Abstract
Synthesis of HIV-1 (-) strong-stop DNA is initiated following annealing of the 3' 18 nucleotides (nt) of tRNA(3)(Lys) 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(3)(Lys) 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(3)(Lys) but not by R18. We present data supporting the conclusion that NC promotes extended interactions between the anticodon stem and variable loop of tRNA(3)(Lys) 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.
Highlights
REQUIREMENT FOR RNA SEQUENCES DOWNSTREAM OF THE PRIMER BINDING SITE ABROGATED BY NUCLEOCAPSID PROTEIN-DEPENDENT PRIMER-TEMPLATE INTERACTIONS*
Effect of RNA Template Length on (Ϫ) SSDNA Synthesis Primed by the D18, R18, and tRNA3Lys Primers—To investigate the potential role of sequences downstream of the primer binding site (PBS) in the synthesis of (Ϫ) SSDNA (Fig. 1A), we analyzed primer extension efficiency with 14 in vitro transcribed RNA templates containing varying lengths of downstream sequences (Fig. 1B)
We have investigated the possible role of Human immunodeficiency virus type 1 (HIV-1) RNA template sequences immediately downstream of the PBS in the initiation of (Ϫ) SSDNA synthesis, using a reconstituted in vitro system
Summary
Vol 278, No 16, Issue of April 18, pp. 14185–14195, 2003 Printed in U.S.A. REQUIREMENT FOR RNA SEQUENCES DOWNSTREAM OF THE PRIMER BINDING SITE ABROGATED BY NUCLEOCAPSID PROTEIN-DEPENDENT PRIMER-TEMPLATE INTERACTIONS*. We report that in the absence of NC, at least 24 bases immediately downstream of the PBS are required for efficient (Ϫ) SSDNA synthesis when RNA primers (native tRNA3Lys or an 18-nt RNA complementary to the PBS (R18)), but not an all DNA (D18) primer, are used These findings, as well as results of assays with chimeric DNA-RNA primers, are correlated with CD spectra and melting studies of 18-nt primer:PBS duplexes, underscoring the important role of helical conformation and thermal stability for priming activity. Mutational analysis supports the hypothesis that the nucleic acid chaperone activity of NC facilitates stable formation of extended interactions between the 3Ј anticodon stem and variable loop of tRNA3Lys and bases upstream of the A-rich loop in the RNA template and that these interactions modulate initiation of reverse transcription
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