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Supplementary Figure 2 from RASSF1C, an Isoform of the Tumor Suppressor RASSF1A, Promotes the Accumulation of β-Catenin by Interacting with βTrCP

The Ras-association domain family 1 (RASSF1) gene has seven different isoforms; isoform A is a tumor-suppressor gene (RASSF1A). The promoter of RASSF1A is inactivated in many cancers, whereas the expression of another major isoform, RASSF1C, is not affected. Here, we show that RASSF1C, but not RASSF1A, interacts with βTrCP. Binding of RASSF1C to βTrCP involves serine 18 and serine 19 of the SS18GYXS19 motif present in RASSF1C but not in RASSF1A. This motif is reminiscent of the canonical phosphorylation motif recognized by βTrCP; however, surprisingly, the association between RASSF1C and βTrCP does not occur via the βTrCP substrate binding domain, the WD40 repeats. Overexpression of RASSF1C, but not of RASSF1A, resulted in accumulation and transcriptional activation of the β-catenin oncogene, due to inhibition of its βTrCP-mediated degradation. Silencing of RASSF1A by small interfering RNA was sufficient for β-catenin to accumulate, whereas silencing of both RASSF1A and RASSF1C had no effect. Thus, RASSF1A and RASSF1C have opposite effects on β-catenin degradation. Our results suggest that RASSF1C expression in the absence of RASSF1A could play a role in tumorigenesis. [Cancer Res 2007;67(3):1054–61]

Supplementary Figure 3 from RASSF1C, an Isoform of the Tumor Suppressor RASSF1A, Promotes the Accumulation of β-Catenin by Interacting with βTrCP

Supplementary Figure 1 from RASSF1C, an Isoform of the Tumor Suppressor RASSF1A, Promotes the Accumulation of β-Catenin by Interacting with βTrCP

LEDGF/p75 (LEDGF) main cellular cofactor of HIV-1 integrase (IN), acts as tethering factor to target integration of HIV in actively transcribed genes. Recently a class of IN inhibitors based on inhibition of LEDGF-IN interaction has been developed. We describe here a new series of IN-LEDGF allosteric inhibitors (INLAIs), with potent anti-HIV-1 activity in the one-digit nanomolar range. These compounds inhibited IN-LEDGF interaction while enhancing IN-IN aberrant multimerization by allosteric mechanism. Compounds of this series were fully active on HIV-1 mutants resistant to IN strand transfer inhibitors (INSTIs) and other class of anti-HIV drugs, confirming that they belong to a new class of antiretrovirals. These compounds displayed most potent antiretroviral activity at post-integration due to aberrant IN polymerization responsible of infectivity defect of viral particles produced. INLAI-resistant mutants were selected by dose escalation method and the most detrimental mutation found was T174I. The impact of these resistance mutations was analyzed by fold shift EC50with regard to wild type virus and the replication capacity of mutated viruses were determined. Crystal structure of BDM-2, the lead compound of this series in complex with IN catalytic core domain (CCD) was elucidated. No antagonism was observed between BDM-2 and a panel of 16 antiretroviral drugs from different classes. In conclusion, the overall virologic profile of BDM-2 warrants the recently completed single ascending dose phase I trial (ClinicalTrials.govId:NCT03634085) and supports further clinical investigation for potential use in combination with other antiretroviral drugs.Author summaryIntegrase-LEDGF allosteric inhibitors are a new class of antiretrovirals recently developed that target the interaction of HIV-1 Integrase with its cellular cofactor LEDGF/p75 required for HIV-1 integration in actively transcribed genes. The great interest of developing such new class of antiretroviral is to add to the anti-HIV drug arsenal compounds that are fully active on resistant viruses to all other classes of drugs currently used in clinic. However, none of these inhibitors are currently in clinical use or in late clinical trials. Here we describe a series of highly potent Integrase allosteric inhibitors that can be considered as precursors of a new class of antiretroviral drugs, with fully conserved activity on viruses resistant to currently used anti-HIV drugs, and a lead compound that has no antagonism with a large panel of present anti-HIV drugs and that was successfully validated in a phase I clinical trial.

