmerman ES, Ardon O, Monteiro-Filho CM, Arganaraz ER, Planelles V: HIV-1 Vpr activates the G2 checkpoint through manipulation of the ubiquitin proteasome system. Virol J 2007, 4:57. See annotation to Ref. [6]. 10. Schrofelbauer B, Hakata Y, Landau NR: HIV-1 Vpr function is mediated by interaction with the damage-specific DNAbinding protein DDB1. Proc Natl Acad Sci U S A 2007, 104:41304135. See annotation to Ref. [6]. 11. Hrecka K, Gierszewska M, Srivastava S, Kozaczkiewicz L, Swanson SK, Florens L, Washburn MP, Skowronski J: Lentiviral Vpr usurps CUL4–DDB1[VprBP] E3 ubiquitin ligase to Current Opinion in Virology 2012, 2:755–763
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modulate cell cycle. Proc Natl Acad Sci U S A 2007, 104:11778-11783. See annotation to Ref. [6].
29. Ayinde D, Maudet C, Transy C, Margottin-Goguet F: Limelight on two HIV/SIV accessory proteins in macrophage infection: is Vpx overshadowing Vpr? Retrovirology 2010, 7:35.
12. Wen X, Duus KM, Friedrich TD, de Noronha CM: The HIV1 protein Vpr acts to promote G2 cell cycle arrest by engaging a DDB1 and cullin4A-containing ubiquitin ligase complex using VprBP/DCAF1 as an adaptor. J Biol Chem 2007, 282:27046-27057. See annotation to Ref. [6].
30. Wei W, Guo H, Han X, Liu X, Zhou X, Zhang W, Yu XF: A novel DCAF1-binding motif required for Vpx-mediated degradation of nuclear SAMHD1 and Vpr-induced G2 arrest. Cell Microbiol 2012.
13. Bergamaschi A, Ayinde D, David A, Le Rouzic E, Morel M, Collin G, Descamps D, Damond F, Brun-Vezinet F, Nisole S et al.: The human immunodeficiency virus type 2 Vpx protein usurps the CUL4A–DDB1 DCAF1 ubiquitin ligase to overcome a postentry block in macrophage infection. J Virol 2009, 83:4854-4860. 14. Srivastava S, Swanson SK, Manel N, Florens L, Washburn MP, Skowronski J: Lentiviral Vpx accessory factor targets VprBP/ DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection. PLoS Pathog 2008, 4:e1000059. First demonstration that Vpx usurps the CRL4A (DCAF1) E3 ligase to enable efficient reverse transcription in macrophages. 15. Sharova N, Wu Y, Zhu X, Stranska R, Kaushik R, Sharkey M, Stevenson M: Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction. PLoS Pathog 2008, 4:e1000057. First demonstration that mature macrophages harbour an antiviral restriction factor that is antagonized by Vpx. 16. Jackson S, Xiong Y: CRL4s: the CUL4-RING E3 ubiquitin ligases. Trends Biochem Sci 2009, 34:562-570. 17. Lee J, Zhou P: DCAFs, the missing link of the CUL4–DDB1 ubiquitin ligase. Mol Cell 2007, 26:775-780. 18. Leupin O, Bontron S, Strubin M: Hepatitis B virus X protein and simian virus 5 V protein exhibit similar UV-DDB1 binding properties to mediate distinct activities. J Virol 2003, 77:6274-6283. 19. Precious B, Childs K, Fitzpatrick-Swallow V, Goodbourn S, Randall RE: Simian virus 5 V protein acts as an adaptor, linking DDB1 to STAT2, to facilitate the ubiquitination of STAT1. J Virol 2005, 79:13434-13441.
31. Belzile JP, Abrahamyan LG, Gerard FC, Rougeau N, Cohen EA: Formation of mobile chromatin-associated nuclear foci containing HIV-1 Vpr and VPRBP is critical for the induction of G2 cell cycle arrest. PLoS Pathog 2010, 6:e1001080. Evidence suggesting that the host protein(s) targeted by HIV-1 Vpr to induce a G2 arrest might be chromatin-associated. 32. Belzile JP, Richard J, Rougeau N, Xiao Y, Cohen EA: HIV-1 Vpr induces the K48-linked polyubiquitination and proteasomal degradation of target cellular proteins to activate ATR and promote G2 arrest. J Virol 2010, 84:3320-3330. 33. Schrofelbauer B, Yu Q, Zeitlin SG, Landau NR: Human immunodeficiency virus type 1 Vpr induces the degradation of the UNG and SMUG uracil-DNA glycosylases. J Virol 2005, 79:10978-10987. 34. Ahn J, Vu T, Novince Z, Guerrero-Santoro J, Rapic-Otrin V, Gronenborn AM: HIV-1 Vpr loads uracil DNA glycosylase-2 onto DCAF1, a substrate recognition subunit of a cullin 4A-ring E3 ubiquitin ligase for proteasome-dependent degradation. J Biol Chem 2010, 285:37333-37341. 35. Wen X, Casey Klockow L, Nekorchuk M, Sharifi HJ, de Noronha CM: The HIV1 protein Vpr acts to enhance constitutive DCAF1-dependent UNG2 turnover. PLoS One 2012, 7:e30939. 36. Goh WC, Rogel ME, Kinsey CM, Michael SF, Fultz PN, Nowak MA, Hahn BH, Emerman M: HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo. Nat Med 1998, 4:65-71. 37. Richard J, Sindhu S, Pham TN, Belzile JP, Cohen EA: HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell-mediated killing. Blood 2010, 115:1354-1363.
