ABSTRACT
HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69â»75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site.
Subject(s)
Gene Products, vpr/chemistry , Gene Products, vpr/metabolism , HIV Infections/virology , HIV Integrase Inhibitors/pharmacology , HIV Integrase/metabolism , HIV-1/physiology , Peptides/pharmacology , Amino Acid Sequence , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Peptides/chemical synthesis , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein MultimerizationABSTRACT
Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.
Subject(s)
Apoptosis , Gene Products, vpr/physiology , HIV-1/physiology , Mitochondria/physiology , Cell-Free System , Gene Products, vpr/chemistry , Humans , Jurkat Cells , Permeability , Proto-Oncogene Proteins c-bcl-2/physiology , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
Among the proteins encoded by human and simian immunodeficiency viruses (HIV and SIV) at least three, Vif, Vpu and Vpr, subvert cellular ubiquitin ligases to block the action of anti-viral defenses. This review focuses on Vpr and its HIV2/SIV counterparts, Vpx and Vpr, which all engage the DDB1.Cullin4 ubiquitin ligase complex through the DCAF1 adaptor protein. Here, we discuss the multiple functions that have been linked to Vpr expression and summarize the current knowledge on the role of the ubiquitin ligase complex in carrying out a subset of these activities.
Subject(s)
Carrier Proteins/metabolism , Cullin Proteins/metabolism , DNA-Binding Proteins/metabolism , Gene Products, vpr/metabolism , HIV-1/metabolism , Amino Acid Sequence , Gene Products, vpr/chemistry , HIV-1/pathogenicity , HIV-1/physiology , Humans , Molecular Sequence Data , Protein Serine-Threonine Kinases , Ubiquitin-Protein Ligases , Virus ReplicationABSTRACT
Shortly after infection by human immunodeficiency virus (HIV), two complexes are formed in a stepwise manner in the cytoplasm of infected cells: the reverse transcription complex that later becomes the preintegration complex. Both complexes include, in addition to cellular proteins, viral RNA or DNA and several proteins, such as reverse transcriptase (RT), integrase (IN), and viral protein R (Vpr). These proteins are positioned in close spatial proximity within these complexes, enabling mutual interactions between the proteins. Physical in vitro interactions between RT and IN that affect their enzymatic activities were already reported. Moreover, we found recently that HIV-1 RT-derived peptides bind and inhibit HIV-1 IN and that an IN-derived peptide binds and inhibits HIV-1 RT. Additionally, HIV-1 Vpr and its C-terminal domain affected in vitro the integration activity of HIV-1 IN. Here, we describe the associations of Vpr-derived peptides with RT and IN. Of a peptide library that spans the 96-residue-long Vpr protein, three partially overlapping peptides, derived from the C-terminal domain, bind both enzymes. Two of these peptides inhibit both RT and IN. Another peptide, derived from the Vpr N-terminal domain, binds IN and inhibits its activities, without binding and affecting RT. Interestingly, two sequential C-terminal peptides (derived from residues 57-71 and 61-75 of full-length Vpr) are the most effective inhibitors of both enzymes. The data and the molecular modeling presented suggest that RT and IN are inhibited as a result of steric hindrance or conformational changes of their active sites, whereas a second mechanism of blocking its dimerization state could be also attributed to the inhibition of IN.
Subject(s)
Gene Products, vpr/chemistry , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/pharmacology , Amino Acid Sequence , Catalytic Domain , DNA Primers , Drug Evaluation, Preclinical/methods , HIV Integrase/chemistry , HIV Integrase/metabolism , HIV Integrase Inhibitors/metabolism , HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/metabolism , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Peptides/pharmacology , Protein Conformation , Reverse Transcriptase Inhibitors/metabolism , Ribonuclease H/antagonists & inhibitors , Sequence Homology, Amino AcidABSTRACT
The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication.