Background HIV-1 Integrase (IN) interacts with the cellular co-factor LEDGF/p75 and tethers the HIV preintegration complex to the host genome enabling integration. Recently a new class of IN inhibitors was described, the IN-LEDGF allosteric inhibitors (INLAIs). Designed to interfere with the IN-LEDGF interaction during integration, the major impact of these inhibitors was surprisingly found on virus maturation, causing a reverse transcription defect in target cells. Results Here we describe the MUT-A compound as a genuine INLAI with an original chemical structure based on a new type of scaffold, a thiophene ring. MUT-A has all characteristics of INLAI compounds such as inhibition of IN-LEDGF/p75 interaction, IN multimerization, dual antiretroviral (ARV) activities, normal packaging of genomic viral RNA and complete Gag protein maturation. MUT-A has more potent ARV activity compared to other INLAIs previously reported, but similar profile of resistance mutations and absence of ARV activity on SIV. HIV-1 virions produced in the presence of MUT-A were non-infectious with the formation of eccentric condensates outside of the core. In studying the immunoreactivity of these non-infectious virions, we found that inactivated HIV-1 particles were captured by anti-HIV-specific neutralizing and non-neutralizing antibodies (b12, 2G12, PGT121, 4D4, 10-1074, 10E8, VRC01) with efficiencies comparable to non-treated virus. Autologous CD4+ T lymphocyte proliferation and cytokine induction by monocyte-derived dendritic cells (MDDC) pulsed either with MUT-A-inactivated HIV or non-treated HIV were also comparable. Conclusions Although strongly defective in infectivity, HIV-1 virions produced in the presence of the MUT-A INLAI have a normal protein and genomic RNA content as well as B and T cell immunoreactivities comparable to non-treated HIV-1. These inactivated viruses might form an attractive new approach in vaccine research in an attempt to study if this new type of immunogen could elicit an immune response against HIV-1 in animal models. Electronic supplementary material The online version of this article (10.1186/s12977-017-0373-2) contains supplementary material, which is available to authorized users.

Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, whereas inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Because these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125-IN can be fully dissociated: MUT-A–induced IN multimerization and the formation of eccentric condensates in viral particles, which are responsible for inhibition of virus maturation, were lost, whereas inhibition of the IN-LEDGF/p75 interaction and consequently integration was fully retained. Hence, the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the Ala-125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125-IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation.

The HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the accuracy of reverse transcription. This role of Vpr was related to the recruitment of the nuclear form of the uracil DNA glycosylase (UNG2) enzyme into virus particles, but several conflicting findings have been reported regarding the role of UNG2 encapsidation on viral infectivity. Here, we report that the catalytic activity of UNG2 was not required for influencing HIV-1 mutation, and this function of UNG2 was mapped within a 60-amino-acid domain located in the N-terminal region of the protein required for direct interaction with the p32 subunit of the replication protein A (RPA) complex. Importantly, enforced recruitment of overexpressed UNG2 into virions resulted in a net increase of virus infectivity, and this positive effect on infectivity was also independent of the UNG2 enzymatic activity. In contrast, virus infectivity and replication, as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of either endogenous UNG2 or RPA p32. Taken together, these results demonstrate that incorporation of UNG2 into virions has a positive impact on HIV-1 infectivity and replication and positively influences the reverse transcription process through a nonenzymatic mechanism involving the p32 subunit of the RPA complex.

