RNase H activity is required for high-frequency repeat deletion during Moloney murine leukemia virus replication.
It has been postulated that retroviral recombination, like strong stop template switching, requires the RNase H activity of reverse transcriptase. To address this hypothesis, Moloney murine leukemia virus-based vectors, which were designed to test the recombination-related property of direct repeat deletion, were encapsidated in virions engineered to contain phenotypic mixtures of wild-type and RNase H catalytic site point mutant reverse transcriptase. Integrated provirus titers per milliliter were determined for these phenotypically mixed virions, and vector proviruses were screened to determine what percentage contained repeat deletions. The results revealed a steady decline in direct repeat deletion frequency that correlated with decreases in functional RNase H, with greater than fourfold decreases in repeat deletion frequency observed when 95% of virion reverse transcriptase was RNase H defective. Parallel experiments were performed to address effects of molar excesses of RNase H relative to functional DNA polymerase. These experiments demonstrated that increasing the stoichiometry of RNase H relative to the amount of functional DNA polymerase had minimal effects on direct repeat deletion frequency. DNA synthesis was error prone when directed principally by RNase H mutant reverse transcriptase, suggesting a role for RNase H catalytic integrity in the fidelity of intracellular reverse transcription.
Brincat
Jennifer L
JL
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620, USA.
Pfeiffer
Julie K
JK
Telesnitsky
Alice
A
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000502:physiology; D017353:Gene Deletion; D006801:Humans; D007763:Lac Operon; D051379:Mice; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011533:Proviruses / Q000502:physiology; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D016914:Ribonuclease H / Q000378:metabolism*; D013698:Templates, Genetic; D014779:Virus Replication / Q000502:physiology
D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H