The role of the large T antigen in the in vitro replication of BPyV.
MSc(R) thesis, University of Glasgow.
Full text available as:
Bovine Polyomavirus (BPyV) is a member of Polyomaviridae, a family of double stranded DNA viruses. When the virus was initially discovered it was thought to be of simian origin (Wognum et al., 1984), but further research indicated that the virus was of bovine origin and that primate cultures had been contaminated through the use of foetal bovine serum (Parry et al., 1983). Studies have also shown that cattle populations world-wide have been exposed to the virus, which does not cause significant pathogenicity (Nairn et al., 2003). In addition, 71% of veterinary surgeons, 50% of cattle farmers and 40% of abattoir workers tested positive for the presence of antibodies to BPyV (Parry et al., 1986). BPyV
Very little investigative work has been carried out on bovine polyomavirus but the entire sequence has been published and shows similarities in genome organization to the non-rodent polyoma viruses, such as SV-40 (Schuurman et al., 1990). In contrast to other polyomaviruses, BPyV has a relatively small genome although the significance of this has not been determined. A reliable in vitro cultivation assay for BPyV has been established; however the mode of replication is significantly different to reported polyomaviruses. BPyV requires 6-8 weeks of culture before virus replication is detected, even by sensitive methods such as quantitative polymerase chain reaction (QPCR) (Nairn et al., 2003).
The main reasons for the concern surrounding BPyV are the exposure risk, the ability of the virus to jump species and its cell transforming capacity described by Schuurman et al (1992) in rodent cells transformed with BPyV. To investigate these observations, the aim of this thesis was to understand the role of BPyV large T antigen, which is known to be directly involved in the replication of BPyV.
During this thesis levels of BPyV viral genome and large T mRNA were assessed by QPCR and Reverse Transcriptase-QPCR (RT-QPCR) during an 8 week cultivation period. High titres of BPyV were only detected in cells supplemented with 1% FCS. BPyV was unable to grow in cells supplemented with 10% FCS, with only low titres of virus being detected within the first 3 weeks. A direct correlation between the large T mRNA and viral replication in cells supplemented with 1% FCS was determined, with large T mRNA being detected immediately prior to exponential viral replication. In addition, cell cultures were assessed using SDS-PAGE and Western Blot techniques to detect proteins specific to large T mRNA and cyclin A (an S-Phase promoting factor). Results indicate that the large T antigen is able to force the cell to enter S-Phase, when both cellular and viral DNA are replicated together.
Detection of large T antigen was inconclusive during the cultivation period, although later experiments attempting to over-express the large T antigen did show the presence of this protein. During this experiment, the over-expression of the large T antigen was shown to be toxic to cells, with cells showing vacuolation and death. In addition, an individual clone of BPyV was isolated and sequenced. The consensus sequence was translated into amino acid, and several differences observed, one in particular that could affect the replication initiation and efficiency.
Actions (login required)