Date of Graduation
2002
Document Type
Dissertation/Thesis
Abstract
Retroviruses exhibit high genetic variability, which is generated by mutations and recombination during viral replication. This can lead to rapid emergence of viral variants resistant to antiretroviral drugs. A better understanding of retroviral replication may lead to the development of improved antiviral drug therapy and effective vaccines to retrovirus-related diseases. The fact that retroviral nucleocapsid protein (NC) is multifunctional and plays crucial roles in viral replication makes it a good target for antiviral drug development. By studying how NC affects reverse transcriptase (RT) template switching, the in vivo role of NC during reverse transcription can be explored. Using a sensitive in vivo direct repeat deletion assay, this dissertation demonstrated that mutations in NC increased RT template switching in vivo, which provided the first evidence that murine leukemia virus (MLV) NC affects RT template switching by increasing processive viral DNA synthesis during reverse transcription. When a predicted stable secondary structure is present in the RNA template, the NC mutants further increased the frequency of template switching up to 30-fold, indicating that one important role of NC is, by virtue of its secondary structure melting activity, to facilitate RT reverse transcription through regions of template containing secondary structures that might serve as RT pause sites. Both zinc finger motif and basic residues of MLV NC contribute to this activity. In addition to NC, structural determinants of RT, particularly the RNase H domain, are known to affect RT template switching. In other related studies, we observed that the spleen necrosis virus (SNV) exhibited a two-fold higher frequency of RT template switching compared to MLV, indicating that closely related viruses may display different frequencies of RT template switching. The protein components of the SNV that are responsible for the high frequency of internal template switching were most likely associated with polymerase-independent RNase H activity of SNV. In the course of investigating the importance of RNase H during reverse transcription, a reverse transcriptase mutator was identified—a conservative point mutation in MLV RNase H led to a fivefold higher mutation rate in an in vivo fidelity assay. Further characterization of the nature of the mutations identified an unusual association of misincorporations by Y586F with A-tract sequences that induce DNA bending. The dissertation will provide new insights into mechanisms of viral DNA synthesis, RT template switching and fidelity.
Recommended Citation
Zhang, Wen-Hui, "Understanding retroviral replication: Roles of nucleocapsid and RNase H during reverse transcription in vivo." (2002). Graduate Theses, Dissertations, and Problem Reports. 10100.
https://researchrepository.wvu.edu/etd/10100