Macrophages are targets of human immunodeficiency virus (HIV) infection and play crucial roles in viral dissemination and pathogenesis (23, 24, 70). HIV-infected macrophages contribute to HIV spread to CD4 T lymphocytes and to the establishment of cellular virus reservoirs (2, 25, 48, 60). Identification of the mechanisms controlling HIV-1 replication in macrophages may lead to new therapeutic strategies. Microenvironmental stimuli can both enhance and inhibit HIV-1 replication in macrophages (29). Several cellular factors have been suggested to restrict HIV-1 infection in undifferentiated monocytes or to reduce macrophage permissivity to infection, including members of the APOBEC3 cytidine deaminase and TRIM families, the small isoform of the transcription factor C/EBPβ (CCAT enhancer-binding protein β), PPAR (for peroxisome proliferator-activated receptor), and more recently, microRNAs (8, 51, 61, 64, 71, 74). Despite these findings, no restriction factors that can be manipulated to render macrophages resistant to HIV-1 replication have clearly emerged. We have previously shown that the engagement of activating immunoglobulin G Fc receptors (FcγR) by immune complexes (IC) on monocyte-derived macrophages (hereafter called macrophages) restricts HIV-1 reverse transcription and integration, whereas viral entry, nuclear import, and gene expression from integrated proviruses are not inhibited (19, 52). Interestingly, FcγR-mediated inhibition is not limited to HIV-1 but also includes target related lentiviruses such as HIV-2, simian immunodeficiency virus (SIV)mac, and SIVagm, suggesting that this may be a common mechanism of lentivirus control (19). Engagement of activating FcγR on macrophages triggers signaling pathways, including phospholipase C, phosphatidylinositol-3 kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase (19). This leads to intracellular calcium augmentation, cytoskeleton remodeling and phagocytosis, as well as activation of transcription factors such as NF-κB, NFAT, and AP-1 (21, 33). Therefore, FcγR-mediated signaling could affect postentry steps of HIV-1 replication by modulating the expression of genes encoding molecules that interfere with reverse transcription or genome integration and/or by acting on the incoming virus through modifications of the cellular environment. The present study was designed to test these hypotheses. We examined whether FcγR aggregation by IC could modulate the expression of host factors that can interfere with early postentry steps of HIV replication. We studied restriction factors of the APOBEC3 and TRIM families (43, 63, 65), as well as host proteins recruited in the HIV-1 reverse transcription and preintegration complexes (RTC/PIC). We found that the cyclin-dependent kinase inhibitor (CKI) p21Cip1/Waf1 (hereafter referred to as p21) is induced by FcγR aggregation. Interestingly, p21 has been recently involved in the resistance to HIV infection in primitive hematopoietic cells (81). We show here that p21 inhibits the replication of HIV-1 and related primate lentiviruses in macrophages.

Many viruses subvert the host ubiquitin-proteasome system to optimize their life cycle. We recently documented such a mechanism for the human immunodeficiency virus type 1 Vpr protein, which promotes cell cycle arrest by recruiting the DCAF1 adaptor of the Cul4A-DDB1 ubiquitin ligase, a finding now confirmed by several groups. Here we examined the impact of Cul4A-DDB1(DCAF1) on Vpr stability. We show that the Vpr(Q65R) mutant, which is defective in DCAF1 binding, undergoes proteasome-mediated degradation at a higher rate than wild-type Vpr. DCAF1 overexpression stabilizes wild-type Vpr and leads to its cytoplasmic accumulation, whereas it has no effect on the Vpr(Q65R) mutant. Conversely, small interfering RNA-mediated silencing of DCAF1 decreases the steady state amount of the viral protein. Stabilization by DCAF1, which is conserved by Vpr species from human immunodeficiency virus type 2 and the SIVmac strain, results in increased G(2) arrest and requires the presence of DDB1, indicating that it occurs through assembly of Vpr with a functional Cul4A-DDB1(DCAF1) complex. Furthermore, in human immunodeficiency virus type 1-infected cells, the Vpr protein, issued from the incoming viral particle, is destabilized under DCAF1 or DDB1 silencing. Together with our previous findings, our data suggest that Cul4A-DDB1(DCAF1) acts at a dual level by providing Vpr with the equipment for the degradation of specific host proteins and by counter-acting its proteasome targeting by another cellular E3 ubiquitin ligase. This protection mechanism may represent an efficient way to optimize the activity of Vpr molecules that are delivered by the incoming virus before neosynthesis takes place. Targeting the Vpr-DCAF1 interaction might therefore present therapeutic interest.

The Ras-association domain family 1 (RASSF1) gene has seven different isoforms; isoform A is a tumor-suppressor gene (RASSF1A). The promoter of RASSF1A is inactivated in many cancers, whereas the expression of another major isoform, RASSF1C, is not affected. Here, we show that RASSF1C, but not RASSF1A, interacts with βTrCP. Binding of RASSF1C to βTrCP involves serine 18 and serine 19 of the SS18GYXS19 motif present in RASSF1C but not in RASSF1A. This motif is reminiscent of the canonical phosphorylation motif recognized by βTrCP; however, surprisingly, the association between RASSF1C and βTrCP does not occur via the βTrCP substrate binding domain, the WD40 repeats. Overexpression of RASSF1C, but not of RASSF1A, resulted in accumulation and transcriptional activation of the β-catenin oncogene, due to inhibition of its βTrCP-mediated degradation. Silencing of RASSF1A by small interfering RNA was sufficient for β-catenin to accumulate, whereas silencing of both RASSF1A and RASSF1C had no effect. Thus, RASSF1A and RASSF1C have opposite effects on β-catenin degradation. Our results suggest that RASSF1C expression in the absence of RASSF1A could play a role in tumorigenesis. [Cancer Res 2007;67(3):1054–61]

