[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 07/2014; 9(7):e103552. DOI:10.1371/journal.pone.0103552 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
International Journal of Hypertension 01/2013; 2013(14):513047. DOI:10.1155/2013/513047
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 12/2011; 86(5):2533-44. DOI:10.1128/JVI.05163-11 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 G2 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 G2 arrest. Our results suggest that this motif may be predictive of the ability of Vpr proteins from other SIV lineages to mediate
G2 arrest. We took advantage of the characterization of a subset of G2 arrest-defective, but DCAF1 binding-proficient mutants, to investigate whether Vpr interferes with cell viability independently
of its ability to induce G2 arrest. These mutants inhibited cell colony formation in HeLa cells and are cytotoxic in lymphocytes, unmasking a G2 arrest-independent cytopathic effect of Vpr. Furthermore these mutants do not block cell cycle progression at the G1 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 10/2009; 4(10):e7514. DOI:10.1371/journal.pone.0007514 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophages are major targets of human immunodeficiency virus type 1 (HIV-1). We have previously shown that aggregation of activating immunoglobulin G Fc receptors (FcgammaR) by immune complexes inhibits reverse transcript accumulation and integration of HIV-1 and related lentiviruses in monocyte-derived macrophages. Here, we show that FcgammaR-mediated restriction of HIV-1 is not due to enhanced degradation of incoming viral proteins or cDNA and is associated to the induction of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1) (p21). Small interfering RNA-mediated p21 knockdown rescued viral replication in FcgammaR-activated macrophages and enhanced HIV-1 infection in unstimulated macrophages by increasing reverse transcript and integrated DNA levels. p21 induction by other stimuli, such as phorbol myristate acetate and the histone deacetylase inhibitor MS-275, was also associated with preintegrative blocks of HIV-1 replication in macrophages. Binding of p21 to reverse transcription/preintegration complex-associated HIV-1 proteins was not detected in yeast two-hybrid, pulldown, or coimmunoprecipitation assays, suggesting that p21 may affect viral replication independently of a specific interaction with an HIV-1 component. Consistently, p21 silencing rescued viral replication from the FcgammaR-mediated restriction also in simian immunodeficiency virus SIV(mac)- and HIV-2-infected macrophages. Our results point to a role of p21 as an inhibitory factor of lentiviral infection in macrophages and to its implication in FcgammaR-mediated restriction.
Journal of Virology 09/2009; 83(23):12253-65. DOI:10.1128/JVI.01395-09 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) genomes encode several auxiliary proteins that
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, has remained unexplained, and it is all the more enigmatic that HIV type 1 (HIV-1), which has no Vpx counterpart,
can infect macrophages. Here, we underscore DCAF1 as a critical host effector of Vpx in its ability to mediate infection and
long-term replication of HIV-2 in human macrophages. Vpx assembles with the CUL4A-DDB1 ubiquitin ligase through DCAF1 recruitment.
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 Cul4A-DDB1DCAF1 ligase to inactivate an evolutionarily conserved factor, which restricts macrophage infection by HIV-2 and closely related
Journal of Virology 04/2009; 83(10):4854-60. DOI:10.1128/JVI.00187-09 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 betaTrCP subunit of the SCF(betaTrCP) ubiquitin ligase complex through its constitutively phosphorylated DS52GXXS56 motif. In this process, Vpu was found to escape degradation, while inhibiting the degradation of betaTrCP natural targets such as beta-catenin and IkappaBalpha. We further addressed this Vpu inhibitory function with respect to the degradation of Emi1 and Cdc25A, two betaTrCP 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 betaTrCP-binding motif. This phosphorylation event triggers Vpu degradation by a betaTrCP-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.
[Show abstract][Hide abstract] ABSTRACT: 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 betaTrCP. Binding of RASSF1C to betaTrCP involves serine 18 and serine 19 of the SS(18)GYXS(19) motif present in RASSF1C but not in RASSF1A. This motif is reminiscent of the canonical phosphorylation motif recognized by betaTrCP; however, surprisingly, the association between RASSF1C and betaTrCP does not occur via the betaTrCP substrate binding domain, the WD40 repeats. Overexpression of RASSF1C, but not of RASSF1A, resulted in accumulation and transcriptional activation of the beta-catenin oncogene, due to inhibition of its betaTrCP-mediated degradation. Silencing of RASSF1A by small interfering RNA was sufficient for beta-catenin to accumulate, whereas silencing of both RASSF1A and RASSF1C had no effect. Thus, RASSF1A and RASSF1C have opposite effects on beta-catenin degradation. Our results suggest that RASSF1C expression in the absence of RASSF1A could play a role in tumorigenesis.
Cancer Research 03/2007; 67(3):1054-61. DOI:10.1158/0008-5472.CAN-06-2530 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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 alpha-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 alpha-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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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 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.
[Show abstract][Hide abstract] ABSTRACT: The genomes of human and simian immunodeficiency viruses (HIV and SIV) encode the gag, pol and env genes and contain at least six supplementary open reading frames termed tat, rev, nef, vif, vpr, vpx and vpu. While the tat and rev genes encode regulatory proteins absolutely required for virus replication, nef, vif, vpr, vpx and vpu encode for small proteins referred to "auxiliary" (or "accessory"), since their expression is usually dispensable for virus growth in many in vitro systems. However, these auxiliary proteins are essential for viral replication and pathogenesis in vivo. The two vpr- and vpx-related genes are found only in members of the HIV-2/SIVsm/SIVmac group, whereas primate lentiviruses from other lineages (HIV-1, SIVcpz, SIVagm, SIVmnd and SIVsyk) contain a single vpr gene. In this review, we will mainly focus on vpr from HIV-1 and discuss the most recent developments in our understanding of Vpr functions and its role during the virus replication cycle.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 lacZalpha 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.
Journal of Virology 03/2003; 77(3):2071-80. DOI:10.1128/JVI.77.3.2071-2080.2003 · 4.44 Impact Factor