The long term objectives of this research are to determine the mechanism responsible for induction and regression of walleye dermal sarcoma (WDS). This tumor is associated with an unusually large (13kb) retrovirus, walleye dermal sarcoma virus (WDSV). Walleye dermal sarcomas develop and regress on a seasonal basis providing a unique model to study retroviral- induced disease. Northern blot analysis and sequence information of WDSV indicates that it is a complex retrovirus which contains at least three accessory genes. The study of viral gene expression of WDSV is essential to the understanding of the regulatory mechanisms involved in tumor development and regression. The basal promoter activity of the WDSV LTR will be determined by measuring the activity of reporter gene constructs (LTR-CAT) in uninfected and WDSV-infected fish cell lines. Analysis of viral encoded functions involved in transactivation of the WDSV LTR will employ a recently developed cell culture system for WDSV infection. The consensus sequences of the WDSV LTR involved in transcriptional regulation and transactivation will be identified using LTR deletion mutants. A major aim of this proposal is to establish a transcriptional map of WDSV utilizing RT-PCR of mRNA and subsequent cloning and sequencing of these cDNAs. The identification and characterization of these accessory gene transcripts will provide the reagents necessary to conduct studies on the proteins encoded by these genes and their involvement in viral gene expression. These experiments should provide the foundation for further investigation of the role of viral accessory genes in tumorigenesis.?GRANT=F32AI09301 The mechanisms by which retrovirus assembles are largely unknown. While progress has been made toward the understanding of the roles of the domains of the gag polyprotein precursor in assembly little is known about how this protein interacts with the cell during this process. Furthermore, the processes of capsid envelopment are often more poorly understood. The assembly and budding of the prototypical D-type retrovirus, Mason-Pfizer monkey virus, will be investigated. This virus offers a distinct advantage for the proposed studies since, unlike the C-type viruses, such as HIV, the processes of capsid assembly and envelopment are temporally and spatially separated in the cell. This separation will allow the examination of these two stages of assembly individually in vitro. Since preliminary experiments have shown the feasibility of in vitro translation for assembly studies this system will be employed to examine the incorporation of protein and RNA into the immature capsid. Analysis of M-PMV gag mutants and HIV/M-PMV chimeras will be performed to delineate domains necessary for efficient cytoplasmic assembly. The assembly of mutant viruses in vitro and in vivo will be compared in order to determine the influence of cellular transport mechanisms upon the process. In addition, the role of cytoplasmic components such as chaperonins in capsid assembly will be investigated. This system, will also be employed for the analysis of capsid envelopment in vitro. Again, the role of cellular components will be investigated. A detailed description of the mechanisms of retrovirus assembly will facilitate the development of therapeutic inhibitors of replication. Moreover, an in vitro assembly system will provide a convenient assay to screen such potential inhibitors.
| Quackenbush, S L; Holzschu, D L; Bowser, P R et al. (1997) Transcriptional analysis of walleye dermal sarcoma virus (WDSV). Virology 237:107-12 |
