The nucleocapsid protein specifically anneals tRNALys-3 onto a noncomplementary primer binding site within the HIV-1 RNA genome in vitro

AUTOR(ES)

Chan, Barden

FONTE

The National Academy of Sciences of the USA

RESUMO

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-TΨC 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.

Documentos Relacionados

• On the importance of the primer activation signal for initiation of tRNAlys3-primed reverse transcription of the HIV-1 RNA genome
• The connection domain in reverse transcriptase facilitates the in vivo annealing of tRNALys3 to HIV-1 genomic RNA
• Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes
• Destabilization of tRNA3Lys from the primer-binding site of HIV-1 genome by anti-A loop polyamide nucleotide analog
• Cleavage of the HIV replication primer tRNALys,3 in human cells expressing bacterial anticodon nuclease.