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Nucleic Acids Research 2006 34(10):2853-2863; doi:10.1093/nar/gkl360
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Published online 24 May 2006

Nucleic Acids Research, 2006, Vol. 34, No. 10 2853-2863
© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Article

Analysis of HIV-1 replication block due to substitutions at F61 residue of reverse transcriptase reveals additional defects involving the RNase H function

Dibyakanti Mandal, Chandravanu Dash1, Stuart F. J. Le Grice1 and Vinayaka R. Prasad*

Department of Microbiology and Immunology, Albert Einstein College of Medicine 1300 Morris Park Avenue, Bronx, NY 10461, USA 1 HIV Drug Resistance Program, National Cancer Institute Frederick, MD, USA

*To whom correspondence should be addressed. Tel: +1 718 430 2517; Fax: +1 718 430 8976; Email: prasad{at}aecom.yu.edu

Received March 9, 2006. Revised March 30, 2006. Accepted April 24, 2006.

We reported previously that substitutions F61L, F61W, F61Y and F61A in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase affect strand displacement synthesis [T. S. Fisher, T. Darden and V. R. Prasad (2003) J. Mol. Biol., 325, 443–459]. We have now determined the effect of these mutations on HIV replication. All mutant viruses were replication defective. Measuring replication intermediates in infected cells did not reveal a specific block as all mutants displayed reduced DNA synthesis (wild-type>F61L>F61W>F61Y>F61A). Analysis of 2-LTR circle junctions revealed that F61W and F61Y mutants generated increased aberrant circle junctions. Circle junctions corresponding to F61Y included 3'-PPT insertions suggesting ribonuclease H defect. In vitro assays mimicking PPT primer generation indicated that F61L, F61W and F61Y mutant RTs were unaffected, while F61A mutant cleaved both at PPT/U3 junction and at +6 with similar efficiencies. In assays measuring cleavage at the RNA/DNA junction to remove the PPT primer, all mutants were significantly affected with F61Y and F61A being most severely impaired. Our results show that (i) replication block of most mutants is due to more than one biochemical defect; (ii) mutations in polymerase domain can affect the function of a distal domain; and (iii) virological analyses of RT mutations can yield insight into structure–function relationship that is otherwise not obvious.


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