How the HIV1 Vpr protein initiates the host cell response leading to cell cycle arrest in G(2) has remained unknown. Here, we show that recruitment of DCAF1/VprBP by Vpr is essential for its cytostatic activity, which can be abolished either by single mutations of Vpr that impair DCAF1 binding, or by siRNA-mediated silencing of DCAF1. Furthermore, DCAF1 bridges Vpr to DDB1, a core subunit of Cul4 ubiquitin ligases. Altogether these results point to a mechanism where Vpr triggers G(2) arrest by hijacking the Cul4/DDB1(DCAF1) ubiquitin ligase. We further show that, Vpx, a non-cytostatic Vpr-related protein acquired by HIV2 and SIV, also binds DCAF1 through a conserved motif. Thus, Vpr from HIV1 and Vpx from SIV recruit DCAF1 with different physiological outcomes for the host cell. This in turn suggests that both proteins have evolved to preserve interaction with the same Cul4 ubiquitin ligase while diverging in the recognition of host substrates targeted for proteasomal degradation.

We analyzed the role of human immunodeficiency virus (HIV)-1 matrix protein (MA) during the virus replication afferent phase. Single-round infection of H9 T lymphocytes showed that the combined mutation of MA Lys residues 26–27 in MA reported nuclear localization signal (NLS)-1 [ Haffar, O.K., Popov, S., Dubrovsky, L., Agostini, I., Tang, H., Pushkarsky, T., Nadler, S.G., Bukrinsky, M., 2000. Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex. J. Mol. Biol. 299 (2): 359–368 ] impaired infectivity, abrogated 2-LTR-circle formation and significantly reduced integration. However, the mutation did not affect viral DNA docking to chromatin in either interphasic or mitotic cells, indicating that MA N-terminal basic domain should not represent a major determinant of HIV-1 nuclear import in T lymphocytes. These data point to a previously unreported role of MA in the late, post-chromatin-binding, afferent phase of HIV-1 replication cycle.

β-arrestins (βarrs) are two highly homologous proteins that uncouple G protein-coupled receptors from their cognate G proteins, serve as adaptor molecules linking G protein-coupled receptors to clathrin-coat components (AP-2 complex and clathrin), and act as scaffolding proteins for ERK1/2 and JNK3 cascades. A striking difference between the two βarrs (βarr1 and βarr2) is that βarr1 is evenly distributed throughout the cell, whereas βarr2 shows an apparent cytoplasmic localization at steady state. Here, we investigate the molecular determinants underlying this differential distribution. βarr2 is constitutively excluded from the nucleus by a leptomycin B-sensitive pathway because of the presence of a classical leucine-rich nuclear export signal in its C terminus (L395/L397) that is absent in βarr1. In addition, using a nuclear import assay in yeast we showed that βarr2 is actively imported into the nucleus, suggesting that βarr2 undergoes constitutive nucleocytoplasmic shuttling. In cells expressing βarr2, JNK3 is mostly cytosolic. A point mutation of the nuclear export signal (L395A) in βarr2, which was sufficient to redistribute βarr2 from the cytosol to the nucleus, also caused the nuclear relocalization of JNK3. These data indicate that the nucleocytoplasmic shuttling of βarr2 controls the subcellular distribution of JNK3.

The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

The karyophilic properties of the human immunodeficiency virus, type I (HIV-1) pre-integration complex (PIC) allow the virus to infect non-dividing cells. To better understand the mechanisms responsible for nuclear translocation of the PIC, we investigated nuclear import of HIV-1 integrase (IN), a PIC-associated viral enzyme involved in the integration of the viral genome in the host cell DNA. Accumulation of HIV-1 IN into nuclei of digitonin-permeabilized cells does not result from passive diffusion but rather from an active transport that occurs through the nuclear pore complexes. HIV-1 IN is imported by a saturable mechanism, implying that a limiting cellular factor is responsible for this process. Although IN has been previously proposed to contain classical basic nuclear localization signals, we found that nuclear accumulation of IN does not involve karyopherins alpha, beta1, and beta2-mediated pathways. Neither the non-hydrolyzable GTP analog, guanosine 5'-O-(thiotriphosphate), nor the GTP hydrolysis-deficient Ran mutant, RanQ69L, significantly affects nuclear import of IN, which depends instead on ATP hydrolysis. Therefore these results support the idea that IN import is not mediated by members of the karyopherin beta family. More generally, in vitro nuclear import of IN does not require addition of cytosolic factors, suggesting that cellular factor(s) involved in this active but atypical pathway process probably remain associated with the nuclear compartment or the nuclear pore complexes from permeabilized cells.