Subject(s)
Epitopes, T-Lymphocyte/immunology , Gene Products, vpr/chemistry , Genes, vpr , HIV Infections/immunology , HIV-1/genetics , Polymorphism, Genetic , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Gene Products, vpr/genetics , Gene Products, vpr/immunology , HIV Infections/virology , HIV-1/immunology , Humans , Molecular Sequence Data , Sequence AlignmentABSTRACT
HIV-1 Vif, Vpr, and Vpu proteins have a profound effect on efficient viral replication and pathogenesis. This study describes the genotypic characterisation of vif , vpr and vpu from 20 South African HIV-1 subtype C primary isolates, and extensive analysis and comparison of known motifs. All HIV-1 subtype C Vif, Vpr and Vpu proteins revealed the presence of highly conserved structural and functional motifs similar to other sub-types, for example, the Vif-APOBEC3G interaction domains. However, several differences were noted when these sequences were compared to subtype B, such as the presence of the LRLL motif which has been implicated in targeting subtype C Vpu predominantly to the cell surface, instead of the Golgi apparatus. A better understanding of the structure/function relationship of these proteins may lead to the development of new classes of antiviral drugs. These results indicate that antiviral drugs that target the conserved functional domains within Vif, Vpr or Vpu could be active against all circulating subtypes, including HIV-1 subtype C.
Subject(s)
Genes, vif , Genes, vpr , Genes, vpu , HIV Infections/genetics , HIV-1/genetics , Gene Products, vif/chemistry , Gene Products, vpr/chemistry , Human Immunodeficiency Virus Proteins , Humans , Phylogeny , Sequence Alignment , Sequence Analysis, Protein , Viral Regulatory and Accessory Proteins/chemistry , vif Gene Products, Human Immunodeficiency Virus , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
The three-dimensional (3-D) structure of human immunodeficiency virus type 2 (HIV-2) Vpr/Vpx was predicted by homology modeling based on the NMR structure of human immunodeficiency virus type 1 (HIV-1) Vpr. The three proteins similarly have three major amphipathic alpha-helices. In contrast to HIV-1 Vpr, Vpr/Vpx of HIV-2 have a long N-terminal loop and clustered prolines in the second half of the C-terminal loop. HIV-2 Vpx uniquely contains a long region between the second and third major helices, and bears several glycines in the first half of the C-terminal loop. Instead of the glycines, there is a group of hydrophilic amino acids and arginines in the corresponding regions of the two Vprs. To compare the cytopathogenic potentials of HIV-1 Vpr and HIV-2 Vpr/Vpx, we examined the production of luciferase as a marker of cell damage. We further analyzed the characteristics of cells transduced with vpr/vpx genes driven by an inducible promoter. The results obtained clearly show that structurally similar, but distinct, HIV Vpr/Vpx proteins are detrimental to target cells.
Subject(s)
Cytopathogenic Effect, Viral/physiology , Gene Products, vpr/chemistry , Gene Products, vpr/metabolism , HIV-1 , HIV-2 , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Amino Acid Sequence , Gene Expression Regulation , Gene Products, vpr/genetics , HIV-1/chemistry , HIV-1/pathogenicity , HIV-2/chemistry , HIV-2/pathogenicity , HeLa Cells , Humans , Molecular Sequence Data , Protein Conformation , Sequence Alignment , Viral Regulatory and Accessory Proteins/genetics , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
HIV-1 Vpr is a highly conserved accessory protein that is involved in many functions of the virus life cycle. Vpr facilitates the entry of the HIV pre-integration complex through the nuclear pore, induces G2 cell cycle arrest, regulates cell apoptosis, increases transcription from the long terminal repeat and enhances viral replication. Vpr contains a Leu/Ile-rich domain (amino acids 60-81) in its C-terminal part, which is critical for dimerization. The sequence comprising residues 52-96 is implicated in properties of the protein such as DNA interaction and apoptosis via interaction with the adenine nucleotide translocator. To understand the specific interactions of Vpr-(52-96), the ability of this peptide to dimerize via a leucine-zipper mechanism has been investigated, by NMR and fluorescence spectroscopy. In contrast with results from a study performed in the presence of trifluoroethanol, our results, obtained in 30% (v/v) [2H]acetonitrile, show that Vpr-(52-96) in solution still forms an a-helix spanning residues 53-75, but dimerizes in an antiparallel orientation, through hydrophobic interactions between leucine and isoleucine residues and stacking between His71 and Trp54. Moreover, to demonstrate the physiological relevance of the dimer structure, fluorescence spectroscopy experiments have been performed in a Mes buffer, which confirmed the formation of the dimer in aqueous solution and highlighted the spatial proximity between Trp54 and His71. Surprisingly, the leucine-zipper structure shown in the present work for Vpr-(52-96) mimics the structure of full-length Vpr-(1-96), and this could explain why some of the properties of Vpr-(52-96) and Vpr-(1-96) are identical, while some are even enhanced for Vpr-(52-96), particularly in the case of DNA transfection experiments.