20. Li T, Robert EI, van Breugel PC, Strubin M, Zheng N: A promiscuous alpha-helical motif anchors viral hijackers and substrate receptors to the CUL4–DDB1 ubiquitin ligase machinery. Nat Struct Mol Biol 2010, 17:105-111.
38. Ward J, Davis Z, DeHart J, Zimmerman E, Bosque A, Brunetta E, Mavilio D, Planelles V, Barker E: HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response. PLoS Pathog 2009, 5:e1000613.
21. Zhao LJ, Wang L, Mukherjee S, Narayan O: Biochemical mechanism of HIV-1 Vpr function. Oligomerization mediated by the N-terminal domain. J Biol Chem 1994, 269:32131-32137.
39. Lopez-Larrea C, Suarez-Alvarez B, Lopez-Soto A, LopezVazquez A, Gonzalez S: The NKG2D receptor: sensing stressed cells. Trends Mol Med 2008, 14:179-189.
22. Zhang S, Feng Y, Narayan O, Zhao LJ: Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein–protein interaction with a novel human cytoplasmic protein VprBP. Gene 2001, 263:131-140.
40. Khamsri B, Murao F, Yoshida A, Sakurai A, Uchiyama T, Shirai H, Matsuo Y, Fujita M, Adachi A: Comparative study on the structure and cytopathogenic activity of HIV Vpr/Vpx proteins. Microbes Infect 2006, 8:10-15.
23. Huang J, Chen J: VprBP targets Merlin to the Roc1-Cul4A– DDB1 E3 ligase complex for degradation. Oncogene 2008, 27:4056-4064.
41. Mahnke LA, Belshan M, Ratner L: Analysis of HIV-2 Vpx by modeling and insertional mutagenesis. Virology 2006, 348:165-174.
24. Kaur M, Khan MM, Kar A, Sharma A, Saxena S: CRL4–DDB1VPRBP ubiquitin ligase mediates the stress triggered proteolysis of Mcm10. Nucleic Acids Res 2012, 40:7332-7346.
42. Ahn J, Hao C, Yan J, DeLucia M, Mehrens J, Wang C, Gronenborn AM, Skowronski J: HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1. J Biol Chem 2012, 287:12550-12558.
25. Park JH, Bang SW, Kim SH, Hwang DS: Knockdown of human MCM10 activates G2 checkpoint pathway. Biochem Biophys Res Commun 2008, 365:490-495. 26. McCall CM, Miliani de Marval PL, Chastain PD 2nd, Jackson SC, He YJ, Kotake Y, Cook JG, Xiong Y: Human immunodeficiency virus type 1 Vpr-binding protein VprBP, a WD40 protein associated with the DDB1-CUL4 E3 ubiquitin ligase, is essential for DNA replication and embryonic development. Mol Cell Biol 2008, 28:5621-5633.
43. Manel N, Hogstad B, Wang Y, Levy DE, Unutmaz D, Littman DR: A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells. Nature 2010, 467:214-217. Demonstration that DCs, which are refractory to HIV-1 infection, harbour a cytoplasmic sensor for HIV-1, whose activation mediates an antiviral response.
27. Le Rouzic E, Benichou S: The Vpr protein from HIV-1: distinct roles along the viral life cycle. Retrovirology 2005, 2:11.
44. Kaushik R, Zhu X, Stranska R, Wu Y, Stevenson M: A cellular restriction dictates the permissivity of nondividing monocytes/macrophages to lentivirus and gammaretrovirus infection. Cell Host Microbe 2009, 6:68-80.
28. Dehart JL, Planelles V: Human immunodeficiency virus type 1 Vpr links proteasomal degradation and checkpoint activation. J Virol 2008, 82:1066-1072.
45. Goujon C, Riviere L, Jarrosson-Wuilleme L, Bernaud J, Rigal D, Darlix JL, Cimarelli A: SIVSM/HIV-2 Vpx proteins promote retroviral escape from a proteasome-dependent restriction
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pathway present in human dendritic cells. Retrovirology 2007, 4:2. 46. Laguette N, Sobhian B, Casartelli N, Ringeard M, Chable-Bessia C, Segeral E, Yatim A, Emiliani S, Schwartz O, Benkirane M: SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 2011, 474:654-657. Identification of SAMHD1 as the Vpx-regulated restriction factor responsible for inhibition of HIV-1 infection in DCs. 47. Hrecka K, Hao C, Gierszewska M, Swanson SK, Kesik Brodacka M, Srivastava S, Florens L, Washburn MP, Skowronski J: Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature 2011, 474:658-661. Identification of SAMHD1 as the antiviral factor restricting HIV-1 in macrophages. This paper further links Vpx-mediated SAMHD1 antagonism to the recruitment of the CRL4A (DCAF1) E3 ligase. 48. Li N, Zhang W, Cao X: Identification of human homologue of mouse IFN-gamma induced protein from human dendritic cells. Immunol Lett 2000, 74:221-224. 49. Rice GI, Bond J, Asipu A, Brunette RL, Manfield IW, Carr IM, Fuller JC, Jackson RM, Lamb T, Briggs TA et al.: Mutations involved in Aicardi-Goutieres syndrome implicate SAMHD1 as regulator of the innate immune response. Nat Genet 2009, 41:829-832. 50. Yan N, Regalado-Magdos AD, Stiggelbout B, Lee-Kirsch MA, Lieberman J: The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1. Nat Immunol 2010, 11:1005-1013. 51. Laguette N, Benkirane M: How SAMHD1 changes our view of viral restriction. Trends Immunol 2012, 33:26-33. 52. Goldstone DC, Ennis-Adeniran V, Hedden JJ, Groom HC, Rice GI, Christodoulou E, Walker PA, Kelly G, Haire LF, Yap MW et al.: HIV1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature 2011, 480:379-382. Showed that SAMHD1 is a dGTP-regulated deoxynucleoside triphosphate triphosphohydrolase. 53. Powell RD, Holland PJ, Hollis T, Perrino FW: Aicardi-Goutieres syndrome gene and HIV-1 restriction factor SAMHD1 is a dGTP-regulated deoxynucleotide triphosphohydrolase. J Biol Chem 2011, 286:43596-43600.
