Human T-cell leukemia virus type II (HTLV-II) has been associated with several cases of rare chronic T-cell leukemia, and has recently been found in a significant proportion of American intravenous drug abusers. HTLV-II encodes two trans-acting proteins, Tax and Rex, that regulate the viral life-cycle, and that may contribute to the malignant transformation of infected host cells. Recent studies in our laboratory have shown that Rex increases cytoplasmic levels of gag/pol mRNA, and that this effect requires cis-acting sequences in the 5' HTLV-II long terminal repeat (LTR), a Rex-responsive element (RxRE). If this RxRE is deleted, only negative regulation of LTR-linked expression by Rex occurs, which corresponds to a decrease in total LTR-linked mRNA levels. The overall objective of this proposal is to define the precise molecular and biochemical mechanisms of these competing Rex regulatory functions. The effects of Rex on the levels and subcellular distribution of gag/pol, env and tax/rex mRNAs will be measured in transient and stable transfections into lymphoid cells by S(1) nuclease protection or quantitative polymerase chain reaction (PCR) of RNA. Rex effects on HTLV-II mRNAs will also be compared to effects on expression of viral proteins, as determined by radioimmunoprecipitation assay (RIPA). Stable transfections will allow study of the steady-state of Rex regulation, and may provide an in vitro model of latent infection. The mechanisms underlying Rex regulation will be explored by mapping all cis-acting RNA RxREs within the HTLV-II genome, and by defining Rex protein functional domains through mutagenesis. All rex mutants and RxREs will be tested for effects on LTR-linked expression in co-transfections using either the wild-type LTR or LTR mutants containing one possible RxRE linked to the chloramphenicol acetyltransferase (CAT) indicator gene. Potential Rex protein-RxRE RNA interactions will be identified by RNA gel retardation assays using nucleoprotein extracts from HTLV-II-infected and -uninfected lymphoid cells and purified Rex protein from a baculovirus vector. If specific """"""""retarded"""""""" bands are found, site-directed RxRE mutants will be created that disrupt the predicted RxRE RNA secondary structure. Changes in LTR-linked gene expression with RxRE mutants will be compared to changes in RNA gel shift patterns to correlate Rex-RxRE binding with Rex function. Understanding the mechanisms of Rex regulation in HTLV-II will help elucidate not only the controlling steps in the viral life-cycle, but also the processing of RNA in eukaryotic cells.
Showing the most recent 10 out of 12 publications
