Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation.
Retrovirus particles each contain two copies of the viral genome in the form of a noncovalently linked RNA dimer. Earlier studies have mapped a cis-acting region near the 5' end of the human immunodeficiency virus type 1 (HIV-1) genome, termed the psi locus, which appears essential for initiation of genomic dimerization, as well as for interactions with the HIV-1 Gag protein that are thought to target the RNA into nascent virions. This HIV-1 psi locus is proposed to be organized in four independent RNA stem-loops; at least three (SL1, SL3, and SL4) contain binding sites for Gag, and one of these (SL1) is implicated in dimer initiation through a kissing-loop mechanism. In this study, we have created HIV-1 proviruses containing psi mutations that affect in vitro Gag binding, RNA dimerization, or both, and we have characterized the effects of these mutations on viral assembly and infectivity by using a single-step infectious assay. We find that various mutations which eliminate the Gag binding sites in SL1 or SL3 produce marked defects in genomic RNA packaging and viral infectivity. In each case, the reduced genomic content of the mutant virions is associated with an increased content of spliced viral transcripts, suggesting that both SL1 and SL3 contribute to the discrimination between spliced and unspliced RNAs. The structures, but not the specific sequences, of the SL1 and SL3 stems appear critical for RNA packaging. Disruption of the stem or deletion of SL1 also results in abnormal genomic dimerization, as assessed by nondenaturing gel electrophoresis of virion-derived RNA. Virions carrying less extensive mutations in the SL1 loop that are known to prevent in vitro dimerization have impaired infectivity despite normal virion RNA content. This suggests that RNA dimerization is not a prerequisite for genomic packaging but instead serves an independent function in the retroviral infectious cycle.
Clever
J L
JL
Department of Pathology, University of California, San Francisco 94143, USA.
Parslow
T G
TG
D001483:Base Sequence; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000737:chemistry*; D019065:Virus Assembly
D012367:RNA, Viral