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Showed that SAMHD1 is a dGTP-regulated deoxynucleoside triphosphate triphosphohydrolase. 54. Lahouassa H, Daddacha W, Hofmann H, Ayinde D, Logue EC, Dragin L, Bloch N, Maudet C, Bertrand M, Gramberg T et al.: SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates. Nat Immunol 2012, 13:223-228. Demonstration that SAMHD1 blocks lentiviral reverse transcription in myeloid cells by lowering intracellular dNTPs to levels that are below those required for efficient synthesis of viral cDNA. 55. Brandariz-Nunez A, Valle-Casuso JC, White TE, Laguette N, Benkirane M, Brojatsch J, Diaz-Griffero F: Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac. Retrovirology 2012, 9:49. 56. Gramberg T, Sunseri N, Landau NR: Evidence for an activation domain at the amino terminus of simian immunodeficiency virus Vpx. J Virol 2010, 84:1387-1396. 57. Laguette N, Rahm N, Sobhian B, Chable-Bessia C, Munch J, Snoeck J, Sauter D, Switzer WM, Heneine W, Kirchhoff F et al.: Evolutionary and functional analyses of the interaction between the myeloid restriction factor SAMHD1 and the lentiviral Vpx protein. Cell Host Microbe 2012, 11:205-217. Mapping of the interaction domain between Vpx and the C-terminal region of SAMHD1 via the identification of residues under positive selection. This study further demonstrates that while SAMHD1 from different primate species are all restricting HIV-1, Vpx degrades and antagonizes SAMHD1 in a species-specific manner. 58. Lim ES, Fregoso OI, McCoy CO, Matsen FA, Malik HS, Emerman M: The ability of primate lentiviruses to degrade the monocyte restriction factor SAMHD1 preceded the birth of the viral accessory protein Vpx. Cell Host Microbe 2012, 11:194-204. This paper demonstrates that multiple Vpx proteins share the ability to degrade and antagonize SAMHD1 but this ability is often species specific. It also provides evidence that vpr is neofunctionalized to degrade SAMHD1 before the acquisition of a separate vpx gene, thus triggering an evolutionary arm race with SAMHD1. 59. Tristem M, Purvis A, Quicke DL: Complex evolutionary history of primate lentiviral vpr genes. Virology 1998, 240:232-237.
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Lentivirus Vpr and Vpx accessory proteins usurp the cullin4–DDB1 (DCAF1) E3 ubiquitin ligase Bizhan Romani1 and E´ric A Cohen1,2 Myeloid cells display a differential permissivity to primate lentivirus infection that is related to their ability to encode the Vpx and to a lesser extent the Vpr accessory proteins. Vpr is encoded by all primate lentiviruses, including HIV-1 and HIV-2, while its paralog, Vpx, is unique to HIV-2 and a subset of simian lentiviruses. Both proteins usurp the CRL4A (DCAF1) E3 ligase to fulfil their functions. Vpx induces the degradation of SAMHD1, a nucleotide triphosphohydrolase that blocks lentiviral reverse transcription in myeloid cells via depletion of the intracellular pool of dNTPs. Vpr engages CRL4A (DCAF1) to degrade a yet unknown factor(s), whose proteolysis induces a G2 cell-cycle arrest in dividing cells. Although the identification of the host protein(s) targeted for degradation by Vpr will be necessary to understand its actual function, the discovery of SAMHD1 has already shed light into a new mechanism of restriction that limits infection of myeloid cells by HIV-1. Addresses 1 Institut de Recherches Cliniques de Montre´al, Montre´al, Que´bec, Canada H2W 1R7 2 Department of Microbiology and Immunology, Universite´ de Montre´al, Montre´al, Que´bec, Canada H3C 3J7 Corresponding author: Cohen, E´ric A ([email protected]) Current Opinion in Virology 2012, 2:755–763 This review comes from a themed issue on Virus replication in animals and plants Edited by Peter Nagy and Christopher Richardson For a complete overview see the Issue and the Editorial Available online 10th October 2012 1879-6257/$ – see front matter, # 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2012.09.010
Introduction Viruses have evolved ways to modulate the host cell environment in order to promote efficient viral replication and to disrupt elements of innate or acquired immunity. One strategy particularly favoured by viruses to achieve these goals is to subvert the host ubiquitin machinery in order to induce the proteasomal or lysosomal degradation of specific cellular factors that play crucial roles in inhibiting viral replication and/or inducing immune responses [1,2]. The covalent attachment of ubiquitin molecules to a target protein requires the sequential activity of three enzymes: an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase, which ultimately controls the specificity of the conjugation process since it directly binds the target protein. A www.sciencedirect.com
major class of the estimated 600–1000 E3 ligases encoded by the human genome is represented by the cullin-ring ubiquitin ligases (CRLs). CRLs are multi-subunit complexes composed of a catalytic core containing the invariant ROC/RBX ring finger protein and a substrate recognition module comprising various adaptor proteins, nucleated around a cullin scaffold protein [3] (Figure 1). When usurping the host ubiquitin machinery, viruses most often target the E3 ubiquitin ligase component. Primate lentiviruses are no exception to this principle. Human immunodeficiency virus (HIV) and several simian immunodeficiency virus (SIV) lineages encode two accessory proteins, viral infectivity factor (Vif) and viral protein U (Vpu) that hijack the host ubiquitin machinery in order to respectively degrade two potent antiviral host proteins — also called restriction factors — the cytidine deaminases APOBEC3F/3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F/ 3G) and Tetherin (BST2, CD317), which act as potent inhibitors of early (reverse transcription) and late (viral release) events