Accumulating data suggest a co-factor role for HHV-6 in the progression of AIDS and in AIDS-related malignancies. Of significance, HHV-6 and HIV-1 can co-infect the same CD4+ target T-cells resulting in the stimulation of HIV-1 replication. Moreover, HHV-6 infection can induce expression of CD4 in CD8+ T-lymphocytes, thus rendering these cells permissive for HIV-1 infection. Our studies have identified a 3.9 kbp Sa/I-L DNA fragment of HHV-6 strain U1102 from the direct repeat region which transactivates human immunodeficiency virus type 1 -long terminal repeat (HIV-1 LTR). Sa/I-L also transforms NIH 3T3 cells and immortalizes primary hamster embryo fibroblasts. The transactivating and transforming activities have been co-localized to a 1.9 kbp Sa/I-L-Hindlll subfragment, (designated Sa/I-L-SH) containing a 357 amino acid (aa) open reading frame, (designated ORF-1). ORF-1 can reactivate tat-defective HIV-1 provirus from latently infected cells, suggesting that ORF-1 is the HHV-6 gene putatively responsible for HIV-1 reactivation during AIDS progression in vivo. Purified ORF-1 protein is sufficient to transactivate the HIV-1 LTR. To extend our observations, this proposal is designed to dissect the essential regions within Sa/I-L-SH required for transformation and transactivation and to determine their relevance within the context of the whole virus during infection.
Our specific aims are to: 1) Characterize the mechanism by which ORF-1 transactivates HIV-1 as well as the minimal HHV-6 transactivator responsive element(s) within the HIV-1 promoter by mutational analysis. Specific emphasis will be placed on the role of ORF-1 within the context of the whole virus to transactivate latent HIV-1. 2) Characterize the minimal transforming region within the Sa/I-L-SH fragment both as an independent entity and in the context of the whole virus by in vitro cell transformation and tumorigenicity assays, and 3) Determine the presence and expression of ORF-1 with AIDS-related and non-AIDS lymphoid malignancies by Southern blot, polymerase chain reaction, and in situ hybridization analyses. These studies will provide significant new information about the biologic relevance of specific HHV-6 gene(s) in the progression of AIDS and AIDS-related malignancies by determining the significance of the ORF-1 transforming and transactivating domains in the context of the viral genome.
