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Journal of Virology, October 2003, p. 11150-11157, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11150-11157.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mutations in the 5' End of the Human Immunodeficiency Virus Type 1 Polypurine Tract Affect RNase H Cleavage Specificity and Virus Titer

Mary Jane McWilliams,1 John G. Julias,1 Stefan G. Sarafianos,2 W. Gregory Alvord,3 Edward Arnold,2 and Stephen H. Hughes1*

HIV-Drug Resistance Program,1 Data Management Services, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, Maryland 21702-1201,3 Center for Advanced Biotechnology and Medicine and Rutgers University Chemistry Department, Piscataway, New Jersey 08854-56382

Received 2 May 2003/ Accepted 21 July 2003

The RNase H activity of retroviral reverse transcriptases (RTs) degrades viral genomic RNA after it has been copied into DNA, removes the tRNA used to initiate negative-strand DNA synthesis, and generates and removes the polypurine tract (PPT) primer used to initiate positive-strand DNA synthesis. The cleavages that remove the tRNA and that generate and remove the PPT primer must be specific to generate linear viral DNAs with ends that are appropriate for integration into the host cell genome. The crystal structure of human immunodeficiency virus type 1 (HIV-1) RT in a complex with an RNA/DNA duplex derived from the PPT revealed that the 5' end of the PPT deviates from traditional Watson-Crick base pairing. This unusual structure may play a role in the proper recognition of the PPT by HIV-1 RT. We made substitution mutations in the 5' end of the PPT and determined their effects on virus titer. The results indicated that single and double mutations in the 5' end of the PPT had modest effects on virus replication in a single-cycle assay. More complex mutations had stronger effects on virus titer. Analysis of the two-long-terminal-repeat circle junctions derived from infecting cells with the mutant viruses indicated that the mutations affected RNase H activity, resulting in the retention of PPT sequences on viral DNA. The mutants tested preferentially retained specific segments of the PPT, suggesting an effect on cleavage specificity. These results suggest that structural features of the PPT are important for its recognition and cleavage in vivo.

* Corresponding author. Mailing address: HIV Drug Resistance Program, NCI-Frederick, P.O. Box B, Bldg. 539, Rm. 130A, Frederick, MD 21702-1201. Phone: (301) 846-1619. Fax: (301) 846-6966. E-mail: hughes{at}ncifcrf.gov.

Journal of Virology, October 2003, p. 11150-11157, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11150-11157.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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