of the lentiviral infection cycle [4]. In both instances, these viral proteins target specific CRLs, namely cullin1-SKP1 (bTrCP) in the case of Vpu and cullin5 (elonginB/C) for Vif [5]. Recently, we and several other investigators demonstrated that a third accessory protein encoded by all primate immunodeficiency viruses, Viral protein R (Vpr), as well as its paralog, Vpx, exclusively encoded by HIV-2 and several SIV lineages, also exert their functions by recruiting an E3 ubiquitin ligase complex composed of cullin4A (CUL4A), damaged DNA binding protein 1 (DDB1) and a member of the DCAF (DDB1–cullin4-associated-factor) family called VPRBP (viral protein R binding protein, also known as DCAF1) [6,7,8,9,10,11,12,13,14,15] (Figure 1). In this review, we highlight the latest discoveries regarding how the CUL4A–DDB1 (DCAF1) (CRL4A (DCAF1)) E3 ubiquitin ligase is exploited by these two genetically related accessory proteins to fulfil what appears to be very distinct functions during viral infection. We also discuss outstanding questions regarding SAMHD1, the recently discovered Vpx cellular target that limits lentiviral infection in myeloid cells, the identity of the yet unknown host protein(s) targeted by Vpr, and the mechanisms via which Vpr and Vpx engage the CRL4A (DCAF1) E3 ligase to degrade host proteins.
The CUL4A–DDB1 (DCAF1) E3 ubiquitin ligase The evolutionary conserved CUL4 E3 ubiquitin ligase (CRL4) family, in concert with its large b-propeller Current Opinion in Virology 2012, 2:755–763
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Figure 1
NEDD8 Cul4A DDB1 H box
Substrate recognizing module
ROC
Catalytic core E2
WD40
? DCAF1
Ub
Vpr
Target? Ub
Ub
Ub Ub
Proteasomal degradation of the target
Activation of ATR
G2 cell cycle arrest Current Opinion in Virology
Proposed model for the mode of induction of G2 arrest by HIV-1 Vpr. The cullin4A–DDB1 (DCAF1) E3 ubiquitin ligase is a multisubunit complex that comprises a catalytic core and a substrate recognition module nucleated around a cullin4A (Cul4A) scaffold protein (pink). The catalytic core consists of a small RING protein (ROC, purple) that binds and activates and E2 enzyme (dark brown), which transfers ubiquitin (Ub, red) to a substrate. The substrate recognition module comprises a WD40-containing DCAF1 substrate specificity receptor (yellow) bound to the three-b-propeller protein, DDB1 (dark blue) via its WD40 domain and a putative structural H box element, as indicated. Vpr (orange) is proposed to bind the E3 ubiquitin ligase complex through DCAF1 to recruit a yet unknown protein target(s) (blue) whose ubiquitination and proteaosomal degradation results in activation of ATR and ultimately in induction of a G2 cell cycle arrest. Covalent conjugation of the ubiquitinlike modifier NEDD8 (light brown) activates cullin4A.
DDB1 adaptor, regulates a diverse set of cellular processes including development, transcription, replication and DNA repair [16]. Mass spectrometric analyses of the CUL4A–DDB1 complex have revealed physical interactions with a set of more than 50 WD40-containing proteins, referred to as DCAFs that act as substrate specificity receptors [17]. At present, more than twodozen substrates have been shown to be regulated by these complexes, a large fraction of those being chromatin-associated proteins [16]. In addition to controlling basic cellular processes, early studies revealed that CRL4A ligases are hijacked by several pathogenic viruses, including members of paramyxovirus and hepadnavirus families, to ubiquitinate host proteins, which are not normally targeted by this E3 ligase family. Hence, proteins V and X encoded by paramyxovirus simian virus Current Opinion in Virology 2012, 2:755–763
5 (SV5) and hepatitis B virus (HBV), respectively, interact directly with DDB1 to redirect the CUL4A–DDB1 E3 ubiquitin ligase towards new substrates [18]. While the biological implications of HBV X engagement to CRL4A remain poorly understood, SV5 V protein hijacking of the CRL4A E3 ligase complex was shown to promote the ubiquitination and subsequent proteasomal degradation of STAT1, so that cellular antiviral responses are impeded via inhibition of interferon (IFN) production [19]. Importantly, recent structural studies have provided key insights into how these viral proteins interact with DDB1 despite their sequence divergence. They reveal that binding to DDB1 is mediated through an alpha-helical motif, also called H box, which is indeed shared by the various cellular DCAFs. Thus, these studies have identified a common structural element that is crucial for anchoring these viral hijackers as well as cellular substrate-recruiting adapters to CRL4A E3 ubiquitin ligase complexes [20]. The DCAF1/VprBP WD40-containing substrate receptor that is part of the CRL4A recruited by Vpr and Vpx was identified less than two decades ago as an HIV-1 Vprinteracting protein [21,22]. However, the cellular target(s) normally recruited by DCAF1 remained elusive until recently. Huang and Chen reported that CRL4A (DCAF1) induces the rapid degradation of the tumour suppressor Merlin (NF2, neurofibromin 2) following serum starvation [23]. More recently, DCAF1 was also found to act as the substrate recognition subunit of the CRL4A complex that mediates the stress-induced proteolysis of Mcm10, a replication factor that plays an essential role in initiation and elongation of DNA replication [24]. Interestingly, mutations in Mcm10 have been shown to lead to stalled replication and cell cycle arrest, while depletion of the protein resulted in activation of the G2 checkpoint pathway [25]. However, depletion of DCAF1 was also found to reduce the rate of DNA replication and to impede cellular proliferation suggesting a crucial role for DCAF1 in regulating factors involved in DNA replication and the cell cycle [11,26].
