The Presence of Human Immunodeficiency Virus Type 1 Vpr Correlates with a Decrease in the Frequency of Mutations in a Plasmid Shuttle Vector

The human immunodeficiency virus type 1 (HIV-1) Vpr protein induces cell cycle arrest at the border of G2 and M similar to the arrest caused by agents which damage DNA. We determined whether the presence of Vpr would affect the ability of cells to repair DNA. We developed a shuttle vector system to analyze the effect of Vpr upon the repair of UV-damaged DNA. Our results demonstrated that the presence of Vpr decreased the rate of deletions in this system. Of note, cells arrested in G2 by other genotoxic agents also increased the frequency of DNA repair of UV-damaged shuttle vectors. We did not observe any direct effect of Vpr upon the rate of double-strand break repair and/or nucleotide excision repair of genomic DNA in cells. Our results suggest a role for HIV-1 Vpr in altering the frequency of DNA repair, a property which may have importance for HIV-1 replication and pathogenesis. The genome of human immunodeficiency virus (HIV), the etiological agent of AIDS, contains nine genes, five of which have been determined to be essential for replication in vitro. The remaining four genes, vpr, vpu, vif, and nef, have consequently been dubbed nonessential accessory genes, and they play various roles, either directly, in enhancing the efficiency of the replication cycle of HIV, or indirectly, in enhancing survival in the presence of the immune system in vivo and potentially in the causation of disease (10, 11, 27). Vpr encodes an 11-kDa protein that is incorporated into the virion via an interaction with the carboxy-terminal portion of the Gag precursor protein (17). Vpr has been proposed to promote nuclear localization of the viral preintegration complex following infection (5, 15), although this role has been disputed (13). This function of Vpr has been suggested to increase the efficiency of replication of HIV in nondividing cells (3, 7). Furthermore, Vpr has been ascribed additional roles in enhancement of transcriptional activity on various promoters, including the HIV long terminal repeat (6), and in the rescue of viral expression from latently infected cell lines (20), although the roles of these effects on the viral life cycle have yet to be determined. We and others have reported that the HIV type 1 (HIV-1) vpr gene product causes cell cycle arrest in HIV-infected target cells (14, 16, 32, 33). We further proposed that cell cycle arrest induced by Vpr may play a role in pathogenesis by inhibiting the clonal expansion and effective immune response of T cells (16, 31). Phenotypic characterization of the Vpr-mediated arrest revealed that the cells arrested in the second gap (G2) phase of the cell cycle, prior to initiation of mitosis (M). The Cdc2 kinase, a major component controlling the transition of cells from G2 to M, was found to be predominantly in the inactive state (16, 30). This point of arrest can also be triggered in a cell following certain types of DNA damage. Alkylating agents and ionizing radiation cause cells to arrest or delay in G2 and prevent or postpone the onset of mitosis, depending upon the severity of the damage. This pause in cell cycle progression has been suggested as a period in which repair to the DNA is carried out, after which the cell resumes cell cycle progression into mitosis (21, 24, 26). We identified a phenotypic similarity between Vpr-mediated cell cycle arrest and arrest caused by DNA-damaging agents (30). The attributes identified included growth arrest in the G2 phase of the cell cycle with hyperphosphorylation of Cdc2 kinase and the ability of methylxanthines to reverse cell cycle arrest by both Vpr and nitrogen mustard. We proposed, therefore, that Vpr may mediate growth arrest either through (i) directly causing DNA damage, (ii) interfering with DNA damage repair processes, or (iii) modulating a DNA damage detection pathway (checkpoint). In this study we determined whether the presence of Vpr influences the rate of DNA damage and repair in cells. We examined the effect of Vpr on both cellular DNA and exogenously introduced nonviral templates. Our results indicate that the presence of Vpr results in a reduction in the frequency of deletions in a shuttle vector system. Vpr, however, did not detectably affect nucleotide excision repair or double-strand break repair on cellular DNA.