The virion-associated Vpr protein of human immunodeficiency virus type 1 (HIV-1) alters cell cycle progression from the G2 phase, influences the virus in vivo mutation rate, and participates in the nuclear translocation of viral DNA. While many Vpr-interacting proteins have been identified, the functional relevance of these interactions remains to be thoroughly documented. We have explored the contribution of the interaction of HIV-1 Vpr with HHR23A, a cellular protein implicated in DNA repair, to the known phenotypes of Vpr. The association of Vpr with HHR23A required the core region of Vpr, which encompasses the two alpha-helical structures of the protein. No binding of HHR23A was detected with the Vpr and Vpx proteins of other primate lentiviruses. HIV-1 Vpr variants containing single amino acid substitutions in each alpha-helix and deficient for binding to HHR23A were isolated. The functional characterization of these Vpr variants indicated that binding to HHR23A did not correlate with the ability of Vpr to induce cell cycle arrest, even though it was previously proposed that HHR23A is a mediator of the Vpr-induced G2 arrest. Also, the Vpr‐HHR23A interaction did not influence the HIV-1 in vivo mutation rate. Finally, Vpr and HHR23A are both localized in the nucleus, but no correlation was observed between the nuclear targeting of Vpr and the interaction with HHR23A. Further analysis is needed to determine the functional role(s) of the Vpr-HHR23A association during the HIV-1 life cycle. © 2001 Academic Press

The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with beta-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene.

A complete renin-angiotensin system (RAS) is locally expressed in the brain and fulfills important functions. Angiotensin II, the major biologically active peptide of the RAS, acts via binding to two main receptor subtypes designated AT1 and AT2. The present paper focuses on AT2 receptors, which have been reported to have neuroprotective effects on stroke, degenerative diseases, and cognitive functions. Our group has identified a family of AT2 receptor interacting proteins (ATIPs) comprising three major members (ATIP1, ATIP3, and ATIP4) with different intracellular localization. Of interest, all ATIP members are expressed in brain tissues and carry a conserved domain able to interact with the AT2 receptor intracellular tail, suggesting a role in AT2-mediated brain functions. We summarize here current knowledge on the ATIP family of proteins, and we present new experimental evidence showing interaction defects between ATIP1 and two mutant forms of the AT2 receptor identified in cases of mental retardation. These studies point to a functional role of the AT2/ATIP1 axis in cognition.

Vpr, a small HIV auxiliary protein, hijacks the CUL4 ubiquitin ligase through DCAF1 to inactivate an unknown cellular target, leading to cell cycle arrest at the G(2) phase and cell death. Here we first sought to delineate the Vpr determinants involved in the binding to DCAF1 and to the target. On the one hand, the three α-helices of Vpr are necessary and sufficient for binding to DCAF1; on the other hand, nonlinear determinants in Vpr are required for binding to the target, as shown by using protein chimeras. We also underscore that a SRIG motif conserved in the C-terminal tail of Vpr proteins from HIV-1/SIVcpz and HIV-2/SIVsmm lineages is critical for G(2) arrest. Our results suggest that this motif may be predictive of the ability of Vpr proteins from other SIV lineages to mediate G(2) arrest. We took advantage of the characterization of a subset of G(2) arrest-defective, but DCAF1 binding-proficient mutants, to investigate whether Vpr interferes with cell viability independently of its ability to induce G(2) arrest. These mutants inhibited cell colony formation in HeLa cells and are cytotoxic in lymphocytes, unmasking a G(2) arrest-independent cytopathic effect of Vpr. Furthermore these mutants do not block cell cycle progression at the G(1) or S phases but trigger apoptosis through caspase 3. Disruption of DCAF1 binding restored efficiency of colony formation. However, DCAF1 binding per se is not sufficient to confer cytopathicity. These data support a model in which Vpr recruits DCAF1 to induce the degradation of two host proteins independently required for proper cell growth.

