Antiviral agent based on the non-structural protein targeting the maturation process of HIV-1: expression and susceptibility of chimeric Vpr as a substrate for cleavage by HIV-1 protease

The processing of precursor proteins (Gag and Gag-pol) by the viral protease is absolutely required in order to generate infectious particles. This prompted us to consider novel strategies that target viral maturation. Towards this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor proteins. We previously reported that virus particles derived from HIV-1 proviral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending on the fusion partner. As an extension of that work, the potential of chimeric Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-based assay. Chimeric Vpr molecules were modified such that the Flag epitope is removed following cleavage, thus allowing us to determine the efficiency of protease cleavage. Following incubation with the protease, the resultant products were analyzed by radioimmunoprecipitation using antibodies directed against the Flag epitope. Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of virus particles containing chimeric Vpr by immunoblot showed reactivities to antibodies against the Flag epitope similar to the data observed in vitro. These results suggest that the pseudosubstrate approach may provide another avenue for developing antiviral agents.     

A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles.

Incorporation of Vpr into human immunodeficiency virus type 1 (HIV-1) virions is mediated by the Gag protein, independently of other viral components. We have coexpressed Vpr and Gag constructs in a vaccinia virus expression system in order to map the region of Gag involved in Vpr packaging. Deletion of the carboxyl-terminal p6 region of Gag impaired the ability of Gag to package Vpr. To confirm the role of p6 in Vpr packaging, Rous sarcoma virus (RSV)-HIV chimeras containing HIV-1 p6 were constructed. Although RSV Gag does not package Vpr into virus particles, a chimera containing HIV-1 p6 is sufficient for Vpr incorporation. To map the region of p6 involved in Vpr packaging, a series of p6 point mutations and deletion mutations was analyzed. Mutations in the N-terminal p6 proline-rich domain, for which preliminary evidence shows a marked decrease in virion incorporated RNA, did not affect Vpr incorporation. Deletion of residues 1 to 31 of HIV-1 p6 did not affect Vpr packaging, but residues 35 to 47, including an (LXX)4 domain, were required for Vpr incorporation into virus particles.     

Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles.

The human immunodeficiency virus type 1 (HIV-1) particles consists of two molecules of genomic RNA as well as molecules originating from gag, pol, and env products, all synthesized as precursor proteins. The 96-amino-acid Vpr protein, the only virion-associated HIV-1 regulatory protein, is not part of the virus polyprotein precursors, and its incorporation into virus particles must occur by way of an interaction with a component normally found in virions. To investigate the mechanism of incorporation of Vpr into the HIV-1 virion, Vpr- proviral DNA constructs harboring mutations or deletions in specific virion-associated gene products were cotransfected with Vpr expressor plasmids in COS cells. Virus released from the transfected cells was tested for the presence of Vpr by immunoprecipitation with Vpr-specific antibodies. The results of these experiments show that Vpr is trans-incorporated into virions but at a lower efficiency than when Vpr is expressed from a proviral construct. The minimal viral genetic information necessary for Vpr incorporation was a deleted provirus encoding only the pr55gag polyprotein precursor. Incorporation of Vpr requires the expression but not the processing of gag products and is independent of pol and env expression. Direct interaction of Vpr with the Pr55gag precursor protein was demonstrated by coprecipitation experiments with gag product-specific antibodies. Overall, these results indicate that HIV-1 Vpr is incorporated into the nascent virion through an interaction with the Gag precursor polyprotein and demonstrate a novel mechanism by which viral protein can be incorporated into virus particles.     

Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis.

The product of the vpr open reading frame of human immunodeficiency virus type 1 (HIV-1) is a 15-kDa, arginine-rich protein that is present in virions in molar quantities equivalent to that of Gag. We report here the results of our investigations into the mechanism by which Vpr is incorporated into virions during assembly in infected cells. For these studies we used an expression vector encoding a Vpr molecule fused at its amino terminus to a nine-amino-acid peptide from influenza virus hemagglutinin. The tagged Vpr expression vector and a vpr mutant HIV-1 provirus were used to cotransfect COS cells, and the resulting virions were tested for the presence of the tagged protein on immunoblots probed with monoclonal antibody against the hemagglutinin peptide. The COS-produced virions were found to contain readily detectable amounts of tagged Vpr and smaller amounts of a putative tagged Vpr dimer. Infectivity of the particles was not altered by incorporation of tagged Vpr. Our results using this system in combination with mutant HIV-1 proviruses suggested that incorporation of Vpr into virions requires the carboxy-terminal Gag protein of HIV-1 (p6) but not gp160, Pol, or genomic viral RNA. In addition, analysis of mutated, tagged Vpr molecules suggested that amino acids near the carboxy terminus (amino acids 84 to 94) are required for incorporation of Vpr into HIV-1 virions. The single cysteine residue near the carboxy terminus was required for production of a stable protein. Arginine residues tested were not important for incorporation or stability of tagged Vpr. These results suggested a novel strategy for blocking HIV-1 replication.     

