RNase H requirements for the second strand transfer reaction of human immunodeficiency virus type 1 reverse transcription.
Retroviral reverse transcriptase (RT) enzymes are responsible for transcribing viral RNA into double-stranded DNA. An in vitro assay to analyze the second strand transfer event during human immunodeficiency virus type 1 (HIV-1) reverse transcription has been developed. Model substrates were constructed which mimic the viral intermediate found during plus-strand strong-stop synthesis. Utilizing wild-type HIV-1 RT and a mutant E478Q RT, the requirement for RNase H activity in this strand transfer event was analyzed. In the presence of Mg2+, HIV-1 RT was able to fully support the second strand transfer reaction in vitro. However, in the presence of Mg2+, the E478Q RT mutant had no detectable RNase H activity and was unable to support strand transfer. In the presence of Mn2+, the E478Q RT yields the initial endoribonucleolytic cleavage at the penultimate C residue of the tRNA primer yet does not support strand transfer. This suggests that subsequent degradation of the RNA primer by the RNase H domain was required for strand transfer. In reactions in which the E478Q RT was complemented with exogenous RNase H enzymes, strand transfer was supported. Additionally, we have shown that HIV-1 RT is capable of supporting strand transfer with substrates that mimic tRNAHis as well as the authentic tRNA3Lys.
Smith
C M
CM
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Smith
J S
JS
Roth
M J
MJ
D001483:Base Sequence; D002413:Cations, Divalent; D004279:DNA, Viral / Q000096:biosynthesis*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D008274:Magnesium; D008345:Manganese; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012346:RNA, Transfer, Amino Acyl; D012354:RNA, Transfer, His; D012367:RNA, Viral; D016914:Ribonuclease H / Q000378:metabolism*; D014158:Transcription, Genetic
D002413:Cations, Divalent; D004279:DNA, Viral; D012346:RNA, Transfer, Amino Acyl; D012354:RNA, Transfer, His; D012367:RNA, Viral; D005973:Glutamine; D018698:Glutamic Acid; D008345:Manganese; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D008274:Magnesium