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 Pr55 gag. 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.

It is currently well established that HIV-1 Vpr augments viral replication in primary human macrophages. In its virion-associated form, Vpr has been suggested to aid efficient translocation of the proviral DNA into the cell nucleus. Although Vpr growth-arrests dividing T cells, the relevance of this biological activity in nondividing macrophages is unclear. Here we use Vpr-mutants to demonstrate that the molecular determinants involved in G2-arresting T cells are also involved in increasing viral transcription in macrophages, even though these cells are refractive to the diploid DNA status typical of G2 phase. Our results suggest that the two phenotypes, namely the nuclear localization and the G2-arrest activity of the protein, segregate functionally among the late and early functions of Vpr. The nuclear localization property of Vpr correlates with its ability to effectively target the proviral DNA to the cell nucleus early in the infection, whereas the G2-arrest phenotype correlates with its ability to activate viral transcription after establishment of an infection. These two functions may render Vpr's role essential and not accessory under infection conditions that closely mimic the in vivo situation, that is, primary cells being infected at low viral inputs.

The accessory protein, Vpr, is a virion-associated protein that is required for HIV-1 replication in macrophages and regulates viral gene expression in T cells. Vpr causes arrest of cell cycle progression at G2/M, presumably through its effect on cyclin B1.Cdc2 activity. Here, we show that the ability of Vpr to activate HIV transcription correlates with its ability to induce G2/M growth arrest, and this effect is mediated by the p300 transcriptional co-activator, which promotes cooperative interactions between the Rel A subunit of NF-kappaB and cyclin B1.Cdc2. Vpr cooperates with p300, which regulates NF-kappaB and the basal transcriptional machinery, to increase HIV gene expression. Similar effects are seen in the absence of Vpr with a kinase-deficient Cdc2, and overexpression of p300 increases levels of HIV Vpr+ replication. Taken together, these data suggest that p300, through its interactions with NF-kappaB, basal transcriptional components, and Cdks, is modulated by Vpr and regulates HIV replication. The regulation of p300 by Vpr provides a mechanism to enhance viral replication in proliferating cells after growth arrest by increasing viral transcription.