Viral protein R, an enigmatic HIV accessory protein The genome of primate lentiviruses encodes a group of proteins comprising Vif, Vpr, Vpx, Vpu, and Nef — the so-called accessory proteins — whose functions are to modify the host cell environment to facilitate viral replication and to evade innate and adaptive antiviral immune responses [5]. Of these, Vpr remains one of the least understood accessory proteins in terms of its role in viral replication and pathogenesis. Vpr is a small phosphorylated nuclear protein that is capable of shuttling between the nucleus and the cytoplasm [27]. This protein, which is conserved across all primate lentiviruses, including HIV-1 and HIV-2, is packaged into progeny virions through an interaction with the Gag precursor protein, thus pointing to an early role during the viral life cycle. Most of the www.sciencedirect.com
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information regarding the roles of Vpr in virus replication has been derived from the study of HIV-1 Vpr. While many functions have been attributed to Vpr, the most widely accepted are induction of an arrest at the G2 phase of the cell cycle in dividing cells and enhancing infection in terminally differentiated myeloid cells, such as monocyte-derived macrophages (MDM) [28,29]. The functional relevance of Vpr-mediated G2 arrest remains unclear since efficient HIV-1 replication can be observed during ex vivo infection of dividing CD4+ T cells even in the absence of Vpr. However, one key advance in the field was the finding that the mechanism underlying Vprmediated G2 arrest involved the engagement of the CRL4A (DCAF1) E3 ubiquitin ligase by Vpr [6,7,8,9,10,11,12] (Figure 1). This recruitment, which was dependent on a physical interaction with the substrate-recruiting receptor DCAF1, was required to establish an intracellular environment that mimicked a DNA damage response initiated by the Ataxia Telangectasia and Rad3-related (ATR) protein kinase, ultimately resulting in the activation of a G2 checkpoint pathway [28]. While the architecture and the composition of the E3 ligase complexed with Vpr are still not entirely defined, mutagenic studies of Vpr have highlighted the existence of at least two functional domains (Figure 2). The first domain is involved in engaging DCAF1 and is dependent on conserved residues located within the third alpha helix of the protein and perhaps a conserved motif (Wx4Fx2Fx3AFxH; where x are residues that are highly variable and F corresponds to residues with bulky hydrophobic side chains) within the first helix [6,7,8,9,11,30]. The second one encompasses the C-terminal region of the protein, a domain of Vpr that is dispensable for DCAF1 binding, yet is essential for the induction of a G2 arrest [7,8,9,31]. The simplest model to explain these findings is that Vpr would recruit the E3 ligase complex through DCAF1 to induce the ubiquitination of a yet unknown cellular substrate(s), whose proteasomal degradation would lead to ATR
activation and G2 arrest (Figure 1). Indeed, results reported by our group revealed that Vpr could induce the lysine (K)48-linked poly-ubiquitination and proteasomal degradation of as-yet-unknown cellular proteins. Furthermore, Vpr-mediated K48-polyubiquitination and proteasomal degradation of these putative substrates were found necessary for the activation of the ATR pathway [32]. While the identity of the host protein(s) targeted by Vpr remains unknown, indirect evidence suggests that it might be chromatin-associated. Specifically, Vpr was found to associate with chromatin through its C-terminus and form mobile nuclear structures, designated as Vpr nuclear foci, where indeed DCAF1 and DNA repair foci components such as g-histone 2AX, 53BP1 and RPA32 co-localized [31]. Interestingly, inhibition of ATR kinase activity or depletion of DCAF1, two processes that prevent Vpr-mediated G2 arrest, did not inhibit the formation of Vpr nuclei foci, suggesting that formation of these Vpr-containing nuclear structures may represent a crucial early event in the process of Vprmediated ATR activation and G2 arrest. It is still unclear whether Vpr binds its substrate(s) directly on chromatin or alternatively via a potential cellular co-factor(s) that would be in proximity or even physically interacting with the substrate as it is the case for the SV5 V protein, which targets STAT1 by binding STAT2 [19]. The latter scenario would indeed account for the presence of Vpr nuclear foci in G2, when in all probability the putative Vpr cellular target(s) is degraded. Importantly, whether Vpr acts as an adaptor to recruit a new substrate(s) to the CRL4A (DCAF1) E3 ligase for ubiquitination and proteasomal degradation, a model analogous to that used by other HIV accessory proteins such as Vif or Vpx (see below), or whether Vpr forcibly enhances the ubiquitination of a natural substrate(s) targeted by DCAF1 remains unresolved. In that regard, it is interesting to note that uracil DNA glycosylases, UNG2 and SMUG1, two proteins previously shown to be targeted by HIV-1 Vpr for degradation but not linked to Vpr-mediated G2 arrest [33,34], might be novel natural substrates of CRL4A (DCAF1). In that particular context, Vpr was
Figure 2
α helix I
SAMHD1-binding domain
α helix II
α helix III
Predicted Vpr targetbinding domain
Elements involved in DCAF1 binding Current Opinion in Virology
Amino acid sequence alignment of Vpr and Vpx proteins encoded by HIV-1/HIV-2 and mapping of the functional regions of the proteins. Amino acid residues that are conserved in all the three sequences are highlighted in green while those conserved in two of the sequences are shown in red. Sequences were aligned using ClustalX (1.83) and highlighted using GeneDoc (2.7). Amino acids that constitute or predicted to form a-helices are indicated in purples boxes. The SAMHD1 binding site of HIV-2 Vpx at the N-terminus and the predicted target-binding site of HIV-1 Vpr at the Cterminus are also indicated. Position of HIV-1/2 Vpr a-helices were indicated as previously reported [40], while those of HIV-2 Vpx were shown as predicted by Mahnke et al. [41]. Gaps are indicated by (–). GeneBank accession numbers for HIV-1 pNL4.3 and HIV-2 ROD are U26942.1 and M15390.1, respectively. www.sciencedirect.com
