Our group is principally concerned with the molecular biology of HIV pathogenesis, especially the pathogenesis of pediatric HIV disease. The course of HIV disease in pediatric patients differs substantially from the course of disease in adults. Because some of the factors that help modulate the course of HIV disease depend on the host and host cells, we are therefore particularly interested in trying to understand the involvement of host cell factors in HIV replication and the effect of HIV infection on the host cell. The hypothesis guiding much of our work holds that there is one set of cellular conditions that is ideal for normal cellular growth and replication, that another set of conditions is ideal for viral replication, and that viruses (particularly HIV) have evolved ways of altering their host cells to enhance viral replication. During the past year, we have used cDNA microarray technology to identify cellular genes with altered expression during HIV replication and to attribute the changes in cellular gene expression to particular stages of the viral replication cycle and individual viral gene products. For example, we have studied the cellular gene expression patterns that accompany activation of cells latently infected with HIV into a lytic infectious cycle, and have shown that a clear, temporally ordered pattern of cellular gene expression accompanies the completion of the infection cycle. We are also examining the effects of the HIV accessory gene Vpr on host cell physiology. In another project, we are studying the gene expression program of the Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8, HHV-8) and are using our studies of the KSHV gene expression program to gain additional insights into the replication and pathogenesis strategies of the virus. We have constructed small arrays containing the entire complement of KSHV ORFs, including certain alternatively spliced messages and use this technology to develop an initial description of the KSHV transcription program during the KSHV lytic replication cycle. We have extended that initial work by refining our description of the transcription program through the use of inhibitors of viral protein and DNA synthesis. We have also started to conduct further experiments that involve the construction of cell lines that contain a latent copy of the KSHV genome plus an inducible copy of a selected KSHV gene. Inducing expression of those selected genes then demonstrates how that selected gene helps modulate the expression of the entire KSHV genome. In collaboration with the laboratory of Jae Jung, we demonstrated that the KSHV ORF50 potently activates the expression of essentially all of the KSHV genome, but that the kinetics of activation is different for different KSHV genes, with the presumed early regulatory genes generally showing increasing in expression soon after the induction of ORF50, while the expression of the late viral structural genes increases later. ORF50 may thus lie at the apex of the KSHV gene expression regulatory pyramid. We are currently studying additional cell lines that inducibly express other selected KSHV genes in an effort to define in more detail the KSHV gene regulation machinery. We are also actively engaged in developing new therapies for pediatric HIV disease and in using newly available therapies to investigate key issues in pediatric HIV pathogenesis. This work includes, a phase I pediatric study of the nucleotide analogue reverse transcriptase inhibitor tenofovir, a study of a novel viral genotyping and dose adjustment antiretroviral agent therapeutic drug monitoring strategy in children, a long term study of the toxicity, tolerability, and immune reconstitution effects of HIV protease inhibitor therapy in children, and a pediatric therapeutic HIV vaccine trial done in collaboration with the Vaccine Research Center, NIAID, NIH. This clinical research is described more completely in the summary of the pediatric HIV group in HAMB.
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