RNase H requirements for the second strand transfer reaction of human immunodeficiency virus type I reverse transcription

Christine M. Smith, Jeffrey S. Smith, Monica J. Roth

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

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 vital 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 tRNA(His) as well as the authentic tRNA3/(Lys).

Original languageEnglish (US)
Pages (from-to)6573-6581
Number of pages9
JournalJournal of virology
Volume73
Issue number8
DOIs
StatePublished - 1999

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Fingerprint Dive into the research topics of 'RNase H requirements for the second strand transfer reaction of human immunodeficiency virus type I reverse transcription'. Together they form a unique fingerprint.

Cite this