Subject(s)
Gene Products, vpr/chemistry , Leucine Zippers , Dimerization , Hydrogen-Ion Concentration , Protein Structure, SecondaryABSTRACT
Viral integrase (IN) and Vpr are both components of the human immunodeficiency virus type 1 (HIV-1) pre-integration complex. To investigate whether these proteins interact within this complex, we investigated the effects of Vpr and its subdomains on IN activity in vitro. When a 21mer oligonucleotide was used as a donor and acceptor, both Vpr and its C-terminal DNA-binding domain [(52-96)Vpr] inhibited the integration reaction, whereas the (1-51)Vpr domain did not affect IN activity. Steady-state fluorescence anisotropy showed that both full-length and (52-96)Vpr bind to the short oligonucleotide, thereby extending previous observations with long DNA. The concentrations of the two proteins required to inhibit IN activity were consistent with their affinities for the oligonucleotide. The use of a 492 bp mini-viral substrate confirmed that Vpr can inhibit the IN-mediated reaction. However, the activity of (52-96)Vpr differed notably since it stimulated specifically integration events involving two homologous mini-viral DNAs. Order of addition experiments indicated that the stimulation was maximal when IN, (50-96)Vpr and the mini-viral DNA were allowed to form a complex. Furthermore, in the presence of (50-96)Vpr, the binding of IN to the mini-viral DNA was dramatically enhanced. Taken together, these data suggest that (52-96)Vpr stimulates the formation of a specific complex between IN and the mini-viral DNA.
Subject(s)
DNA, Viral/genetics , Gene Products, vpr/metabolism , HIV Integrase/metabolism , HIV-1/genetics , Amino Acid Sequence , Binding Sites/genetics , Electrophoretic Mobility Shift Assay , Fluorescence Polarization , Gene Products, vpr/chemistry , Gene Products, vpr/genetics , HIV-1/enzymology , HIV-1/metabolism , Humans , Kinetics , Molecular Sequence Data , Oligonucleotides/genetics , Oligonucleotides/metabolism , Protein Binding , Recombination, Genetic , Virus Integration/genetics , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
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.
Subject(s)
Gene Products, vpr/metabolism , HIV-1/physiology , Virus Replication , Gene Products, vpr/chemistry , Gene Products, vpr/genetics , Genes, vpr , HIV-1/genetics , HIV-1/pathogenicity , Humans , Models, Molecular , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
The human immunodeficiency virus type 1 (HIV-1) genome encodes a highly conserved regulatory gene product, Vpr (96 residues, 14kDa), which is incorporated into virions. In the infected cells, Vpr, expressed late in the virus cycle, is believed to function in the early phases of HIV-1 replication, such as nuclear migration of pre-integration complex, transcription of the proviral genome, viral multiplication by blocking cells in G2 phase and regulation of apoptosis phenomenon. Vpr has a critical role in long term AIDS disease by inducing infection in non-dividing cells such as monocytes and macrophages. To gain insight into the structure-function relationships of Vpr, the (1-96)Vpr protein was synthesized with 22 labeled amino acids. Its 3D structure was analyzed in the presence of CD(3)CN and in pure water at low pH and refined by restrained simulated annealing. The structure of the protein is characterized by three well-defined alpha-helices: 17-33, 38-50 and 56-77 surrounded by flexible N and C-terminal domains. In contrast to the structure obtained in the presence of TFE, the three alpha-helices are folded around a hydrophobic core constituted of Leu, Ile, Val and aromatic residues as illustrated by numerous long range NOEs. This structure accounts for the interaction of Vpr with different targets.
Subject(s)
Gene Products, vpr/chemistry , HIV-1/chemistry , Nuclear Magnetic Resonance, Biomolecular , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data , Protein Conformation , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
Protein import into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly clear that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Human immunodeficiency virus type 1 (HIV-1) Vpr is one such protein which does not have an identifiable canonical NLS and yet efficiently localizes to the nuclear compartment. Here, we use confocal microscopy to demonstrate that mutations in the putative central hydrophobic helix of Vpr result in the retention of the protein in highly localized ring-like structures around the nuclear periphery with striking impairment in their ability to enter the nuclear interior. By characterizing other biological activities associated with this protein, such as its ability to incorporate into budding virions and its ability to arrest cells in G2, we show that this helical domain is specific for the nuclear translocation of the protein with very little effect on these other functions. Interestingly, however, perturbation of this helical motif also perturbs the protein's ability to augment viral replication in primary human macrophages indicating that the integrity of this secondary structure is essential for optimal infection in these non-dividing cells.
