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Journal of Virology, November 1998, p. 9353-9358, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Roles of the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein in Annealing and Initiation versus Elongation in Reverse Transcription of Viral Negative-Strand Strong-Stop DNA

Liwei Rong,1 Chen Liang,1 Mayla Hsu,1 Lawrence Kleiman,1 Patrice Petitjean,2 Hugues de Rocquigny,2 Bernard P. Roques,2 and Mark A. Wainberg1,*

McGill University AIDS Centre, Lady Davis Institute---Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2,1 and Département de Pharmacochimie Moléculaire et Structurale, U266 INSERM-URA D1500 CNRS, UER des Sciences Pharmaceutiques et Biologiques, 75270 Paris Cedex 06, France2

Received 17 February 1998/Accepted 16 July 1998

To study the initiation of human immunodeficiency virus type 1 reverse transcription, we have used the viral nucleocapsid protein (NC7) to anneal tRNA3Lys primer onto viral genomic RNA and have then eliminated NC7 from this primer-template complex by digestion with proteinase K and phenol-chloroform extraction of residual protein. Our data show that saturating concentrations of NC7 resulted in the formation of an active tRNA-template complex that yielded enhanced production of full-length negative-strand strong-stop DNA [(-)ssDNA] and that this complex remained active even after the elimination of NC7. While both of the two Zn finger motifs found within NC7 were essential for efficient elongation, NC protein that contained a point mutation in the first Zn finger or that was devoid of both Zn fingers yielded primer-template complexes that could still be initiated in 1-base-extension assays. In contrast, the use of heat annealing to produce primer-template complexes resulted in proportions of full-length (-)ssDNA lower than those seen with NC protein, and the addition of NC protein to such preformed primer-template complexes was able to reverse this defect only to a marginal extent.

* Corresponding author. Mailing address: McGill AIDS Centre, Lady Davis Institute/Jewish General Hospital, 3755 Cote Ste-Catherine Rd., Montreal, Quebec, Canada H3T 1E2. Phone: (514) 340-8260. Fax: (514) 340-7537. E-mail: mdwa{at}musica.mcgill.ca.

Journal of Virology, November 1998, p. 9353-9358, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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