In vitro synthesis of long DNA products in reactions with HIV-RT and nucleocapsid protein.
In vitro reaction conditions using HIV reverse transcriptase (RT) and nucleocapsid protein (NC) that allowed efficient synthesis of single-stranded DNA products over a thousand nucleotides in length from genomic HIV RNA were characterized. Consistent with previous reports, the reactions required high concentrations of NC and RT. Long products were produced as a result of frequent strand transfer between RNA templates, averaging at least one transfer per 300 nucleotides synthesized. No change in RT processivity was observed in the reactions in the presence versus absence of NC. Synthesis of long products required formation of a high molecular mass aggregate between NC and nucleic acids. The aggregate formed rapidly and pelleted with low speed centrifugation. The aggregate was accessible to RT as pre-formed aggregates synthesized long products when RT was added. NC finger mutants lacking either finger one or two or with the finger positions switched were all effective in promoting long products. This suggests that the aggregation/condensation but not helix-destabilizing activity of NC was required. We propose that these high molecular mass aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC to close proximity, thereby mimicking the role of the capsid environment within the host cell.
Anthony
Reshma M
RM
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
Destefano
Jeffrey J
JJ
D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D008957:Models, Genetic; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase