Sampa Mukherjee

Interaction of Virion Protein Vpr of Human Immunodeficiency Virus Type 1 with Cellular Transcription Factor Sp1 and trans-Activation of Viral Long Terminal Repeat

Acquired immunodeficiency syndrome (AIDS) is a result of replication of the human immunodeficiency virus type 1 (HIV-1) predominantly in CD4 T lymphocytes and macrophages. However, most of these cells in vivo are immunologically quiescent, a condition restricting HIV-1 replication. Vpr is an HIV-1 virion protein suspected to enhance HIV-1 replication in vivo. We demonstrate in this report that Vpr specifically activates HIV-1 long terminal repeat (LTR)-directed transcription. This effect is most pronounced on a minimal promoter from HIV-1 LTR containing the TATA box and binding motifs for the ubiquitous cellular transcription factor Sp1. Evidence is presented that Vpr interacts with Sp1 when Sp1 is bound to the Sp1 motifs within the HIV-1 LTR. Both Vpr-Sp1 interaction and Vpr trans-activation require a central Leu/Ile-rich domain in Vpr. Our findings suggest that Vpr trans-activation through Sp1 is most critical for the immediate early transcription of HIV-1 when other positive regulators, such as NF-κB, are limited or inactive, a condition presumably present in vivo. By interacting with Sp1, Vpr also has the potential to influence cellular gene expression and cellular functions. Thus, therapeutic approaches directed toward blocking the Vpr trans-activation function could prove valuable in treating AIDS.

Characterization of a leucine-zipper-like domain in Vpr protein of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) replicates productively in vitro in CD4(+)-T cells and/or macrophages. In the host, however, HIV-1 replication may be restricted by the quiescence of susceptible cells. Vpr is a 15-kDa late viral gene product, which is assembled in the virion and suspected to enhance HIV-1 replication in the infected host. We demonstrated previously that Vpr interacted specifically with the cellular transcription factor Sp1, and activated transcription from the HIV-1 long-terminal-repeat. Both Vpr-Sp1 interaction and trans-activation by Vpr required a central Leu/Ile-rich domain (LR domain, aa 60-81) in Vpr. This domain of Vpr was also found critical for Vpr interaction with another cellular protein of 180 kDa. We now provide biochemical evidence that the Vpr LR-domain has a leucine-zipper-like structure. The leucine-zipper structure has been found in a variety of cellular transcription factors, which use the leucine-zipper domain to form a specific dimer before they can bind to DNA through an upstream basic domain. The LR domain of HIV-1 Vpr, when fused to the basic domain of the cellular transcription factor CREB, was capable of supporting specific DNA binding by the CREB basic domain. Point mutational analysis of the Leu/Ile residues in the LR domain suggested that multiple Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues may be involved in maintaining the leucine-zipper-like structure. Mutagenesis in the context of the full-length Vpr also helped identify Leu/Ile residues critical for Vpr interaction with the cellular 180-kDa protein. These results suggested that the leucine-zipper-like domain may be an important functional determinant for HIV-1 Vpr.

Initial characterization of viral sequences from a SHIV-inoculated pig-tailed macaque that developed AIDS.

Abstract: In this study, we report on the derivation of a pathogenic SIV‐HIV chimeric virus (SHIV) and the initial characterization of the viral sequences from the first (macaque PPc) of a series of pig‐tailed macaques that developed CD4+ T cell loss and AIDS. Viral genes were amplified by PCR from the brain, lymphoid, and kidney tissues and their sequences compared to the original SHIV used to initiate passages in macaques. Our results show that the vpu gene, which was nonfunctional in the original SHIV, now coded for functional protein in macaque PPc. The tat and rev genes had no consensus changes but the nef gene had 4–5 consensus changes, depending on the tissue examined. The gp 120 gene had the highest number of nucleotide and amino acid substitution rates that varied from 0.64% to 1.44% and 1.17% to 3.71%, respectively, again depending on the tissue examined. These results suggest that a constellation of changes accumulated at the genomic level during the derivation of a SHIV that was pathogenic for pig‐tailed macaques.

Pathogenic Simian/Human Immunodeficiency Virus SHIV KU Inoculated into Immunized Macaques Caused Infection, but Virus Burdens Progressively Declined with Time

ABSTRACT Using the simian immunodeficiency virus/human immunodeficiency virus (SHIV)-macaque model of AIDS, we had shown in a previous report that a live, nonpathogenic strain of SHIV, further attenuated by deletion of the vpu gene and inoculated orally into adult macaques, had effectively prevented AIDS following vaginal inoculation with pathogenic SHIVKU. Examination of lymph nodes from the animals at 18 weeks postchallenge had shown that all six animals were persistently infected with challenge virus. We report here on a 2-year follow-up study on the nature of the persistent infections in these animals. DNA of the vaccine virus was present in the lymph nodes at all time points tested, as far as 135 weeks postchallenge. In contrast, the DNA of SHIVKU became undetectable in one animal by week 55 and in three others by week 63. These four macaques have remained negative for SHIVKU DNA as far as the last time point examined at week 135. Quantification of the total viral DNA concentration in lymph nodes during the observation period showed a steady decline. All animals developed neutralizing antibody and cytotoxic-T-lymphocyte responses to SHIVKU that persisted throughout the observation period. Vaccine-like viruses were isolated from two animals, and a SHIVKU-like virus was isolated from one of the two macaques that remained positive for SHIVKU DNA. There was no evidence of recombination between the vaccine and the challenge viruses. Thus, immunization with the live vaccine not only prevented disease but also contributed to the steady decline in the virus burdens in the animals.

Selective transmission of R5-tropic HIV type 1 from dendritic cells to resting CD4+ T cells.

In an in vitro coculture model of monocyte-derived, cultured human dendritic cells (DC) with autologous CD4(+) resting T cells, CCR5 (R5)-tropic strains of HIV-1, but not CXCR4 (X4)-tropic strains, were transmitted to resting CD4+ T cells, leading to prolific viral output, although DC were susceptible to infection with either strain. Macrophages, which were also infectable with either R5- or X4-tropic strains, did not transmit infection to CD4+ cells. Highly productive HIV infection in this model appeared to be a consequence of heterokaryotic syncytium formation between infected DC and T cells since syncytia formation developed only in R5-infected DC/CD4+ cocultures. These results suggested that the unique microenvironment derived from the fusion between the infected DC and CD4+ cell was highly permissive and selective for replication of R5-tropic viruses. The apparent selectivity for R5-tropic strains in such syncytia was attributable neither to differential DC-mediated activation nor to selective modulation of induction of alpha- or beta-chemokines in the infected DC. This model of HIV replication may provide useful insights into in vitro correlates of HIV pathogenicity.

Neuropathogenesis of chimeric simian human immunodeficiency virus infection in rhesus macaques.

Comparative studies were performed to determine the neuropathogenesis of infection in macaques with simian human immunodeficiency virus (SHIV)89.6P and SHIVKU. Both viruses utilize the CD4 receptor and CXCR4 co‐receptor. However, in addition, SHIV89.6P uses the CCR5 co‐receptor. Both agents are dual tropic for CD4+ T cells and blood‐derived macrophages of rhesus macaques. Following inoculation into macaques, both caused rapid elimination of CD4+ T cells but they varied greatly in mechanisms of neuropathogenesis. Two animals infected with SHIV89.6P developed typical lentiviral encephalitis in which multinucleated giant cell formation, nodular accumulations of microglial cells, activated macrophages and astrocytes, and perivascular accumulations of mononuclear cells were present in the brain. Many of the macrophages in these lesions contained viral RNA. Three macaques infected with SHIVKU and killed on days 6, 11 and 18, respectively, developed a slowly progressive infection in the CNS but macrophages were not productively infected and there were no pathological changes in the brain. Two other animals infected with this virus and killed several months later showed minimal infection in the brain even though one of the two developed encephalitis of unknown etiology. The basic difference in the mechanisms of neuropathogenesis by the two viruses may be related to co‐receptor usage. SHIV89.6P, in utilizing the CCR5 co‐receptor, caused neuropathogenic effects that are similar to other neurovirulent primate lentiviruses.