We are examining human T-cell leukemia virus type 1 (HTLV-1) with respect to virus particle assembly and maturation, virus infectivity, transmission, and replication, and virus-induced changes in cellular gene expression that lead to oncogenic transformation. We have developed infectious molecular clones of HTLV-1 and have constructed virus mutants and virus vectors to study individual steps in the viral infectious cycle. It was shown that HTLV-1 particles are approximately 4000-fold less infectious in cell-free infections compared to HIV-1 when the two viruses were pseudotyped with the same envelope protein. We are now examining the step in the infection process that confers this difference. These vector systems also permitted a rapid and quantitative method to screen for antiviral agents directed against HTLV-1. We determined the inhibition values for HTLV-1 replication by various nucleoside analogs. We also characterized a new proteolytic processing site in the HTLV-1 polyprotein which defined the amino terminus of reverse transcriptase. Recombinant HTLV-1 reverse transcriptase is currently being expressed from baculovirus vectors for in vitro protein characterization. With respect to virus gene expression, we have examined the expression levels of putative regulatory proteins encoded in open reading frames in the 3' end of the virus genome. The products of these genes have not been detected in virus infected cells but there is strong evidence that they are produced in vivo. We examined the levels of expression of the alternatively spliced mRNAs that encode these proteins using real-time quantitative RT-PCR methods and splice site-specific primers. These mRNAs were expressed at levels 100-fold to 10,000-fold lower than the virus mRNAs that encode structural and regulatory proteins. Similar PCR strategies are being applied to detect and quantify the products of virus reverse transcription early after HTLV-1 infection. Changes in T-cell gene expression that result from HTLV-1 infection have been investigated using DNA microarray technology, RNase protection and real-time quantitative RT-PCR. We found that HTLV-1 transformed T-cells express specific type 2 cytokines including IL-9, IL-13 and IL-5. The HTLV-1 induced expression of IL-13 was investigated in detail in cells infected in vitro and in lymphocytes from infected individuals. The expression of IL-13 in HTLV-1 infected cells may have important implications for the pathogenesis of HTLV-1 associated disease. A second general area of investigation in the lab concerns the regulation of alternative pre-mRNA splicing by retrovirus encoded regulatory proteins. we have shown that lentivirus Rev proteins interact with viral mRNA and with cellular splicing factors. We developed an in vitro splicing system coupled with real-time RT-PCR methods to monitor and quantify alternative splicing. We are applying this methodology to address what factors and cis-acting elements control alternative splicing in the lentivirus model system. (Project transferred to BRL)
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