Subject(s)
Gene Products, vpr/chemistry , Gene Products, vpr/metabolism , HIV-1/physiology , Protein Structure, Secondary , Amino Acid Sequence , Amino Acids/chemistry , Animals , Apoptosis , Biological Transport , COS Cells , Cell Nucleus/metabolism , Cell Nucleus/virology , Cells, Cultured , DNA Fragmentation , G2 Phase , Gene Expression , Gene Products, vpr/genetics , HIV-1/genetics , Humans , Macrophages/virology , Molecular Sequence Data , Mutagenesis , Structure-Activity Relationship , Virion , Virus Replication , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
The human immunodeficiency virus type 1 (HIV-1) Vpr protein is a virion-associated protein that localizes in the nucleus of infected cells. Vpr has been shown to facilitate HIV infection of non-dividing cells such as macrophages by contributing to the nuclear translocation of the pre-integration complex. More recently, Vpr expression has been shown to induce an accumulation of cells at the G2 phase of the cell-cycle. We have previously reported that Vpr stimulates reporter gene expression directed from the HIV-1 long terminal repeat (LTR) as well as from heterologous viral promoters. However, the mode of action of Vpr-mediated transactivation remains to be precisely defined. We report here that, for a constant amount of transfected DNA, the level of chloramphenicol acetyltransferase (CAT) mRNA is increased in Vpr-expressing cells using either HIV-1 or a murine leukemia virus (MLV) SL3-3 LTR-CAT reporter construct. Moreover, this Vpr-mediated transactivation requires that promoters direct a minimal level of basal expression. Our mutagenic analysis indicates that the transactivation mediated by Vpr is not dependent on the ability of the protein to localize in the nucleus or to be packaged in the virions. Interestingly, all transactivation-competent Vpr mutants were still able to induce a cell-cycle arrest. Conversely, transactivation-defective mutants lost the ability to mediate cell-cycle arrest, implying a functional relationship between these two functions. Overall, our results indicate that the G2 cell-cycle arrest mediated by Vpr creates a cellular environment where the HIV-1 LTR is transcriptionally more active.
Subject(s)
Gene Products, vpr/genetics , Gene Products, vpr/metabolism , HIV-1/genetics , HIV-1/metabolism , Transcriptional Activation , Amino Acid Sequence , Animals , Base Sequence , Chloramphenicol O-Acetyltransferase/genetics , DNA Primers/genetics , DNA, Viral/genetics , G2 Phase , Gene Products, vpr/chemistry , Genes, Reporter , Genes, Viral , HIV Long Terminal Repeat , Humans , Jurkat Cells , Leukemia Virus, Murine/genetics , Mice , Molecular Sequence Data , Mutation , Phenotype , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
The HIV-1 regulatory protein Vpr (96 amino acid residues) is incorporated into the virus particle through a mechanism involving its interaction with the C-terminal portion of Gag. Vpr potentiates virus replication by interrupting cell division in the G2 phase and participates in the nuclear transport of proviral DNA. The domain encompassing the 40 C-terminal residues of Vpr was shown to be involved in cell cycle arrest and binding of nucleocapsid protein NCp7, and suggested to promote nuclear provirus transfer. Accordingly, we show here that the synthetic 52-96 but not 1-51 sequences of Vpr interact with HIV-1 RNA. Based on these results, the structure of (52-96)Vpr was analysed by two-dimensional 1H-NMR in aqueous TFE (30%) solution and refined by restrained molecular dynamics. The structure is characterized by a long (53-78) amphipathic alpha-helix, followed by a less defined (79-96) C-terminal domain. The Leu60 and Leu67 side-chains are located on the hydrophobic side of the helix, suggesting their involvement in Vpr dimerization through a leucine zipper-type mechanism. Accordingly, their replacement by Ala eliminates Vpr dimerization in the two hybrid systems, while mutations of Ile74 and Ile81 have no effect. This was confirmed by gel filtration measurements and circular dichroism, which also showed that the alpha-helix still exists in (52-96)Vpr and its Ala60, Ala67 mutant in the presence and absence of TFE. Based on these results, a model of the coiled-coil Vpr dimer has been described, and its biological relevance as well as that of the structural characteristics of the 52-96 domain for the different functions of Vpr, including HIV-1 RNA binding, are discussed.
