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Journal of Virology, January 2001, p. 672-686, Vol. 75, No. 2
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.2.672-686.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

In Vitro Analysis of Human Immunodeficiency Virus Type 1 Minus-Strand Strong-Stop DNA Synthesis and Genomic RNA Processing

Mark D. Driscoll,dagger Marie-Pierre Golinelli,Dagger and Stephen H. Hughes*

ABL-Basic Research Program, National Cancer Institute---Frederick Research and Development Center, Frederick, Maryland 21702-1201

Received 6 July 2000/Accepted 25 October 2000

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA3Lys primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5' end of the RNA that remained annealed to the 3' end of the minus-strand strong-stop DNA only if NC was present in the reaction.

* Corresponding author. Present address: HIV Drug Resistance Program, NCI---Frederick, P.O. Box B, Frederick, MD 21702-1201. Phone: (301) 846-1619. Fax: (301) 846-6966. E-mail: hughes{at}ncifcrf.gov.

dagger Present address: Molecular Staging, Guilford, CT 06437.

Dagger Present address: HIV Drug Resistance Program, National Cancer Institute---FCRDC, Frederick, MD 21702-1201.

Journal of Virology, January 2001, p. 672-686, Vol. 75, No. 2
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.2.672-686.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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