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J. Biol. Chem., Vol. 278, Issue 16, 14185-14195, April 18, 2003

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Efficient Initiation of HIV-1 Reverse Transcription in Vitro
REQUIREMENT FOR RNA SEQUENCES DOWNSTREAM OF THE PRIMER BINDING SITE ABROGATED BY NUCLEOCAPSID PROTEIN-DEPENDENT PRIMER-TEMPLATE INTERACTIONS^*

Yasumasa Iwatani^§, Abbey E. Rosen^¶, Jianhui Guo, Karin Musier-Forsyth^¶, and Judith G. Levin^**

From the  Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Bethesda, Maryland 20892 and the ^¶ Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455

Synthesis of HIV-1 () strong-stop DNA is initiated following annealing of the 3' 18 nucleotides (nt) of tRNA to the primer binding site (PBS) near the 5' terminus of viral RNA. Here, we have investigated whether sequences downstream of the PBS play a role in promoting efficient () strong-stop DNA synthesis. Our findings demonstrate a template requirement for at least 24 bases downstream of the PBS when tRNA or an 18-nt RNA complementary to the PBS (R18), but not an 18-nt DNA primer, are used. Additional assays using 18-nt DNA-RNA chimeric primers, as well as melting studies and circular dichroism spectra of 18-nt primer:PBS duplexes, suggest that priming efficiency is correlated with duplex conformation and stability. Interestingly, in the presence of nucleocapsid protein (NC), the 24 downstream bases are dispensable for synthesis primed by tRNA but not by R18. We present data supporting the conclusion that NC promotes extended interactions between the anticodon stem and variable loop of tRNA and a sequence upstream of the A-rich loop in the template. Taken together, this study leads to new insights into the initiation of HIV-1 reverse transcription and the functional role of NC-facilitated tRNA-template interactions in this process.

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