Subject(s)
Capsid Proteins , Gene Products, vpr/chemistry , Gene Products, vpr/metabolism , Viral Proteins , Amino Acid Sequence , Capsid/chemistry , Capsid/metabolism , Circular Dichroism , Dimerization , Gene Products, gag/chemistry , Gene Products, gag/metabolism , Gene Products, vpr/genetics , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Nucleic Acids/chemistry , Nucleic Acids/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Conformation , Protein Structure, Secondary , RNA, Viral/chemistry , RNA, Viral/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Yeasts/genetics , Zinc Fingers , gag Gene Products, Human Immunodeficiency VirusABSTRACT
Among the putative accessory genes of HIV-1, the 96-amino-acid virion-associated vpr gene product has been described to have several novel biological activities. These include cytoplasmic-to-nuclear translocation, which empowers HIV to infect and replicate in non-dividing cells and to increase viral replication, particularly in macrophages. Along with these viral effects, we found that HIV-1 Vpr induces dramatic biological changes in the target cells of HIV infection, including induction of changes in transcriptional patterns, morphological changes, and complete inhibition of proliferation, which collectively was termed differentiation. These changes occur in the absence of other viral gene products, suggesting that Vpr mediates its proviral effects partially or perhaps solely through modulation of the state of the target cell rather than directly on the virus. The inhibition of proliferation in T cell lines has been extended by several groups to demonstrate that the inhibition of proliferation is through G2 cell cycle arrest, further supporting the idea that Vpr acts directly on cellular targets. We have recently described a role for Vpr in modulating the glucocorticoid pathway, which is involved in the regulation of the state of the cell, in cytoplasmic-to-nuclear translocation, and in modulation of host cell transcription. It is important to note that certain anti-glucocorticoid compounds modulate Vpr activity in vitro. These results support the idea that the host cell contains specific receptor molecule(s) through which Vpr mediates its activity. Consequently, Vpr represents a unique target for anti-HIV drug development and has significance for HIV-1 disease progression.
Subject(s)
Gene Products, vpr/metabolism , HIV-1/physiology , Monocytes/virology , T-Lymphocytes/virology , Virus Replication , Amino Acid Sequence , Cell Cycle/drug effects , Cell Differentiation , Cells, Cultured , Gene Products, vpr/chemistry , Gene Products, vpr/pharmacology , Glucocorticoids/metabolism , Humans , Molecular Sequence Data , Peptide Fragments/pharmacology , Proviruses/physiology , Receptors, HIV/physiology , T-Lymphocytes/cytology , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
Human immunodeficiency virus type 1 (HIV-1) replicates productively in vitro in CD4(+)-T cells and/or macrophages. In the host, however, HIV-1 replication may be restricted by the quiescence of susceptible cells. Vpr is a 15-kDa late viral gene product, which is assembled in the virion and suspected to enhance HIV-1 replication in the infected host. We demonstrated previously that Vpr interacted specifically with the cellular transcription factor Sp1, and activated transcription from the HIV-1 long-terminal-repeat. Both Vpr-Sp1 interaction and trans-activation by Vpr required a central Leu/Ile-rich domain (LR domain, aa 60-81) in Vpr. This domain of Vpr was also found critical for Vpr interaction with another cellular protein of 180 kDa. We now provide biochemical evidence that the Vpr LR-domain has a leucine-zipper-like structure. The leucine-zipper structure has been found in a variety of cellular transcription factors, which use the leucine-zipper domain to form a specific dimer before they can bind to DNA through an upstream basic domain. The LR domain of HIV-1 Vpr, when fused to the basic domain of the cellular transcription factor CREB, was capable of supporting specific DNA binding by the CREB basic domain. Point mutational analysis of the Leu/Ile residues in the LR domain suggested that multiple Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues critical for Vpr interaction with the cellular 180-kDa protein. These results suggested that the leucine-zipper-like domain may be an important functional determinant for HIV-1 Vpr.
