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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.

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.

Vpr, an accessory gene product of human immunodeficiency virus type-1, is thought to transport a viral DNA from the cytoplasm to the nucleus in resting macrophages. Previously, we reported that a peptide encompassing amino acids 52-78 of Vpr (C45D18) promotes the nuclear trafficking of recombinant proteins that are conjugated with C45D18. Here, we present evidence that C45D18, when conjugated with a six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide)-block-oligo(4-aminostyrene) (SV: star vector), facilitates gene expression in resting macrophages. Although there was no difference between SV alone and C45D18-SV with respect to gene transduction into growing cells, C45D18-SV resulted in more than 40-fold greater expression of the exogenous gene upon transduction into chemically differentiated macrophages and human quiescent monocyte-derived macrophages. The data suggest that C45D18 contributes to improving the ability of a non-viral vector to transduce macrophages with exogenous genes and we discuss its further application.

Vpr, an accessory gene product of HIV-1, is incorporated into cells when added to the culture medium. Via such function Vpr has been shown to transduce a protein into cells that is expressed as a chimeric protein with Vpr. The domain required for protein transduction, however, remained to be clarified. Here we identified a sequence encompassing 52-78 amino acids of Vpr (C45D18) that enables nuclear trafficking of proteins. When chemically synthesized C45D18 was added to the culture medium of human cord blood mononuclear (CBMN) cells, most cells became positive for the incorporated C45D18. Furthermore, recombinant proteins conjugated with the C45D18 were efficiently transduced and transported to regions corresponding to the nucleus. Incorporation of C45D18-conjugated protein was observed within a few hours after addition of the protein, independent of cellular growth. Although it is well known that Tat-derived peptide has a transducing activity, C45D18 was more active than Tat peptide for trafficking proteins into cells. Taking together with results from FACS analysis revealing that more than 90% of CBMN cells were positive for X-gal staining after treatment of C45D18-conjugated beta-galactosidase, we propose that C45D18 translocates bioactive macromolecules directly into the nucleus.

The paradigm of HIV-1 infection includes the diminution of CD4(+) T cells, loss of immune function, and eventual progression to AIDS. However, the mechanisms that drive host T cell depletion remain elusive. One HIV protein thought to participate in this destructive cascade is the Vpr gene product. Accordingly, we review the biology of the HIV-1 viral protein R (Vpr) an apoptogenic HIV-1 accessory protein that is packaged into the virus particle. In this review we focus specifically on Vpr's ability to induce host cell apoptosis. Recent evidence suggests that Vpr implements a unique mechanism to drive host cell apoptosis, by directly depolarizing the mitochondria membrane potential. Vpr's attack on the mitochondria results in release of cytochrome c resulting in activation of the caspase 9 pathway culminating in the activation of caspase 3 and the downstream events of apoptosis. Vpr may interact with the adenine nucleotide translocator (ANT) to prompt this cascade. The role of Vpr-induced apoptosis in HIV pathogenesis is considered.

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.

HIV-1 Vpr is a virion-associated protein that can cause growth arrest when produced inside the cell but when added externally it can cause cell death. Employing the yeast model system, the C-terminal domain, in particular the sequence HFRIGCRHSRIG (Vpr(71-82)), is essential for both the growth arrest and cytocidal activities. Conservation of this sequence in HIV-2 and SIV suggests that these residues may be functionally important. Using site-directed mutagenesis we show that the most highly conserved aa residues, His71 and Gly75, were important for the cell cycle inhibitory effects. In contrast, we show that the wild-type Vpr(71-82) peptide and three variants of this peptide with Gly75 changed to Ser, Ala, and Ile all exhibited the same cytocidal activity suggesting that the intracellular and extracellular effects are unrelated., (Copyright 1999 Academic Press.)

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., (Copyright 1999 Academic Press.)

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., (Copyright 1998 Academic Press.)

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., (Copyright 1998 Academic Press Limited.)

Vpr, one of the accessory gene products of the human immunodeficiency virus-1 (HIV-1) genome, exhibits diverse biological characteristics. Vpr functions as a transcriptional activator of HIV and heterologous promoters. It is capable of arresting cells in cell cycle progression and plays a crucial role in the infection of macrophages. Despite the wealth of information available on the biological aspects of Vpr, the structure of Vpr remains poorly understood. To gain insight into the structure-function relationship of Vpr, peptides corresponding to putative helical regions of Vpr were synthesized and their structures determined by circular dichroism (CD) spectroscopy. The CD studies confirmed the predicted helical structures of these peptides. Based on the data, a hypothetical model for the structure of Vpr was proposed which displays an anti-parallel alpha-helix core structure reminiscent of a helix-loop-helix motif. These findings are consistent with the results from mutational studies of Vpr and provide a plausible structural basis to further investigate the multiple functions of Vpr as a viral protein.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) is incorporated into the virion by the Gag polyprotein precursor Pr55gag. The importance of the p6gag sequence at the C-terminal end of Pr55gag has a crucial role in Vpr incorporation. To identify the Gag sequences directly involved in Vpr binding, we compared the Vpr binding affinities of the 71 amino acid nucleocapsid protein p7, the C-terminal peptide (35-71) p7C and p6gag by affinity chromatography. p7 and p7C have the strongest Vpr binding activities compared to p6gag. These results suggest that the nucleocapsid protein and its C-terminal domain may be important for the incorporation of Vpr into the mature HIV-1 virion and the subsequent localisation of viral nucleic acid to the cell nucleus by Vpr.