The objective of this study is to investigate the cofactor role of human cytomegalovirus (HCMV) infection in HIV-induced pediatric AIDS, particularly will focus on investigating the effects of HCMV infection on the replication and transmission of HIV during pregnancy. The proposed approaches use cell culture systems and the clinical materials and resources from UNC AIDS Clinical Trial Unit to investigate whether and how HCMV infection of placental and fetal cells facilitate HIV reactivation, replication, transmission and subsequently increase the frequency and severity of HIV infection. The hypothesis of this study is that the reactivation of latent HCMV infection in pregnant women during the gestation period will enhance HIV gene expression and increase the frequency of HIV transmission from mother to fetus as well as the severity of congenital HIV infection. The specific approaches to achieve the objective are (1) To investigate the induction and enhancement of the CD4 (HIV receptor) and Fc receptor (an alternate mechanism of HIV infection via immune complex) expression in cells infected with HCMV. Cells to be studied include umbilical endothelial cells, placental trophoblasts (JAR trophoblastic tumor cells), Hofbaur macrophages, ML-3 premyelocytic cells and CEM T-cells;(2) To examine the induction of various cytokines (include IL-1b, CSF and TNF-alpha) synthesis in cells infected with HCMV. mRNAs of various cytokines will be monitored by Northern blot, and proteins by Western and ELISA; (3) To investigate the transactivation of HIV LTR promoter sequences and induction of HIV antigen synthesis by HCMV IE genes in cells mentioned in Aim (1). Plasmids containing HCMV IE1, IE2 or IE1&2 regions and promoter deletion mutants of mutants of HIV (LTR)-CAT constructs will be used to asses the mechanism of transactivation and to define the transactivation targets (on HIV). Induction of HIV antigens will be analyzed by Western blot or enzyme assay; and (4) To correlate the status and consequence of HIV infection in mother and fetus with the HCMV infection in pregnant women with AIDS. The presence of HCMV and HIV infections will be monitored by virus isolation, immunochemistry and PCR. Evident of viral infection in placental and fetal cells will be studied by in situ hybridization, PCR and immuno-cytochemistry. Results obtained will be analyzed for statistical significance.
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