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Journal of Virology, August 1999, p. 6573-6581, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

RNase H Requirements for the Second Strand Transfer Reaction of Human Immunodeficiency Virus Type 1 Reverse Transcription

Christine M. Smith, Jeffrey S. Smith,dagger and Monica J. Roth*

Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Received 15 September 1998/Accepted 3 May 1999

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.

* Corresponding author. Mailing address: Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Phone: (732) 235-5048. Fax: (732) 235-4783. E-mail: Roth{at}waksman.rutgers.edu.

dagger Present address: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185.

Journal of Virology, August 1999, p. 6573-6581, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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