The nucleocapsid protein specifically anneals tRNALys-3 onto a noncomplementary primer binding site within the HIV-1 RNA genome in vitro.
HIV type 1 (HIV-1) specifically uses host cell tRNALys-3 as a primer for reverse transcription. The 3' 18 nucleotides of this tRNA are complementary to a region on the HIV RNA genome known as the primer binding site (PBS). HIV-1 has a strong preference for maintaining a lysine-specific PBS in vivo, and viral genomes with mutated PBS sequences quickly revert to be complementary to tRNALys-3. To investigate the mechanism for the observed PBS reversion events in vitro, we examined the capability of the nucleocapsid protein (NC) to anneal various tRNA primer sequences onto either complementary or noncomplementary PBSs. We show that NC can anneal different full-length tRNAs onto viral RNA transcripts derived from the HIV-1 MAL or HXB2 isolates, provided that the PBS is complementary to the tRNA used. In contrast, NC promotes specific annealing of only tRNALys-3 onto an RNA template (HXB2) whose PBS sequence has been mutated to be complementary to the 3' 18 nt of human tRNAPro. Moreover, HIV-1 reverse transcriptase extends this binary complex from the proline-specific PBS. The formation of the noncomplementary binary complex does not occur when a chimeric tRNALys/Pro containing proline-specific D and anticodon domains is used as the primer. Thus, elements outside the acceptor-TPsiC domains of tRNALys-3 play an important role in preferential primer use in vitro. Our results support the hypothesis that mutant PBS reversion is a result of tRNALys-3 annealing onto and extension from a PBS that specifies an alternate host cell tRNA.
Chan
B
B
University of Minnesota, Department of Chemistry, 207 Pleasant Street SE, Minneapolis, MN 55455, USA.
Musier-Forsyth
K
K
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005816:Genetic Complementation Test; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D017931:DNA Primers; D019590:Nucleocapsid Proteins; C034477:RNA primers; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D012313:RNA