This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thomas, D. C.
Right arrow Articles by Pathak, V. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thomas, D. C.
Right arrow Articles by Pathak, V. K.

 Previous Article  |  Next Article 

Journal of Virology, May 2007, p. 4798-4807, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02471-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Determination of the Ex Vivo Rates of Human Immunodeficiency Virus Type 1 Reverse Transcription by Using Novel Strand-Specific Amplification Analysis{triangledown}

David C. Thomas,1,2 Yegor A. Voronin,1,{dagger} Galina N. Nikolenko,1 Jianbo Chen,3 Wei-Shau Hu,3 and Vinay K. Pathak1*

Viral Mutation Section, HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland 21702,1 Basic Research Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702,2 Viral Recombination Section, HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland 217023

Received 9 November 2006/ Accepted 14 February 2007

Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4+ T cells (~68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer (~4 min), plus-strand DNA transfer (~26 min), and initiation of plus-strand DNA synthesis (~9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.

* Corresponding author. Mailing address: Viral Mutation Section, HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute-Frederick, P.O. Box B, Room 334, Frederick, MD 21702. Phone: (301) 846-1710. Fax: (301) 846-6013. E-mail: vpathak{at}ncifcrf.gov

{triangledown} Published ahead of print on 21 February 2007.

{dagger} Present address: Department of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109.

Journal of Virology, May 2007, p. 4798-4807, Vol. 81, No. 9
0022-538X/07/$08.00+0     doi:10.1128/JVI.02471-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Onafuwa-Nuga, A., Telesnitsky, A. (2009). The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination. Microbiol. Mol. Biol. Rev. 73: 451-480 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»