Uracil DNA glycosylase specifically interacts with Vpr of both human immunodeficiency virus type 1 and simian immunodeficiency virus of sooty mangabeys, but binding does not correlate with cell cycle arrest.

The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.     

Nuclear export of Vpr is required for efficient replication of human immunodeficiency virus type 1 in tissue macrophages

Retroviruses must gain access to the host cell nucleus for subsequent replication and viral propagation. Human immunodeficiency virus type 1 (HIV-1) and other primate lentiviruses are distinguished from the gammaretroviruses by their ability to infect nondividing cells such as macrophages, an important viral reservoir in vivo. Rather than requiring nuclear membrane breakdown during cell division, the HIV-1 preintegration complex (PIC) enters the nucleus by traversing the central aqueous channel of the limiting nuclear pore complex. The HIV-1 PIC contains three nucleophilic proteins, matrix, integrase, and Vpr, all of which have been implicated in nuclear targeting. The mechanism by which Vpr can display such nucleophilic properties and yet also be available for incorporation into virions assembling at the plasma membrane is unresolved. We recently characterized Vpr as a nucleocytoplasmic shuttling protein that contains two novel nuclear import signals and an exportin-1-dependent nuclear export signal (NES). We now demonstrate that mutation of this NES impairs the incorporation of Vpr into newly formed virions. Furthermore, we find that the Vpr NES is required for efficient HIV replication in tissue macrophages present in human spleens and tonsils. These findings underscore how the nucleocytoplasmic shuttling of Vpr not only contributes to nuclear import of the HIV-1 PIC but also enables Vpr to be present in the cytoplasm for incorporation into virions, leading to enhancement of viral spread within nondividing tissue macrophages.     

Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme.

The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr.     

Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr.

Human immunodeficiency virus type 1 Vpr is a virion-associated, regulatory protein that is required for efficient viral replication in monocytes/macrophages. The protein is believed to act in conjunction with the Gag matrix protein to allow import of the viral preintegration complex in nondividing cells. In cells, Vpr localizes to the nucleus. Recently, we showed that Vpr prevents the activation of p34cdc2-cyclin B. This results in arrest of Vpr-expressing cells in the G2/M phase of the cell cycle. Here, we use a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal alpha-helical region, the central hydrophobic region, or the carboxy-terminal basic region to define the functional domains of the protein. The results showed cell cycle arrest was largely controlled by the carboxy-terminal basic domain of the protein. In contrast, the amino-terminal alpha-helical region of Vpr was required for nuclear localization and packaging into virions. The carboxy terminus appeared to be unnecessary for nuclear localization. In the alpha-helical region, mutation of Ala-30 to Pro resulted in a protein that localized to the cytoplasm. Surprisingly, fusion of Vpr to luciferase resulted in a molecule that failed to localize to the nucleus. In addition, we show that simian immunodeficiency virus Vpr, but not Vpx, induces G2 arrest. We speculate that Vpr has two sites for interaction with cellular factors: one in the alpha-helical region that specifies nuclear localization and one in the carboxy-terminal domain that is required for Cdc2 inhibition.     

Human immunodeficiency virus type 1 Vpr induces DNA replication stress in vitro and in vivo

The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.     

The Putative Alpha Helix 2 of Human Immunodeficiency Virus Type 1 Vpr Contains a Determinant Which Is Responsible for the Nuclear Translocation of Proviral DNA in Growth-Arrested Cells

Several viral determinants were shown to play a role in the ability of human immunodeficiency virus type 1 (HIV-1) to infect nondividing cells. In particular, Vpr and Gag matrix (MA) were recognized to be involved in the nuclear transport of the viral preintegration complex. The goal of the present study was to evaluate the ability of isogenic HIV-1 viruses harboring different vpr and gag genes to infect nondividing cells. Surprisingly, our results reveal that the introduction of mutations in the MA nuclear localization signal marginally affected the ability of proviruses to establish infection in growth-arrested HeLa or MT4 cells. In contrast, we show that in our experimental system, the absence of Vpr expression leads to a reduction in viral infectivity and production which correlates with a decrease in the synthesis and nuclear transport of proviral DNA as determined by PCR analysis. Moreover, our data demonstrate that this reduction of viral replication is also observed with proviruses containing different mutated Vpr alleles. In particular, the Vpr Q65E mutant, which contains a substitution in the second predicted amphipathic alpha-helical structure located in the central region of the protein, is associated with an impairment of the protein nuclear localization and a concomitant reduction of the nuclear transport of proviral DNA. The results of this study provide evidence that a putative amphipathic alpha-helical structure in the central region of Vpr contains a determinant involved in the nuclear translocation of the preintegration complex in nondividing cells.     

Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R

Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr(52-96)) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr(52-96) was 10- to 1,000-fold more active. Vpr(52-96)-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.     

Reversion of the lethal phenotype of an HIV-1 integrase mutant virus by overexpression of the same integrase mutant protein

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for integration of viral DNA into host cell chromatin. We have reported previously (Priet, S., Navarro, J. M., Gros, N., Querat, G., and Sire, J. (2003) J. Biol. Chem. 278, 4566-4571) that IN also plays a role in the packaging of the host uracil DNA glycosylase UNG2 into viral particles and that the region of IN encompassing residues 170-180 was responsible for the interaction with UNG2 and for its packaging into virions. In this work, we aimed to investigate the replication of HIV-1 viruses rendered deficient in virion-associated UNG2 by single or double point mutations in the region 170-180 of IN. We show that the L172A/K173A IN mutant virus was deficient for UNG2 packaging and was defective for replication because of a blockage at the stage of proviral DNA integration in host cell DNA. In vitro assays using long term repeat mimics, however, demonstrate that the L172A/K173A IN mutant was catalytically active. Moreover, trans-complementation experiments show that the viral propagation of L172A/K173A viruses could be rescued by the overexpression of Vpr.L172A/K173A IN fusion protein in a dose-dependent manner and that this rescue is independent of UNG2 packaging. Altogether, our data indicate that L172A/K173A mutations of IN induce a subtle defect in the function of IN, which nevertheless dramatically impairs viral replication. Unexpectedly, this blockage of replication could be overcome by forcing the packaging of higher amounts of this same mutated integrase. This is the first study reporting that blockage of the integration process of HIV-1 provirus carrying a mutation of IN could be alleviated by increasing amounts of IN even carrying the same mutations.