THE NUCLEOCAPSID PROTEIN SPECIFICALLY ANNEALS TRNA(LYS-3) ONTO A NONCOMPLEMENTARY PRIMER BINDING-SITE WITHIN THE HIV-1 RNA GENOME IN-VITRO

HIV type 1 (HIV-1) specifically uses host cell tRNA(Lys-3) as a primer for reverse transcription. The 3' 18 nucleotides of this tRNA are com plementary to a region on the HIV RNA genome known as the primer bindi ng site (PBS). HIV-1 has a strong preference for maintaining a lysine- specific PBS in vivo, and viral genomes with mutated PBS sequences qui ckly revert to be complementary tu tRNA(Lys-3). To investigate the mec hanism fur thf observed PBS reversion events in vitro, we examined the capability of the nucleocapsid protein (NC) to anneal various tRNA pr imer sequences onto either complementary or noncomplementary PBSs. We show that NC can anneal different full-length tRNAs onto viral RNA tra nscripts derived from the HIV-1 MAL or HXB2 isolates, provided that th e PBS is complementary to the tRNA used. In contrast, NC promotes spec ific annealing of only tRNA(Lys-3) onto an RNA template (HXB2) whose P IGS sequence has been mutated to be complementary to the 3' IS nt of h uman tRNA(Pro). Moreover, HIV-1 reverse transcriptase extends this bin ary complex from the proline-specific PES. The formation of the noncom plementary binary complex does not occur when a chimeric tRNA(Lys/Pro) containing proline-specific D and anticodon domains is used as the pr imer, Thus, elements outside the acceptor-T Psi C domains of tRNA(Lys- 3) play an important role in preferential primer use in vitro. Our res ults support the hypothesis that mutant PBS reversion is a result of t RNA(Lys-3) annealing onto end extension from a PBS that specifies an a lternate host cell tRNA.