Although HIV-1 Vpr displays several functions in vitro, limited information exists concerning their relevance during infection. Here, we characterized Vpr variants isolated from a rapid and a long-term non-progressor (LTNP). Interestingly, vpr alleles isolated from longitudinal samples of the LTNP revealed a dominant sequence that subsequently led to diversity similar to that observed in the progressor patient. Most of primary Vpr proteins accumulated at the nuclear envelope and interacted with host-cell partners of Vpr. They displayed cytostatic and proapoptotic activities, although a LTNP allele, harboring the Q65R substitution, failed to bind the DCAF1 subunit of the Cul4a/DDB1 E3 ligase and was inactive. This Q65R substitution correlated with impairment of Vpr docking at the nuclear envelope, raising the possibility of a functional link between this property and the Vpr cytostatic activity. In contradiction with published results, the R77Q substitution, found in LTNP alleles, did not influence Vpr proapoptotic activity.

Human immunodeficiency virus type 2 (HIV-2), the second causative agent of human AIDS (5), arose from cross-transmission of sooty mangabey simian immunodeficiency virus (SIVsm), a lentivirus that naturally infects sooty mangabeys without inducing overt disease (14). Although AIDS caused by HIV-2 is as lethal as that caused by HIV-1, most HIV-2 carriers remain asymptomatic much longer than HIV-1 carriers (36). On the other hand, cross-transmission of SIVsm to rhesus macaques gave rise to highly pathogenic viral strains, and infected animals develop a disease similar to human AIDS (14). Thus, the SIVsm lentiviral lineage exhibits markedly different pathogenicities in its original and recently acquired hosts. It is currently unknown whether cross-species transmission of primate lentiviruses leads to specific functional changes in viral proteins that might directly modulate pathogenesis in the new host species. The HIV-1 and HIV-2 lentiviral lineages differ in the auxiliary proteins encoded by their genomes, which likely reflects different selective pressures in their adaptation to the host cell environment. Thus, the Vpx protein found in HIV-2 has no counterpart in HIV-1, whereas the genetically related Vpr protein is present in both lineages (47). Both Vpx and Vpr are actively packaged into the virions (1, 40), which suggests a role in early infection steps, i.e., prior to viral synthesis. Lack of Vpx strongly decreases the pathogenicity of SIVsmPBj in infected macaques (17). In vitro, Vpx is dispensable for infection of immortalized T cells but has been shown to be critical for primary macrophage infection (9, 23). In contrast, no cell culture system has so far revealed a strong requirement for Vpr in HIV infection, although Vpr-defective HIV-1 shows attenuated replication in human macrophages (2, 6, 16). Despite the absence of a clear defective phenotype of HIV lacking Vpr, this viral protein has so far attracted more attention than Vpx, presumably because of its intriguing ability to arrest the cell cycle at the G2/M transition (15, 21, 34, 35). We and others recently demonstrated that Vpr recruits DCAF1/VprBP, an adaptor of the CUL4A-DDB1 ubiquitin ligase complex (3, 8, 18, 26, 38, 46). A major role of ubiquitin ligases is the labeling of specific proteins for proteasome-mediated degradation, and the current hypothesis is that Vpr diverts the DCAF1 ubiquitin ligase to induce the degradation of a host protein required for entry into mitosis. We previously showed that Vpx from SIVsm also interacts with DCAF1, although unlike Vpr, it does not arrest the cell cycle (26). The ability to recruit DCAF1 might thus represent an ancient functional acquisition that predates the emergence of the genetically related but functionally distinct Vpr and Vpx genes. Here, we explore this hypothesis and address the potential role of Vpx-DCAF1 interaction in the context of HIV-2 replication in human macrophages.

