MuLV-based vectors pseudotyped with truncated HIV glycoproteins mediate specific gene transfer in CD4+ peripheral blood lymphocytes.

Human immunodeficiency virus (HIV) infection ultimately leads to the destruction of the CD4+ lymphocyte subset and the onset of AIDS. In recent years, several gene therapy procedures making use of retroviral vectors that selectively target HIV susceptible cells have been proposed in order to interfere with HIV productive infection. However, the HIV glycoproteins' inability to be incorporated in other heterologous retroviruses considerably limits true HIV cell tropism of such vectors. We now report the use of murine leukemia virus (MuLV) viral particles harboring a truncated form of the HIV glycoprotein for specific gene delivery. Reporter lacZ gene transfer was determined to be appropriately specific to CD4+ cells when HeLaCD4 cells or peripheral blood lymphocytes (PBLs) were infected with these pseudotyped MuLV virus vectors. In contrast, MuLV viruses harboring amphotropic MuLV envelope glycoproteins displayed a broad and nonspecific infection of PBL subpopulations. This new approach, taking advantage of the ability of truncated HIV envelope glycoproteins to be incorporated into heterologous retroviral particles, may foreseeably be used in future interventions based on the coordinated delivery of therapeutic gene products specifically to cell types susceptible to HIV infection.

HIV transcriptional activation by the accessory protein, VPR, is mediated by the p300 co-activator.

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.

Vpr stimulates viral expression and induces cell killing in human immunodeficiency virus type 1-infected dividing Jurkat T cells.

In this study we investigated the effects of Vpr during human immunodeficiency virus (HIV) infection of proliferating Jurkat T cells by using a vesicular stomatitis virus envelope G glycoprotein pseudotyped HIV superinfection system. We observe that the expression of Vpr results in a severe reduction in the life span of HIV type 1 (HIV-1)-infected dividing T cells in culture. In agreement with a recent report (S. A. Stewart, B. Poon, J. B. M. Jowett, and I. S. Chen, J. Virol. 71:5579-5592, 1997), we show that events characteristic of apoptotic cell death are involved in the Vpr-mediated cytopathic effects. Our results also show that infection with viruses expressing the wild-type vpr gene results in an increase in viral gene expression and production. Interestingly, the effects of Vpr on cell viability and on viral gene expression both correlate with the ability of the protein to induce a cell cycle arrest in the G2/M phase. Mutagenesis analyses show that the C terminus of Vpr is essential for these biological activities. Although the role of Vpr is currently associated with the infection of nondividing cells, our results suggest that Vpr can also directly increase viral replication in vivo in infected dividing T cells. Furthermore, these in vitro observations suggest that Vpr-mediated cytotoxic effects could contribute to the CD4+ depletion associated with AIDS progression.

Human immunodeficiency virus type 1 Vpr localization: nuclear transport of a viral protein modulated by a putative amphipathic helical structure and its relevance to biological activity.

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.

Human immunodeficiency virus type 1 Vpr is a positive regulator of viral transcription and infectivity in primary human macrophages.

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 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.

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.

Affinity labeling of the mu opioid receptor in bovine striatal membranes with [3H]-14 beta-(bromoacetamido)-7,8-dihydromorphine.

[3H]-14 beta-(Bromoacetamido)-7,8-dihydromorphine ([3H]H2BAM) was synthesized and tested for its ability to selectively label mu opioid receptors in bovine striatal membranes. Incubating membranes with N-tosyl-L-phenylalanine chloromethyl ketone and dithiothreitol before the addition of [3H]H2BAM reduced nonspecific [3H]H2BAM binding so that [3H]H2BAM binding to opioid receptors was up to 70% of the total [3H]H2BAM binding and was dependent on [3H]H2BAM concentration, incubation time, and pH of the reaction. At pH 7.5, [3H]H2BAM bound selectively to the mu opioid receptor, but mainly noncovalently. After the initial binding of [3H]H2BAM to the receptor, membranes were washed and then incubated at 37 degrees C in 50 mM Tris-HCl, pH 8.5, for 3 h, a time that resulted in greater than 80% of the [3H]H2BAM associated with the receptor becoming covalently bound to the opioid receptor. The mu-selective peptide [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin inhibited [3H]H2BAM labeling of membranes, while delta- or kappa-selective compounds were ineffective. Both NaCl and the nonhydrolyzable guanine nucleotide analog guanylyl 5'-imidodiphosphate reduced the incorporation of [3H]H2BAM into membranes. When [3H]H2BAM-labeled striatal membranes were separated under reducing conditions on a sodium dodecyl sulfate-polyacrylamide gel, two proteins with molecular weights of 54,000 and 44,000 were specifically labeled. The 54-kDa protein was present in a greater amount than the 44-kDa protein. Both proteins bound to wheat germ agglutinin-Sepharose and concanavalin A-Sepharose, suggesting that both proteins contain multiple carbohydrate moieties. Despite the inclusion of protease inhibitors, the 44-kDa protein may be a proteolytic fragment of the 54-kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)