Reverse transcriptases (RTs) frequently switch templates, leading to an increase in genetic variation and retroviral recombination. We have performed a series of studies in which high-frequency deletion of homologous repeats was used as a model system for probing the mechanism of RT template switching and in vivo reverse transcription. We have determined the effects of the size of the repeats, distance between repeats, RNA secondary structure, intracellular nucleotide pools, and mutations in murine leukemia virus (MLV) RT on the frequency and locations of RT template switching. These studies led to the proposal of a new dynamic copy-choice model for retroviral recombination, which postulated that there is a steady state between the rates of DNA synthesis and RNA degradation and that conditions affecting this steady state influence the frequency of RT template switching. Analysis of direct-repeat deletions has also led to new insights into the in vivo mechanism of reverse transcription. The results of these studies have provided the first demonstration of polymerase-dependent ribonuclease H (RNase H) activity in vivo and indicated that MLV nucleocapsid (NC) protein increases the in vivo rate of DNA synthesis, especially in regions of the template containing secondary structures. We plan to determine the roles of HIV-1 RT, NC, and RNA secondary structure in RT template switching. To gain insights into the mechanisms of in vivo reverse transcription, we are analyzing the role of MLV and HIV-1 NC zinc fingers and basic residues in minus-strand and plus-strand transfer. We are also analyzing the effects of RNA secondary structure on the rate of reverse transcription. Finally, we are investigating whether MLV- and HIV-1-based vectors containing two primer-binding site (PBS) regions can initiate reverse transcription more than once; we are also using this system as a sensitive in vivo assay to analyze the effects of cis-mutations in the PBS regions on the efficiency of DNA synthesis initiation.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010532-01
Application #
6952144
Study Section
(RML)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Delviks-Frankenberry, Krista A; Lengruber, Renan B; Santos, Andre F et al. (2013) Connection subdomain mutations in HIV-1 subtype-C treatment-experienced patients enhance NRTI and NNRTI drug resistance. Virology 435:433-41
Delviks-Frankenberry, Krista A; Nikolenko, Galina N; Pathak, Vinay K (2010) The ""Connection"" Between HIV Drug Resistance and RNase H. Viruses 2:1476-1503
Delviks-Frankenberry, Krista A; Nikolenko, Galina N; Maldarelli, Frank et al. (2009) Subtype-specific differences in the human immunodeficiency virus type 1 reverse transcriptase connection subdomain of CRF01_AE are associated with higher levels of resistance to 3'-azido-3'-deoxythymidine. J Virol 83:8502-13
Nikolenko, Galina N; Delviks-Frankenberry, Krista A; Palmer, Sarah et al. (2007) Mutations in the connection domain of HIV-1 reverse transcriptase increase 3'-azido-3'-deoxythymidine resistance. Proc Natl Acad Sci U S A 104:317-22
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Delviks-Frankenberry, Krista A; Nikolenko, Galina N; Barr, Rebekah et al. (2007) Mutations in human immunodeficiency virus type 1 RNase H primer grip enhance 3'-azido-3'-deoxythymidine resistance. J Virol 81:6837-45
Voronin, Yegor A; Sidorov, Igor A; Pathak, Vinay K (2006) A probability model predicting initiation efficiency of retroviral vectors with two primer-binding sites. J Theor Biol 242:347-55
Krajewski, Krzysztof; Zhang, Yijun; Parrish, Damon et al. (2006) New HIV-1 reverse transcriptase inhibitors based on a tricyclic benzothiophene scaffold: synthesis, resolution, and inhibitory activity. Bioorg Med Chem Lett 16:3034-8
Nikolenko, Galina N; Palmer, Sarah; Maldarelli, Frank et al. (2005) Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: balance between RNase H activity and nucleotide excision. Proc Natl Acad Sci U S A 102:2093-8
Chen, Jianbo; Dang, Que; Unutmaz, Derya et al. (2005) Mechanisms of nonrandom human immunodeficiency virus type 1 infection and double infection: preference in virus entry is important but is not the sole factor. J Virol 79:4140-9

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