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found to promote constitutive DCAF1-dependent UNG2 turnover by augmenting rather than permitting the interaction between UNG2 and the E3 ligase [35]. The functional relevance of Vpr-mediated G2 arrest in HIV-1 replication and pathogenesis will require the identification of the host protein(s) targeted by Vpr. While induction of a G2/M checkpoint in dividing cells could indeed be the function that Vpr was evolved to fulfil, it is also possible that it could be due to another function orchestrated by Vpr. However, the fact that transcription of viral genes is modestly upregulated when cells are in G2 suggests that Vpr-mediated G2 arrest could provide an optimal cellular environment for virus production. Such an effect would probably confer the virus with a selective advantage given the very short half-life of HIV-1-infected T cells [36]. More recent studies have also pointed towards an immuno-modulatory role of Vpr-mediated activation of DNA damage response. Indeed, our group and others have shown that Vpr-mediated activation of ATR leads to an upregulation of ligands of the activating natural killer (NK) cell receptor, NKG2D, in particular ULBP1 and ULBP2, at the surface of HIV-1-infected T cells, an effect that results in increased susceptibility to NK-cell cytolytic activity [37,38]. Although the purpose of upregulating NKG2D ligands remains ambiguous at this time, it is possible that it could further contribute to T cell depletion by rendering infected and Vpr-transducedbystander cells targets for NK-cell mediated killing. Additionally, upregulation of NKG2D ligands could be a contributing factor in the NK-cell dysfunction observed during chronic HIV infection through sustained effector activation, as previously observed in cancer models [39]. That being said, by analogy to the function of other viral accessory proteins, it is quite possible that the function of Vpr is to degrade a host factor that is unfavourable for viral replication or persistence during natural infection in vivo or in a specific cell type. Whether this host factor is targeted by the Vpr/CRL4A ligase complex to enhance HIV-1 infection of macrophages remains unknown. One could envision that degradation of a host factor(s) by Vpr in dividing cells would lead to induction of a G2 checkpoint, while Vpr-mediated degradation of the same cellular target in non-dividing macrophages would enhance HIV-1 replication or/and promote viral persistence. Identification of the host protein (s) targeted for degradation by the Vpr/CRL4A (DCAF1) complex will clearly provide important insights into the actual function of Vpr. Furthermore, the crystal structure of the DDB1DCAF1-Vpr substrate-recruiting module will be necessary to fully understand the architecture of the complex and the role of Vpr in either redirecting the specificity of the E3 ubiquitin ligase or in modulating its activity towards a natural substrate(s). Current Opinion in Virology 2012, 2:755–763
Vpx, an antagonist of SAMHD1 Primate lentiviruses in the phylogenetic group, which comprises HIV-2, its SIV precursor that naturally infects sooty mangabey (SIVsm) and SIVsm-derived rhesus macaques SIV(SIVmac), encode both Vpr, an orthologue of the HIV-1 Vpr protein, and Vpx, a related protein that displays sequence and predicted structural similarities [40,41] (Figure 2). As Vpr, Vpx is packaged into the core of progeny viral particles, is localized in the nucleus of infected cells and engages the CRL4A (DCAF1) E3 ubiquitin ligase through binding to the DCAF1 substrate specificity receptor [13,14,42]. Despite these similarities, the functional outcome of engaging the same E3 ubiquitin ligase is very different for Vpr and Vpx. Vpx expression does not induce a G2 cell cycle arrest. Rather, the protein was found to be crucial for the ability to efficiently infect myeloid cells, such as monocytes, dendritic cells (DC) and MDMs. In contrast, HIV-1 is inefficient at infecting DCs and although HIV-1 Vpr enhances HIV-1 replication in MDM, its effect is quite modest compared to that of Vpx during HIV-2 and SIVsm/mac infections [29]. Interestingly, the fact that both MDMs and DCs preloaded with Vpx-containing virus-like particles (VLPs) became extremely sensitive to HIV-1, suggested that not only could Vpx counteract a restriction against cognate viruses but it also enabled HIV-1 to infect DCs and to more efficiently infect MDMs [14,15,43]. Consistent with the notion that Vpx could overcome a dominant restriction factor in myeloid cells, heterokaryons generated between permissive COS cells and non-permissive/ restrictive MDMs, supported infection by wt SIV but not Vpx-deficient SIV [15]. Studies in myeloid cells designed to determine which step of the virus life cycle is affected by Vpx, revealed that the viral protein targeted the process of reverse transcription [13,14,44,45]. Importantly, the recruitment of the CRL4A (DCAF1) E3 ligase by Vpx was found to be required for enhancement of viral infectivity in myeloid cells. Furthermore, treatment of monocytes or DCs with the proteasome inhibitor MG132 restored partially the infectivity of Vpx-deficient SIVmac particles [45]. Taken together, these findings suggest a model in which Vpx redirects the activity of the CRL4A (DCAF1) to eliminate a host factor that is detrimental to efficient reverse transcription in MDMs and DCs. This assumption has now been readily demonstrated. Using immunoprecipitation combined with mass spectrometry, two groups have recently identified sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) as the cellular restriction factor targeted by Vpx [46,47]. While expression of SAMHD1 was associated with the restrictive or permissive phenotype of various cell types, the correlation was far from perfect, suggesting that additional requirements may be at play. Notably, Vpx was shown to relieve the SAMHD1mediated block of reverse transcription in myeloid cells by diverting the CRL4A (DCAF1) E3 ligase towards the www.sciencedirect.com
Lentivirus Vpr and Vpx accessory proteins Romani and Cohen 759
restriction factor, leading to its rapid and efficient proteasomal degradation (Figure 3).
