The Role of Nucleocapsid and U5 Stem/A-Rich Loop Sequences in tRNA3Lys Genomic Placement and Initiation of Reverse Transcription in Human Immunodeficiency Virus Type 1

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FONTE

American Society for Microbiology

RESUMO

We have studied the effect of mutations in the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) sequence on tRNA3Lys genomic placement, i.e., the in vivo placement of primer tRNA3Lys on the HIV-1 primer binding site (PBS). HIV-1 produced from COS cells transfected with wild-type or mutant proviral DNA was used in this study. We have found that mutations in the amino acid sequences flanking the first Cys-His box in the NC sequence produce the maximum inhibition of genomic placement. A similar finding was obtained when the NC-facilitated annealing of primer tRNA3Lys to the HIV PBS in vitro was studied. However, since the genomic placement of tRNA3Lys occurs independently of precursor protein processing, the NC mutations studied here have probably exerted their effect through one or both of the precursor proteins, Pr55gag and/or Pr160gag-pol. One mutation in the linker region between the two Cys-His boxes, P31L, prevented packaging of both Pr160gag-pol and tRNA3Lys and prevented the genomic placement of tRNA3Lys. Both packaging and genomic placement were rescued by cotransfection with a plasmid coding for wild-type Pr160gag-pol. For other linker mutations [R7R10K11 S, R32G, and S3(32-34)], packaging of Pr160gag-pol and tRNA3Lys was not affected, but genomic placement was, and placement could not be rescued by cotransfection with plasmids coding for either Pr55gag or Pr160gag-pol. After placement, the initiation of reverse transcription within extracellular virions is characterized by a 2-base DNA extension of the placed tRNA3Lys. This process requires precursor processing, and those NC mutations which showed the most inhibition of initiation were in either of the two NC Cys-His boxes. Destabilization of a U5 stem-A-rich loop immediately upstream of the PBS (through deletion of four consecutive A’s in the loop) did not affect the in vivo genomic placement of tRNA3Lys but resulted in the presence in the extracellular virus of longer cDNA extensions of tRNA3Lys, with a corresponding decrease in the presence of unextended and 2-base-extended tRNA3Lys.

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