Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are closely related human retroviruses that transform T lymphocytes in cell culture and persist in infected individuals. HTLV-1 infection is clearly associated with leukemia/lymphoma and neurological disease, whereas HTLV-2 disease association is less compelling. HTLV replication and survival requires the expression of multiple gene products from an unspliced and a series of highly related alternatively spliced mRNA species. To date, the levels of viral gene expression throughout the process of infection, cellular transformation, and pathogenesis have not been experimentally assessed. We posit that having a concise viral gene expression profile will provide important insight into the function of specific viral genes and their role in the biology and pathogenesis of HTLV-1.
We first compiled and generated a series of oligonucleotide primer pairs and probes to quantify both HTLV-1 and HTLV-2 mRNA species using real-time RT-PCR. The combination of amplification followed by hybridization increased the specificity and sensitivity of the assay. The splice site-specific primers used in our experiments allowed differentiation of the closely related alternatively spliced mRNAs. Specifically designed Taqman probes hybridized only to the segment to be amplified and emit signal upon amplification, which results in increased detection specificity. The assay offered a wide dynamic range as seen by our ability to quantify specific cDNA plasmid samples accurately at concentrations from 25 copies to 2.5 x 107copies per reaction. Quantitation of HTLV mRNAs between different cell lines showed variability (gag/pol > tax/rex > env > accessory genes), but the overall levels of each mRNA relative to each other within a cell line were similar. Our analysis also provided important information on some splice acceptor site utilization.
In Chapter 3 we utilized our newly developed real-time RT-PCR assay to determine the kinetics of viral gene expression in three systems: 293T cells transiently transfected with HTLV-1 proviral plasmid, human PBMCs infected with HTLV-1 in vitro, and PBMCs harvested from HTLV-1-inoculated rabbits. The HTLV-1 gene expression profiles in transiently transfected and infected cells were similar; over time, all transcripts increased and then maintained stable levels. The gag/pol, tax/rex, and env mRNAs were detected first and at the highest levels, whereas expression of the accessory genes, including the anti-sense hbz, was significantly lower than tax/rex. In infected rabbits, tax/rex and gag/pol mRNA levels peaked early after inoculation and progressively decreased, which correlated inversely with the proviral load and host antibody response against viral proteins. Interestingly, hbz mRNA was detectable at one week post-infection and increased and stabilized. This study provides the first evidence linking hbz expression to proviral load and the survival of the virus infected cell in the host.
Lastly, in Chapter 4 we further explored the role that the HTLV-1 post-transcriptional regulator, Rex, played in viral gene expression. It was known that Rex utilized specific host machinery to actively export gag/pol unspliced and env incompletely-spliced viral mRNA from the nucleus to the cytoplasm. However, all HTLV-1 sense transcripts contain the Rex response element (RxRE) in their 3’ untranslated region. The contribution of Rex to the export and expression of completely spliced HTLV-1 mRNAs, particularly those encoding the accessory proteins, had been difficult to assess due to their low abundance in cells. Using our quantitative realtime RT-PCR assay we confirmed the effect of Rex on the export and expression level of unspliced gag/pol mRNA, singly-spliced env mRNA, and doubly-spliced tax/rex mRNA. Furthermore, we showed that Rex slightly decreased the overall expression level of singly and doubly spliced mRNAs encoding Tax, HBZ, and the accessory proteins, but had no significant effect on the export of these mRNAs. Rex RNA-pull down experiments demonstrated that Rex associated not only with the gag/pol and env mRNA, but also associated with all other singly and doubly spliced viral mRNAs encoding regulatory and accessory proteins. In contrast, the hbz antisense mRNA, which does not contain the RxRE, was not bound by Rex. Our results are the first to quantitatively measure the effects of Rex on the expression level and export of HTLV-1 accessory protein mRNAs and to show that Rex associates with all the RxRE containing positive sense HTLV-1 mRNA transcripts. The Rex association with HTLV-1 transcripts provides the possibility of post-transcriptional regulations by Rex other than export.