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Journal of Virology, December 2004, p. 13315-13324, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.13315-13324.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Effects of Mutations in the G Tract of the Human Immunodeficiency Virus Type 1 Polypurine Tract on Virus Replication and RNase H Cleavage

John G. Julias,1 Mary Jane McWilliams,2 Stefan G. Sarafianos,3 W. Gregory Alvord,4 Eddy Arnold,3 and Stephen H. Hughes2*

Basic Research Program, SAIC-Frederick, Inc.,1 HIV Drug Resistance Program,2 Data Management Services, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, Maryland,4 Center for Advanced Biotechnology and Medicine (CABM) and Rutgers University Chemistry Department, Piscataway, New Jersey3

Received 27 April 2004/ Accepted 30 June 2004

The RNase H cleavages that generate and remove the polypurine tract (PPT) primer during retroviral reverse transcription must be specific in order to create a linear viral DNA that is suitable for integration. Lentiviruses contain a highly conserved sequence consisting of six guanine residues at the 3' end of the PPT (hereafter referred to as the G tract). We introduced mutations into the G tract of a human immunodeficiency virus type 1-based vector and determined the effects on the virus titer and RNase H cleavage specificity. Most mutations in the G tract had little or no effect on the virus titer. Mutations at the second and fifth positions of the G tract increased the proportion of two-long-terminal-repeat (2-LTR) circle junctions with one or two nucleotide insertions. The second and fifth positions of the G tract make specific contacts with amino acids in the RNase H domain that are important for RNase H cleavage specificity. These complementary data define protein-nucleic acid interactions that help control the specificity of RNase H cleavage. When the G-tract mutants were analyzed in a viral background that was deficient in integrase, in most cases the proportion of consensus 2-LTR circle junctions increased. However, in the case of a mutant with Ts at the second and fifth positions of the G tract, the proportion of 2-LTR circle junctions containing the one-nucleotide insertion increased, suggesting that linear viral DNAs containing an extra base are substrates for integration. This result is consistent with the idea that the 3' end-processing reactions of retroviral integrases may help to generate defined ends from a heterogenous population of linear viral DNAs.

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

Journal of Virology, December 2004, p. 13315-13324, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.13315-13324.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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