which is induced by interferon-g and in response to viral infection [48]. Mutations in SAMHD1 cause Aicardi-Goutie`res syndrome (AGS), a rare and heterogenous genetic disease associated with inflammatory encephalopathy and characterized by inappropriate triggering of innate
The gene encoding human SAMHD1 was initially identified in DCs as a homologue of the mouse MG11 gene, Figure 3
(a)
HIV-1 ΔVpx HIV-2/SIVsm
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dNTP depletion Nuclear pore complex
dNTP depletion SAMHD1 dNTP
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(b) Virion incorporated Vpx
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↑dNTPs
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Cul4A DDB1 DCAF1 Vpx
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SAMHD1 dNTP pools are increased
Current Opinion in Virology
Vpx interacts with the CRL4A (DCAF1) E3 ubiquitin ligase to induce the degradation of SAMHD1, a restriction factor that blocks reverse transcription in myeloid cells by depleting the pool of dNTPs. (a) In non-permissive myeloid cells, SAMHD1 blocks reverse transcription of HIV-1 (which, does not encode Vpx) or Vpx-deficient HIV-2/SIVsm by depleting the intracellular pools of deoxynucleoside triphosphates (dNTP) through its dNTP triphosphohydrolase activity. (b) HIV-2 and its precursor, SIVsm, express Vpx to counteract the SAMHD1-mediated restriction. Vpx diverts the activity of the CRL4A (DCAF1) E3 ligase to degrade SAMHD1 via the proteasome. This degradation increases the intracellular pool of dNTPs to levels that are required for efficient synthesis of the viral DNA by reverse transcription. dN, deoxynucleosides; PPP, triphosphates. www.sciencedirect.com
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immune responses, including overproduction of IFNa [49]. Like TREX1, another gene associated with AGS, SAMHD1 has been proposed to act as a negative regulator of the innate response by preventing the accumulation of reverse transcription products of virus or transposable elements that might otherwise be sensed by the cellular intrinsic type 1 IFN response [50,51]. SAMHD1 contains a SAM domain involved in protein–protein and protein– RNA interactions and a central HD domain, which is predicted to have nucleotidase and phosphodiesterase activities, raising the possibility that SAMHD1 may have a role in nucleotide metabolism. Indeed, structural and biochemical studies of the HD domain revealed that SAMHD1 is a potent dGTP-regulated triphosphohydrolase that converts deoxynucleoside triphosphates (dNTPs) into deoxynucleoside and triphosphate [52,53] (Figure 3a). Importantly, site-specific mutation of the crucial catalytic amino-acids of the HD domain responsible for the triphosphohydrolase activity of SAMHD1 was found to alleviate the restriction of HIV-1 infection in non-permissive cells [46], indicating that the antiviral activity of SAMHD1 would be mediated by an effect on dNTP metabolism. Indeed, in an elegant series of experiments, Lahouassa and colleagues recently provided evidence that SAMHD1 regulates the pool of dNTPs in myeloid cells, thus exerting a block on HIV-1 reverse transcription by reducing intracellular levels of dNTPs to below those required for the synthesis of viral DNA [54]. Degradation of SAMHD1 in non-permissive cells via transduction of Vpx-containing VLPs, resulted in increased dNTP levels and rescued HIV-1 restriction. Consistently, incubation of purified MDMs with extracellular deoxynucleosides to increase the intracellular pool of dNTPs enhanced the ability of DVpx SIVmac to infect cells, indicating that depletion of the dNTP pool is probably the mechanism underlying SAMHD1-mediated restriction of primate lentivirus infection of myeloid cells. This mechanism provides a rationale as to why SAMHD1 restriction functions only in terminally differentiated and non-dividing cells. DCs and MDMs are non-dividing cells that do not have to keep high concentrations of dNTPs. Furthermore, it explains the apparent paradox between the localization of SAMHD1, which is nuclear, and the site where it exerts its restriction on reverse transcription, which is cytosolic (Figure 3a). In fact, recent evidence suggests that Vpx-mediated degradation of SAMHD1 would be initiated in the nucleus. Indeed, while cytoplasmic variants of SAMHD1 were found to potently block lentiviral infection, they were resistant to degradation induced by the nuclear Vpx protein [55] (Figure 3b). Overall, these findings suggest that SAMHD1 is distinct from other host restriction factors identified up to now, such as APOBEC3F/3G, Tetherin or TRIM5, in that it does not appear to exert its antiviral activity by directly targeting a viral component of lentiviruses. Instead, Current Opinion in Virology 2012, 2:755–763
SAMHD1 renders specific target cells non-permissive to infection by depleting the building blocks that are central to lentivirus replication. Whether SAMHD1 restriction activity is limited to cells of the myeloid lineage or also affects other non-dividing cells with low pools of dNTPs, remains to be determined. The molecular mechanism for Vpx-mediated recruitment of SAMHD1 to the CRL4A (DCAF1) E3 ligase complex and the domains on the respective molecules that are involved have provided insights into the architecture of the Vpx–E3 ligase complex. Vpx binding with DCAF1 involves a conserved central helical domain in the Vpx protein, which indeed displays homology with the corresponding putative DCAF1-binding domain of HIV-1/ HIV-2 Vpr [13,14] (Figure 2). Recent genetic and biochemical evidence suggest that in complex with DCAF1, the structure of Vpx may be modulated in such way that the N-terminal domain of the protein forms a new interface to recruit SAMHD1 to the CRL4A (DCAF1) E3 ligase for ubiquitination [42]. This region of Vpx, which was previously suggested to bind a cellular restriction factor, is