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.
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.
Iwatani
Yasumasa
Y
Laboratory of Molecular Genetics, National Institute of Child Health & Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Rosen
Abbey E
AE
Guo
Jianhui
J
Musier-Forsyth
Karin
K
Levin
Judith G
JG
D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000378:metabolism; D002942:Circular Dichroism; D017931:DNA Primers / Q000494:pharmacology; D004277:DNA, Single-Stranded; D054303:HIV Reverse Transcriptase / Q000378:metabolism* / Q000502:physiology; D015497:HIV-1 / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D017931:DNA Primers; D004277:DNA, Single-Stranded; D019590:Nucleocapsid Proteins; D012357:RNA, Transfer, Lys; D012313:RNA; D054303:HIV Reverse Transcriptase