Subject(s)
Gene Products, vpr/chemistry , HIV-1/chemistry , Leucine Zippers , Amino Acid Sequence , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , DNA/metabolism , Gene Products, vpr/genetics , Gene Products, vpr/metabolism , HIV-1/genetics , HIV-1/metabolism , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Point Mutation , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
BACKGROUND: The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W)-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clement, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816-48826, 2002). In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae. RESULTS: Our data clearly shows that integrity of N-terminal alpha-helix I (17-33) and alpha-helix III (53-83) is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides. CONCLUSIONS: Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes alpha-helix I (17-33) and alpha-helix III (53-83). Furthermore, the mechanism(s) underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells.
Subject(s)
Gene Products, vpr/physiology , HIV-1/physiology , Saccharomyces cerevisiae/cytology , Base Sequence , Cell Division , DNA Primers , Gene Products, vpr/chemistry , Gene Products, vpr/genetics , Genome, Viral , HIV-1/genetics , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protein Structure, Secondary , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/virology , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
The structure of the complex of Ras with the Ras-binding domain of its effector RalGDS (RGS-RBD), the first genuine Ras-effector complex, has been solved by X-ray crystallography. As with the Rap-RafRBD complex (Nasser et al., 1995), the interaction is via an inter-protein beta-sheet between the switch I region of Ras and the second strand of the RGS-RBD sheet, but the details of the interactions in the interface are remarkably different. Mutational studies were performed to investigate the contribution of selected interface residues to the binding affinity. Gel filtration experiments show that the Ras x RGS-RBD complex is a monomer. The results are compared to a recently determined structure of a similar complex using a Ras mutant (Huang et al., 1998) and are discussed in relation to partial loss-of-function mutations and the specificity of Ras versus Rap binding.
Subject(s)
GTP-Binding Proteins/chemistry , ras Proteins/chemistry , Crystallography, X-Ray , GTP-Binding Proteins/physiology , Gene Products, vpr/chemistry , Gene Products, vpr/physiology , Models, Molecular , Mutagenesis , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Signal Transduction , ral Guanine Nucleotide Exchange Factor , rap GTP-Binding Proteins , ras Proteins/physiologyABSTRACT
The auxiliary regulatory protein Vpr of HIV-1 possesses several biological activities which are believed to facilitate HIV-1 replication and pathogenesis. In this report, experimental evidence suggests a novel biological activity of Vpr: facilitation of the turnover of Vpr mutants bearing the L64P mutation. This novel activity of Vpr was shared by Vpr molecules from different subtypes of HIV-1. Co-expression of the wild type Vpr with the VprW54A/L64P mutant resulted in normal synthesis of the mutant mRNA but enhanced ubiquitination and turnover of the mutant protein. These results suggest that Vpr may interact with the ubiquitin/proteasome pathway to regulate the stability of viral or cellular proteins.
Subject(s)
Gene Products, vpr/genetics , Gene Products, vpr/metabolism , HIV-1/chemistry , Mutation , Ubiquitins/metabolism , Amino Acid Sequence , Apoptosis/drug effects , Cell Line , Cysteine Proteinase Inhibitors/pharmacology , Gene Expression , Gene Products, vpr/chemistry , Gene Products, vpr/drug effects , HIV-1/genetics , Humans , Kinetics , Leupeptins/pharmacology , RNA, Messenger/biosynthesis , Time Factors , Transfection , vpr Gene Products, Human Immunodeficiency VirusABSTRACT
Identification of immunodominant T-helper-cell determinants after natural infection is an important step in the design of immunogens for potential use in vaccination. Using cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals and a panel of peptides encompassing the sequence of the regulatory protein vpr from HIV-1, we identified the T-helper determinant QLLFIHFRIGCRHSR, which is active in 37.5% of these individuals. To gain insight on the efficacy of this peptide in helping induce neutralizing antibodies against a B-cell determinant (BD), we synthesized constructs containing B- and T-cell determinants and tested them in BALB/c mice, the highest responders to the T-cell determinant moiety among several strains tested. These immunogens induced antibodies against two chosen B-cell determinants from HIV-1IIIB gp160 (amino acids 310-322 from the V3 loop of gp120 and 736-751 from gp41) that were able to neutralize HIV-1 infection in vitro. The highest neutralization titer against HIV-1IIIB was obtained by immunization with the homopolymer of the construct containing the T-cell epitope from vpr and the B-cell epitope from the V3 loop. We believe that the immunodominant T-cell determinant from vpr is a promising epitope to consider in the design of future peptide vaccines.