distinct from the region of HIV/SIV Vpr that is believed to bind its cellular target, which is located in its C-terminal region [28,56] (Figure 2). Whether DCAF1 also participates by binding to SAMHD1 directly or indirectly remains to be demonstrated. Nevertheless, the observation that no stable association was detected with DCAF1 alone discards the possibility that SAMHD1 is a natural substrate of the ligase and that Vpx’s role is to modulate its activity [42]. Rather, it argues that the mechanism used by Vpx to antagonize SAMHD1 is based on diverting the activity the CRL4A (DCAF1) E3 ligase towards the restriction factor to induce its ubiquitination and degradation (Figure 3b). Crystallographic studies will be required to reveal the detailed structural basis of the interactions occurring between DCAF-1, Vpx and SAMHD1. Recent studies have analysed the restriction activity of SAMHD1 from divergent primate species and their potential sensitivities to degradation in presence of Vpx protein from cognate SIVs [57,58]. Although primate SAMHD1 restriction activity towards HIV-1 was maintained through evolution, the sensitivity of primate SAMHD1 to Vpx-mediated degradation was species-specific. Indeed, SIVmac Vpx recruitment of human SAMHD1 to the E3 ligase complex was found to be dependent on an interaction with the C-terminal region of SAMHD1 [42,57]. This region of the protein, which appears to contain an autonomous sequence element recognized by SIVmac Vpx, at least in vitro [42], contains a cluster of amino acid residues that display strong positive selection during primate evolution. This provides a rationale for the species-specific targeting of human SAMHD1 by different SIV Vpx alleles [57]. However, although mutational analysis of the sites under www.sciencedirect.com
Lentivirus Vpr and Vpx accessory proteins Romani and Cohen 761
positive selection in the C-terminus of human SAMHD1 revealed their potential importance for interaction with Vpx, the conservation of identical amino acid residues at a crucial position (V626) in owl monkey and gray mouse lemur SAMHD1 was not predictive of their sensitivities to SIVmac Vpx [57]. This finding raises the possibility that other sites may be involved in SAMHD1 recognition. Indeed, Lim and colleagues have also shown that positively selected residues within the N-terminal domain of SAMHD1 could determine both binding and sensitivity to Vpx-mediated degradation [58]. One unanswered question is why Vpx-encoding HIV-2 and SIVs have evolved mechanisms to degrade SAMHD1 and overcome restriction in DCs and MDMs, while HIV1, which is highly sensitive to SAMHD1, has not? One clue to this important question may lie in a recent observation made by Manel and colleagues. When DCs are rendered permissive to HIV-1 infection through expression of Vpx, these cells sense the infection via the presence of a cryptic sensor that detects newly synthesized viral proteins and, as a result produce IFNs [43]. Thus, the lack of Vpx may have allowed HIV-1 to avoid infecting myeloid cells so that, the activation of cytoplasmic sensors and the induction of inflammatory cytokines can be limited. In contrast, the ability of HIV-2 and some SIVs to establish infection of myeloid cells may have contributed to a better overall control of the invading virus by the immune system of the host. Whether these genetic and functional differences between HIV-1 and HIV-2 account at least in part for the different virulence properties of these two closely related viruses is an open question. Clearly, further investigation will be needed to provide additional insights into the role of SAMHD1 antagonism by Vpx in mediating the innate immune response and in influencing viral pathogenesis.
Concluding remarks Investigations of the evolutionary relationship of SAMHD1 orthologues in different primate species reveal that SAMHD1 antagonism may have resulted from a new function acquired by an ancestral Vpr protein during viral evolution [58]. Indeed, evolutionary analysis suggests that this new function appears to have preceded the acquisition of a Vpx gene in some primate lentiviruses. In fact, present day SIV lineages, such as SIV from African green monkeys (agm) and sykes (syk) monkeys, were found to encode Vpr proteins that display both SAMHD1 antagonism and the capacity to induce a G2 arrest. The new ability of this ancestral Vpr to degrade SAMHD1 is believed to have initiated an evolutionary arm race with SAMHD1, subjecting the protein to high levels of diverse selection during primate evolution. These novel and exciting notions led Lim and colleagues [58] to propose that this co-evolution led to the acquisition of Vpx through a gene duplication or recombination events, as previously proposed [59]. It may have become too costly for Vpr to www.sciencedirect.com
compete at the same time in two separate arm races with different primate host proteins. The selective advantage and functional implications of keeping solely Vpr and its associated G2 arrest activity, as for HIV-1, or encoding both Vpr and Vpx, as in the case of HIV-2, will require further investigation. Clearly, the identification and functional details of the host protein(s) targeted by Vpr will help address these important questions. However, it is reasonable to predict that Vpr and Vpx through their ability to usurp the CRL4A (DCAF1) E3 ligase and target host proteins for degradation, like SAMHD1, could have further effects on lentivirus immune evasion and pathogenesis.
Acknowledgements The authors thank Vibhuti Dave for helpful discussions and apologize to the authors of many interesting studies that could not be cited due to space limitations. EAC is recipient of the Canada Research Chair in Human Retrovirology. This work was supported by grants from the Canadian Institute of Health Research (HET 85519 and MOP 12381) to EAC.
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