Faculty Bibliography

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 15-kDa virion-associated protein that functions as a regulator of cellular processes linked to the HIV life cycle. We report the interaction of a 41-kDa cytosolic viral protein R interacting protein 1 (Rip-1) with Vpr in vitro. Rip-1 displays a wide tissue distribution, including relevant targets of HIV infection. Vpr protein induced nuclear translocation of Rip-1, as did glucocorticoid receptor (GR)-II-stimulating steroids. Importantly, Vpr and Rip-1 coimmunoprecipitated with the human GR as part of an activated receptor complex. Vpr complementation of a vpr mutant virus was also mimicked by GR-II-stimulating steroids. Vpr and GR-II actions were inhibited by mifepristone, a GR-II pathway inhibitor. Together these data directly link the activity of the vpr gene product to the glucocorticoid steroid pathway and provide a biochemical mechanism for the cellular and viral activity of Vpr, as well as suggest that a unique class of antivirals, which includes mifepristone (RU486), may influence HIV-1 replication.

The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics.

In human immunodeficiency virus (HIV)-positive individuals, the vast majority of infected peripheral blood cells and lymph node cells may be latently or nonproductively infected. The vpr open reading frame of HIV-1 encodes a 15-kDa virion-associated protein, Vpr. The vpr gene has been shown to increase virus replication in T cells and monocyte/macrophages in vitro. We have previously reported that vpr expression in various tumor lines leads to growth inhibition and differentiation, indicating that Vpr may function as a regulator of cellular permissiveness to HIV replication. Here we show that Vpr protein is present in significant amounts in the serum of AIDS patients. Purified serum Vpr activated virus expression from five latently infected cell lines, U1, OM.10.1, ACH-2, J1.1, and LL58. Serum Vpr also activated virus expression from resting peripheral blood mononuclear cells of HIV-infected individuals. Together, these findings implicate serum Vpr in the activation of HIV replication in vivo and in the control of latency. Anti-Vpr antibodies inhibited Vpr activity, suggesting that humoral immunity modulates Vpr activity in vivo. These results have broad implications for the virus life cycle and for the prospective control of HIV replication and pathogenesis.

Cell lines from rhabdomyosarcomas, which are tumors of muscle origin, have been used as models of CD4-independent HIV infection. These cell lines can be induced to differentiate in vitro. We report here that the vpr gene of HIV1 is sufficient for the differentiation of the human rhabdomyosarcoma cell line TE671. Differentiated cells are characterized by great enlargement, altered morphology, lack of replication, and high level expression of the muscle-specific protein myosin. We have also observed the morphological differentiation and inhibition of proliferation of two other transformed cell lines. vpr-transfected cells remain fully viable in culture for extended periods. These observations elucidate a potential role for vpr in the virus life cycle and raise the possibility that some aspects of HIV-induced pathologies may be caused by a disturbance of cells by vpr.

A rabbit antiserum reactive with the human complement component C3d/Epstein-Barr virus receptor (complement receptor type 2, CR2) immunoprecipitates a Mr 155,000 murine B-cell surface antigen. The apparent molecular weight and cellular distribution of this murine antigen are similar to those of human CR2. Cells expressing the murine protein bind sheep erythrocytes coated with antibody and murine C1-C3d but do not bind Epstein-Barr virus at all. The monospecific antiserum to human CR2 together with goat F(ab')2 anti-rabbit IgG blocks attachment of the C3d-coated erythrocytes to receptor-bearing murine B lymphocytes. To further characterize murine CR2, a lambda gt11 library from the murine late pre-B-cell line 70Z/3 was screened with human CR2 cDNA. A partial cDNA clone of 3.5 kilobases with 79% amino acid sequence identity to human CR2 in the unique intracytoplasmic region and 63% identity to the sixth human CR2 repeat was obtained. Blot hybridization with the murine cDNA clone identified an RNA species of approximately 4.7 kilobases, similar in size to human CR2 mRNA, from a murine B-cell line but not from a murine T-cell line.

In an attempt to compare B cell chronic lymphocytic leukemia (B-CLL) with its normal cellular counterpart, the cell surface phenotype of 100 cases of B-CLL was determined by using a panel of monoclonal antibodies (MoAbs) directed against B cell-restricted and -associated antigens. The majority of B-CLL cells expressed Ia, B4 (CD19), B1 (CD20), B2 (CD21), surface immunoglobulin (sIg), and T1 (CD5) but lacked C3b (CD35) receptors. In contrast, the overwhelming majority of small unstimulated B cells expressed Ia, B4, B1, B2, sIg, and C3b receptors but lacked detectable T1. Small numbers of weakly sIg+ cells could be identified in peripheral blood and tonsil that coexpressed the B1 and T1 antigens. Approximately 16% of fetal splenocytes coexpressed B1, T1, weak sIg, B2, and Ia but lacked C3b receptors and therefore closely resembled most B-CLL cells. With the phenotypic differences between the majority of small unstimulated B cells and B-CLL cells, we examined normal in vitro activated B cells and B-CLL cells for the expression of B cell-restricted and -associated activation antigens. Of 20 cases examined, virtually all expressed B5, and approximately 50% of the cases expressed interleukin-2 receptors (IL-2R) and Blast-1. Normal B cells were activated with either anti-Ig or 12-0-tetradecanoylphorbol-beta-acetate (TPA) and then were examined for coexpression of B1, T1, and the B cell activation antigens B5 and IL-2R. Only cells activated with TPA coexpressed B1 and T1 as well as B5 and IL-2R. B cells activated with either anti-Ig or TPA proliferated in the presence of IL-2, whereas B-CLL cells did not, although they all expressed the identical 60-kilodalton proteins by immunoprecipitation. These studies are consistent with the notion that B-CLL resembles several minor subpopulations of normal B cells including a population of B cells that are activated in vitro directly through the protein kinase C pathway.

In an attempt to relate the functional events of B cell activation with changes in cell surface molecules, we have used a panel of monoclonal antibodies directed against cell surface antigens expressed on activated but not resting B cells, to determine a sequence of activation antigen expression following anti-immunoglobulin stimulation. Within the first 24 hr of culture with anti-Ig, resting splenic B cells were induced to express B5 and interleukin-2 receptor (IL-2R) and subsequently express T9 and BB1 by 48 hr. Maximum antigen expression was seen by day 3 with the majority of cells expressing B5, IL-2R, T9, and BB1, and fewer numbers of cells expressing Blast-1 and Blast-2. By day 6, the expression of these antigens significantly decreased. Dual fluorochrome staining of anti-Ig activated B cells demonstrated heterogeneity of activation antigen expression, suggesting the existence of subpopulations of activated B cells. In an attempt to relate the non-Hodgkin's lymphomas (NHLs) to this sequence of activation, 69 tumor samples from patients with B cell NHLs were then examined for expression of these activation antigens. Histologically defined subgroups of B cell NHLs demonstrated differential expression of activation antigens with B5, BB1, and T9 exhibiting the widest distribution, whereas IL-2R, Blast-1, and Blast-2 demonstrated more limited expression. The finding that no B cell malignancy phenotypically resembles the small resting B lymphocyte coupled with the observation that virtually all B cell NHLs examined expressed activation antigens suggests that these tumors may be the neoplastic counterparts of subpopulations of activated B lymphocytes.