Incorporation of Vpr into human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor

The 96-amino acid Vpr protein is the major virion-associated accessory protein of the human immunodeficiency virus type 1 (HIV-1). As Vpr is not part of the p55 Gag polyprotein precursor (Pr55(gag)), its incorporation requires an anchor to associate with the assembling viral particles. Although the molecular mechanism is presently unclear, the C-terminal region of the Pr55(gag) corresponding to the p6 domain appears to constitute such an anchor essential for the incorporation of the Vpr protein. In order to clarify the mechanism by which the Vpr accessory protein is trans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55(gag) using the yeast two-hybrid system and the maltose-binding protein pull-down assay. The present study provides genetic and biochemical evidence indicating that the Pr55(gag) can physically interact with the Vpr protein. Furthermore, point mutations affecting the integrity of the conserved L-X-S-L-F-G motif of p6(gag) completely abolish the interaction between Vpr and the Pr55(gag) and, as a consequence, prevent Vpr virion incorporation. In contrast to other studies, mutations affecting the integrity of the NCp7 zinc fingers impaired neither Vpr virion incorporation nor the binding between Vpr and the Pr55(gag). Conversely, amino acid substitutions in Vpr demonstrate that an intact N-terminal alpha-helical structure is essential for the Vpr-Pr55(gag) interaction. Vpr and the Pr55(gag) demonstrate a strong interaction in vitro as salt concentrations as high as 900 mM could not disrupt the interaction. Finally, the interaction is efficiently competed using anti-Vpr sera. Together, these results strongly suggest that Vpr trans-incorporation into HIV-1 particles requires a direct interaction between its N-terminal region and the C-terminal region of p6(gag). The development of Pr55(gag)-Vpr interaction assays may allow the screening of molecules that can prevent the incorporation of the Vpr accessory protein into HIV-1 virions, and thus inhibit its early functions.     

The p6gag domain of human immunodeficiency virus type 1 is sufficient for the incorporation of Vpr into heterologous viral particles.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a virion-associated regulatory protein. Mutagenesis has shown that the virion association of Vpr requires sequences near the C terminus of the HIV-1 Gag polyprotein Pr55gag. To investigate whether Vpr incorporation is mediated by a specific domain of Pr55gag, we examined the ability of chimeric HIV-1/Moloney murine leukemia virus (MLV) Gag polyproteins to direct the incorporation of Vpr. Vpr expressed in trans did not associate with particles formed by the authentic MLV Gag polyprotein or with particles formed by chimeric Gag polyproteins that had the matrix (MA) or capsid (CA) domain of MLV precisely replaced by the corresponding domain of HIV-1HXB2. By contrast, Vpr was efficiently incorporated upon replacement of the C-terminal nucleocapsid (NC) domain of the MLV Gag polyprotein with HIV-1 p15 sequences. Vpr was also efficiently incorporated into particles formed by a MLV Gag polyprotein that had the HIV-1 p6 domain fused to its C terminus. Furthermore, a deletion analysis revealed that a conserved region near the C terminus of the p6 domain is essential for Vpr incorporation, whereas sequences downstream of the conserved region are dispensable. These results show that a virion association motif for Vpr is located within residues 1 to 46 of p6.     

A conserved LXXLF sequence is the major determinant in p6gag required for the incorporation of human immunodeficiency virus type 1 Vpr.

The vpr gene product of human immunodeficiency virus type (HIV-1) is a virion-associated regulatory protein. A transferable virion association motif for Vpr is located in the p6 domain of the HIV-1 Gag polyprotein. To map the sequences in p6 that are involved in Vpr incorporation, we analyzed the ability of mutant forms of p6 to direct the incorporation of Vpr into chimeric viral particles. Our results show that the determinants which govern Vpr incorporation are largely confined to a C-terminal region of the p6 domain. Within this region, three hydrophobic residues in a highly conserved sequence motif (L-X-S-L-F-G) are absolutely required. Remarkably, the transfer of the conserved motif and of a single flanking residue to a heterologous Gag polyprotein was sufficient to transfer the ability to incorporate Vpr at moderate levels. The transfer of residues 32 to 46 of p6 led to Vpr incorporation levels that were comparable to those obtained with full-length HIV-1 Gag protein, indicating that this region contains essentially all the information required for efficient Vpr incorporation.     

Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to modulate the virus mutation rate and for replication in macrophages

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.     

DNA Repair Enzyme Uracil DNA Glycosylase Is Specifically Incorporated into Human Immunodeficiency Virus Type 1 Viral Particles through a Vpr-Independent Mechanism

The Vpr protein, encoded by the human immunodeficiency virus type 1 (HIV-1) genome, is one of the nonstructural proteins packaged in large amounts into viral particles. We have previously reported that Vpr associates with the DNA repair enzyme uracil DNA glycosylase (UDG). In this study, we extended these observations by investigating whether UDG is incorporated into virions and whether this incorporation requires the presence of Vpr. Our results, with highly purified viruses, show that UDG is efficiently incorporated either into wild-type virions or into Vpr-deficient HIV-1 virions, indicating that Vpr is not involved in UDG packaging. Using an in vitro protein-protein binding assay, we reveal a direct interaction between the precursor form of UDG and the viral integrase (IN). Finally, we demonstrate that IN-defective viruses fail to incorporate UDG, indicating that IN is required for packaging of UDG into virions.     

Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation.

The Vpr gene product of human immunodeficiency virus type 1 is a virion-associated protein that is important for efficient viral replication in nondividing cells such as macrophages. At the cellular level, Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Incorporation of Vpr into viral particles requires a determinant within the p6 domain of the Gag precursor polyprotein Pr55gag. In the present study, we have used site-directed mutagenesis to identify a domain(s) of Vpr involved in virion incorporation and nuclear localization. Truncations of the carboxyl (C)-terminal domain, rich in basic residues, resulted in a less stable Vpr protein and in the impairment of both virion incorporation and nuclear localization. However, introduction of individual substitution mutations in this region did not impair Vpr nuclear localization and virion incorporation, suggesting that this region is necessary for the stability and/or optimal protein conformation relevant to these Vpr functions. In contrast, the substitution mutations within the amino (N)-terminal region of Vpr that is predicted to adopt an alpha-helical structure (extending from amino acids 16 to 34) impaired both virion incorporation and nuclear localization, suggesting that this structure may play a pivotal role in modulating both of these biological properties. These results are in agreement with a recent study showing that the introduction of proline residues in this predicted alpha-helical region abolished Vpr virion incorporation, presumably by disrupting this secondary structure (S. Mahalingam, S. A. Khan, R. Murali, M. A. Jabbar, C. E. Monken, R. G. Collman, and A. Srinivasan, Proc. Natl. Acad. Sci. USA 92:3794-3798, 1995). Interestingly, our results show that two Vpr mutants harboring single amino acid substitutions (L to F at position 23 [L23F] and A30F) on the hydrophobic face of the predicted helix coded for relatively stable proteins that retained their ability to translocate to the nucleus but exhibited dramatic reduction in Vpr incorporation, suggesting that this hydrophobic face might mediate protein-protein interactions required for Vpr virion incorporation but not nuclear localization. Furthermore, a single mutation (E25K) located on the hydrophilic face of this predicted alpha-helical structure affected not only virion incorporation but also nuclear localization of Vpr. The differential impairment of Vpr nuclear localization and virion incorporation by mutations in the predicted N-terminal alpha-helical region suggests that this region of Vpr plays a role in both of these biological functions of Vpr.     

Virion-Targeted Viral Inactivation of Human Immunodeficiency Virus Type 1 by Using Vpr Fusion Proteins

Inactivation of progeny virions with chimeric virion-associated proteins represents a novel therapeutic approach against human immunodeficiency virus (HIV) replication. The HIV type 1 (HIV-1) Vpr gene product, which is packaged into virions, is an attractive candidate for such a strategy. In this study, we developed Vpr-based fusion proteins that could be specifically targeted into mature HIV-1 virions to affect their structural organization and/or functional integrity. Two Vpr fusion proteins were constructed by fusing to the first 88 amino acids of HIV-1 Vpr the chloramphenicol acetyltransferase enzyme (VprCAT) or the last 18 C-terminal amino acids of the HIV-1 Vpu protein (VprIE). These Vpr fusion proteins were initially designed to quantify their efficiency of incorporation into HIV-1 virions when produced in cis from the provirus. Subsequently, CD4⁺ Jurkat T-cell lines constitutively expressing the VprCAT or the VprIE fusion protein were generated with retroviral vectors. Expression of the VprCAT or the VprIE fusion protein in CD4⁺ Jurkat T cells did not interfere with cellular viability or growth but conferred substantial resistance to HIV replication. The resistance to HIV replication was more pronounced in Jurkat-VprIE cells than in Jurkat-VprCAT cells. Moreover, the antiviral effect mediated by VprIE was dependent on an intact p6^gag domain, indicating that the impairment of HIV-1 replication required the specific incorporation of Vpr fusion protein into virions. Gene expression, assembly, or release was not affected upon expression of these Vpr fusion proteins. Indeed, the VprIE and VprCAT fusion proteins were shown to affect the infectivity of progeny virus, since HIV virions containing the VprCAT or the VprIE fusion proteins were, respectively, 2 to 3 times and 10 to 30 times less infectious than the wild-type virus. Overall, this study demonstrated the successful transfer of resistance to HIV replication in tissue cultures by use of Vpr-based antiviral genes.     