HIV and SIV genomes encode several auxiliary proteins which have increasingly shown their importance in the virus-host relationship. One of these proteins, Vpx, is unique to the HIV-2/SIVsm lineage and is critical for viral replication in macrophages. The functional basis for this requirement as well as the Vpx mode of action have remained a mystery for quite some time. We have previously shown that HIV-1 Vpr induces cell cycle arrest by recruiting the CUL4A-DDB1DCAF1 ubiquitin ligase. This presumably leads to the degradation of a host factor required for cell cycle progression into mitosis. Vpr and Vpx proteins are evolutionary related and show significant sequence similarity even though they are not functionally redundant. This prompted us to address whether Vpx, like Vpr, recruits the CUL4A-DDB1DCAF1 ubiquitin ligase and whether Vpx uses this functional property to enable efficient macrophage infection by HIV-2. Confirming this hypothesis, our results show that DCAF1 is a critical host effector of Vpx in its ability to mediate infection and long-term replication of HIV-2 in human macrophages. WT Vpx associates with the DDB1 component of the CUL4A ubiquitin ligase through DCAF1 binding. In contrast the Q76R Vpx mutant is unable to bind DCAF1 and recruit DDB1. When placed in the HIV-2 provirus, Q76R Vpx severely diminished viral replication in macrophages. Vpx is incorporated into newly formed virions, suggesting an early function in the next infection cycle. Accordingly, precluding Vpx present in the incoming virions from recruiting DCAF1 in target macrophages leads to a postentry block characterized by defective accumulation of HIV-2 reverse transcripts. In addition Vpx from SIVsm functionally complements Vpx-defective HIV-2 in a DCAF1-binding dependent manner. Altogether, our data point to a mechanism in which Vpx diverts the DCAF1 ubiquitin ligase to inactivate an evolutionary conserved factor (RF: restriction factor), which restricts infection of macrophages by HIV-2 and closely related simian viruses (Figure 1). This function of Vpx challenges the previous idea that Vpx complements the lack of cellular factors necessary for viral replication in macrophages. Vpx therefore acts similarly to other HIV auxiliary proteins (Vif, Vpu, Nef) known to inactivate cellular factors in order to create an advantageous environment for the virus.

Since the first isolation of HIV-1 from a patient with generalized lymphadenopathy in 1983, great progress has been made in understanding the viral life cycle and the functional nuances of each of the nine genes encoded by HIV-1. Considerable attention has been paid to four small HIV-1 open reading frames, vif, vpr, vpu and nef. These genes were originally termed "accessory" because their deletion failed to completely disable viral replication in vitro. More than twenty years after the cloning and sequencing of HIV-1, a great deal of information is available regarding the multiple functions of the accessory proteins and it is well accepted that, collectively, these gene products modulate the host cell biology to favor viral replication, and that they are largely responsible for the pathogenesis of HIV-1. Expression of Vpr, in particular, leads to cell cycle arrest in G(2), followed by apoptosis. Here we summarize our current understanding of Vpr biology with a focus on Vpr-induced G(2) arrest and apoptosis.

Viral protein U (Vpu) of HIV-1 has two known functions in replication of the virus: degradation of its cellular receptor CD4 and enhancement of viral particle release. Vpu binds CD4 and simultaneously recruits the βTrCP subunit of the SCFβTrCP ubiquitin ligase complex through its constitutively phosphorylated DS52GXXS56 motif. In this process, Vpu was found to escape degradation, while inhibiting the degradation of βTrCP natural targets such as β-catenin and IκBα. We further addressed this Vpu inhibitory function with respect to the degradation of Emi1 and Cdc25A, two βTrCP substrates involved in cell-cycle progression. In the course of these experiments, we underscored the importance of a novel phosphorylation site in Vpu. We show that, especially in cells arrested in early mitosis, Vpu undergoes phosphorylation of the serine 61 residue, which lies adjacent to the βTrCP-binding motif. This phosphorylation event triggers Vpu degradation by a βTrCP-independent process. Mutation of Vpu S61 in the HIV-1 provirus extends the half-life of the protein and significantly increases the release of HIV-1 particles from HeLa cells. However, the S61 determinant of regulated Vpu turnover is highly conserved within HIV-1 isolates. Altogether, our results highlight a mechanism where differential phosphorylation of Vpu determines its fate as an adaptor or as a substrate of distinct ubiquitin ligases. Conservation of the Vpu degradation determinant, despite its negative effect on virion release, argues for a role in overall HIV-1 fitness., Author Summary In addition to the structural and catalytic proteins characteristic of retroviruses, HIV-1 encodes auxiliary proteins that govern virus-host interaction. One such auxiliary protein termed Vpu both enhances virion release from human cells and the degradation of CD4, the cellular surface receptor of HIV-1. The latter activity relies on a strategy frequently used by pathogens, i.e., the hijacking of the protein degradation machinery. This process involves the ubiquitin ligases that ensure the selection of the protein subsequently degraded. Vpu interacts both with an ubiquitin ligase and CD4, bridging the latter to the degradation machinery. Here, we show that in addition to subverting one particular ubiquitin ligase to trigger CD4 degradation, Vpu is itself recognized in a phosphorylation-dependent manner by a distinct ubiquitin ligase and degraded. Mutations of Vpu that confer resistance to this degradation process enhance viral particle release, whereas the determinant of Vpu turnover is paradoxically well conserved among HIV-1 isolates. These results suggest that the virus is constrained to limit its own production for long-term persistence.

