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Journal of Virology, May 2004, p. 5402-5413, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5402-5413.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Frequent Dual Initiation in Human Immunodeficiency Virus-Based Vectors Containing Two Primer-Binding Sites: a Quantitative In Vivo Assay for Function of Initiation Complexes

Yegor A. Voronin1,2 and Vinay K. Pathak1*

HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland 21702,1 Department of Biochemistry, West Virginia University, Morgantown, West Virginia 256062

Received 14 November 2003/ Accepted 19 January 2004

We previously demonstrated that murine leukemia virus (MLV)-based vectors containing two primer-binding sites (PBSs) have the capacity to initiate reverse transcription more than once (Y. A. Voronin and V. K. Pathak, Virology 312:281-294, 2003). To determine whether human immunodeficiency virus (HIV)-based vectors also have the capacity to initiate reverse transcription twice, we constructed an HIV type 1 (HIV-1)-based vector containing the HIV-1 PBS, a green fluorescent protein reporter gene (GFP), and a second PBS derived from HIV-2 3' of GFP. Simultaneous initiation of reverse transcription at both the 5' HIV-1 PBS and 3' HIV-2 PBS was predicted to result in deletion of GFP. As in the MLV-based vectors, GFP was deleted in approximately 25% of all proviruses, indicating frequent dual initiation in HIV-based vectors containing two PBSs. Quantitative real-time PCR analysis of early reverse transcription products indicated that HIV-1 reverse transcriptase efficiently used the HIV-2 PBS. To investigate tRNA primer-RNA template interactions in vivo, we introduced several mutations in the HIV-2 U5 region. The effects of these mutations on the efficiency of reverse transcription initiation were measured by quantitative real-time PCR analysis of early reverse transcription products, with initiation at the HIV-1 PBS used as an internal control. Disruption of the lower and upper parts of the U5-inverted repeat stem reduced the efficiency of initiation 20- and 6-fold, respectively. In addition, disruption of the proposed interactions between viral RNA and tRNALys3 thymidine-pseudouridine-cytidine and anticodon loops decreased the efficiency of initiation seven- and sixfold, respectively. These results demonstrate the relative influence of various RNA-RNA interactions on the efficiency of initiation in vivo. Furthermore, the two-PBS vector system provides a sensitive and quantitative in vivo assay for analysis of RNA-RNA and protein-RNA interactions that can influence the efficiency of reverse transcription initiation.

* Corresponding author. Mailing address: HIV Drug Resistance Program, National Cancer Institute—Frederick, Building 535, Room 334, Frederick, MD 21702. Phone: (301) 846-1710. Fax: (301) 846-6013. E-mail: VPATHAK{at}ncifcrf.gov.

Journal of Virology, May 2004, p. 5402-5413, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5402-5413.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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