Human immunodeficiency virus type 1 Nef incorporation into virions does not increase infectivity

The viral protein Nef contributes to the optimal infectivity of human and simian immunodeficiency viruses. The requirement for Nef during viral biogenesis particles suggests that Nef might play a role in this process. Alternatively, because Nef is incorporated into viruses, it might play a role when progeny virions reach target cells. We challenged these hypotheses by manipulating the amounts of Nef incorporated in viruses while keeping its expression level constant in producer cells. This was achieved by forcing the incorporation of Nef into viral particles by fusing a Vpr sequence to the C-terminal end of Nef. A cleavage site for the viral protease was introduced between Nef and Vpr to allow the release of Nef fragments from the fusion protein during virus maturation. We show that the resulting Nef-CS-Vpr fusion partially retains the ability of Nef to downregulate cell surface CD4 and that high amounts of Nef-CS-Vpr are incorporated into viral particles compared with what is seen for wild-type Nef. The fusion protein is processed during virion maturation and releases Nef fragments similar to those found in viruses produced in the presence of wild-type Nef. Unlike viruses produced in the presence of wild-type Nef, viruses produced in the presence of Nef-CS-Vpr do not have an increase in infectivity and are as poorly infectious as viruses produced in the absence of Nef. These findings demonstrate that the presence of Nef in viral particles is not sufficient to increase human immunodeficiency virus type 1 infectivity and suggest that Nef plays a role during the biogenesis of viral particles.     

Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr.

The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.     

Isolation of human immunodeficiency virus type 1 cores: retention of Vpr in the absence of p6(gag)

Mature human immunodeficiency virus type 1 (HIV-1) virions contain a typically cone-shaped core that encases the viral genome. In this study, we established conditions which allowed the efficient isolation of morphologically intact HIV-1 cores from virions. The isolated cores consisted mostly of cones which appeared uniformly capped at both ends but were heterogeneous with respect to the shape of the broad cap as well as the dimensions and angle of the cone. Vpr, a nonstructural virion component implicated in the nuclear import of the viral genome, was recovered in core preparations of HIV-1 and simian immunodeficiency viruses from African green monkeys. Unexpectedly, p6(gag), a structural protein required for the incorporation of Vpr, was absent from HIV-1 core preparations. Taken together, our results indicate that the incorporation of Vpr into the virion core is a conserved feature of primate lentiviruses and that the interactions required for the uptake of Vpr into assembling particles differ from those which confine Vpr within the core.     

Integrase-lexA fusion proteins incorporated into human immunodeficiency virus type 1 that contains a catalytically inactive integrase gene are functional to mediate integration

Purified fusion proteins made up of a retroviral integrase and a sequence-specific DNA-binding protein have been tested in in vitro assays for their ability to direct integration into specific target sites. To determine whether these fusion proteins can be incorporated into human immunodeficiency virus type 1 (HIV-1) and are functional to mediate integration, we used an in trans approach to deliver various integrase-LexA proteins to an integrase-defective virus containing an integrase mutation at aspartate residue 64. Integrase-LexA, integrase-LexA DNA-binding domain, or N- or C-terminally truncated integrase-LexA proteins were fused to the HIV-1 accessory protein, Vpr. Coexpression of the Vpr fusion proteins and an integrase-defective HIV-1 molecular clone by a producer cell line resulted in efficient incorporation of the fusion protein into the integrase-mutated virus. In addition, each of these viruses was infectious and capable of performing integration, as determined by two independent cellular assays that measure reporter gene expression. With the exception of the N-terminally truncated integrase fused to LexA, which was at about 1%, all of the fusion proteins restored integration to a similar level, at 17 to 24% of that of the wild-type virus. The low level observed with the N-terminally truncated integrase fused to LexA is consistent with previous results implying that the N terminus of integrase is involved in multiple steps of the retroviral life cycle. These data indicate that the integrase-fusion proteins retain catalytic function in the integrase-mutated viruses and demonstrate the feasibility of incorporating integrase fusion proteins into HIV-1 for the development of site-directed retroviral vectors.     

Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.