Human immunodeficiency virus type 1 is able to infect nondividing cells, such as macrophages, and the viral Vpr protein has been shown to participate in this process. Here, we investigated the impact of the recruitment into virus particles of the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, on the virus mutation rate and on replication in macrophages. We demonstrate that the interaction of Vpr with UNG2 led to virion incorporation of a catalytically active enzyme that is directly involved with Vpr in modulating the virus mutation rate. The lack of UNG in virions during virus replication in primary monocyte-derived macrophages further exacerbated virus mutant frequencies to an 18-fold increase compared with the 4-fold increase measured in actively dividing cells. Because the presence of UNG is also critical for efficient infection of macrophages, these observations extend the role of Vpr to another early step of the virus life cycle, e.g. viral DNA synthesis, that is essential for replication of human immunodeficiency virus type 1 in nondividing cells.

The evolution of drug resistance is a major complication of human immunodeficiency virus type 1 (HIV-1) chemotherapy. HIV-1 reverse transcriptase (RT) is a major target of antiretroviral therapy and ultimately the target of drug resistance mutations. Previous studies have indicated that drug-resistant HIV-1 RTs can alter HIV-1 mutant frequencies. In this study, we have tested a panel of HIV-1 RT variants for their ability to influence virus mutant frequencies. The RT variants tested included drug-resistant RT variants as well as other variants analyzed in enzyme fidelity studies with the lacZ α gene as a mutation target and/or implicated as being important for enzyme fidelity by structural studies. Combinations of mutations that alone had a statistically significant influence on virus mutant frequencies resulted in different mutant frequency phenotypes. Furthermore, when virus replication occurred in the presence of drugs [e.g., 3′-azido-3′-deoxythymidine, (−)2/,3′-dideoxy-3′-thiacytidine, hydroxyurea, thymidine, or thioguanine] with selected RT variants, virus mutant frequencies increased. Similarly, Vpr variants deficient for binding to the uracil DNA glycosylase repair enzyme were observed to influence HIV-1 virus mutant frequencies when tested alone or in combination with RT variants. In summary, these observations indicate that HIV-1 mutant frequencies can significantly change by single amino acid substitutions in RT and that these effects can be altered by additional mutations in RT, by drugs, and/or by expression of Vpr variants. Such altered virus mutant frequencies could impact HIV-1 dynamics and evolution in small population sizes.

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

Background LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction. Results We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket. Conclusion Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.

Background Macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. We have previously shown that the engagement of the activating Fc receptors of the IgG (FcγR) by immune complexes (IC) suppresses the replication of HIV-1 and related lentiviruses in monocyte-derived macrophages, inhibiting both the accumulation of reverse transcripts and the proviral integration [1].

Background HIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages. Results In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first α-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first α-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors. Conclusion These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.

The most widely recognized property of the HIV-1 Vpr auxiliary protein is its ability to arrest cell cycle progression at the G2 phase, which eventually triggers an apoptotic response. There is a long standing controversy as to whether Vpr might exhibit cytopathic activity independently of its G2 arrest property. We developed a clonogenic assay based on the expression of wild-type and mutant versions of Vpr using an EBV-based episome carrying a hygromycin resistance selection marker. Expression of the wt Vpr protein abrogated the formation of hygromycin-resistant cell colonies, confirming that long-term expression of Vpr is lethal in dividing cells. We previously showed that Vpr promotes G2 arrest through the recruitment of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Intriguingly, several HIV-1 Vpr mutants, which are devoid of G2 arrest properties but conserve DCAF1 binding, strongly reduced the formation of hygromycin-resistant cell colonies. Long term expression of these mutants induced G1 arrest and apoptosis. Thus Vpr expression can cause cell death independently of its G2 arrest activity. Disruption of the DCAF1-binding function of Vpr restored efficiency of colony formation indicating that recruitment of DCAF1 is required for this second cytotoxic activity of Vpr. However, DCAF1 binding is not sufficient to confer cytopathicity since Vpx from HIV-2/SIVsm does not impair colony formation. Altogether our data indicate that Vpr exhibits two dictinct cytotoxic activities in dividing cells, which both require the recruitment of DCAF1. Based on the current view of Vpr mechanism of action, i.e. hijacking of the host ubiquitination machinery, Vpr likely induces the proteasome-mediated degradation of two host proteins which are independently required for proper cell growth. This work was supported by grants from ANRS, Sidaction, Fondation de France and Mairie de Paris.