My current research interests are focused on the mechanisms of retroviral reverse transcription, reverse transcriptase (RT) template switching, and fidelity of viral DNA synthesis. My broad research interests also include retroviral RNA packaging, virion structure and assembly, and development of retroviruses as tools for gene therapy. Retroviral populations exhibit high levels of genetic variation and evolutionary potential, which can lead to rapid emergence of viral variants resistant to antiretroviral drugs. An important mechanism for generating mutations in retroviral genomes is error-prone DNA synthesis by the virally encoded RT. Frequent template switching by RT between copackaged viral RNAs further increases retroviral variation by assortment of mutations. Several different aspects of the structure of RT may affect the RT fidelity. Some of these structural features include the YXDD box, amino acids that directly or indirectly define the substrate dNTP-binding site, the primer grip, and the thumb and RNase H domains. Using a rapid in vivo assay that we have developed, we are currently assessing the importance of various structural determinants of RT to the fidelity of DNA synthesis. Two obligatory template-switching events, minus-strand transfer and plus-strand transfer, are required for completion of viral DNA synthesis. Other internal template-switching events occur frequently during reverse transcription and can lead to the generation of deletions and recombination. To understand the mechanism of RT template switching, we have developed various in vivo assays using retroviral vectors containing directly repeated sequences. Using the template-switching properties of RTs, we have also developed novel self-activating and self-inactivating retroviral vectors that improve the safety and efficacy of gene therapy. We have shown that the template-switching properties of retroviruses can be used to efficiently delete the viral RNA-packaging signal to further inactivate the retroviral vectors during the process of reverse transcription. RT template switching can also be used to functionally reconstitute genes during reverse transcription. We are currently exploring these approaches to improve suicide gene therapy for the treatment of cancers.
| Halvas, E K; Svarovskaia, E S; Pathak, V K (2000) Role of murine leukemia virus reverse transcriptase deoxyribonucleoside triphosphate-binding site in retroviral replication and in vivo fidelity. J Virol 74:10349-58 |
| Svarovskaia, E S; Delviks, K A; Hwang, C K et al. (2000) Structural determinants of murine leukemia virus reverse transcriptase that affect the frequency of template switching. J Virol 74:7171-8 |
| Halvas, E K; Svarovskaia, E S; Pathak, V K (2000) Development of an in vivo assay to identify structural determinants in murine leukemia virus reverse transcriptase important for fidelity. J Virol 74:312-9 |
| Halvas, E K; Svarovskaia, E S; Freed, E O et al. (2000) Wild-type and YMDD mutant murine leukemia virus reverse transcriptases are resistant to 2',3'-dideoxy-3'-thiacytidine. J Virol 74:6669-74 |
