期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：37
DOI：10.1073/pnas.2112475118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
HIV-1 replication is critically dependent on the selective incorporation of viral RNA genomes into assembling virions. Although RNA elements that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency have until now remained unknown. The present studies show that genome selection is achieved by a bipartite mechanism that requires both dimerization-dependent exposure of packaging signals within the 5′ leader of the viral RNA and structural sequestration of the 5′ cap. Cap sequestration likely prevents capture by the cellular RNA processing and translation machinery, a mechanism that may help explain why some cellular RNA polymerase III transcripts that lack 5′ caps can parasitize virions. Cap sequestration may be commonly employed among RNA viruses that package 5′-capped genomes.
HIV-1 selectively packages two copies of its 5′-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5′ leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (Ψ
CES) is unexpectedly poorly packaged when assayed in competition with the intact 5′ leader. Ψ
CES lacks a 5′-tandem hairpin element that sequesters the 5′ cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5′ ribozyme to Ψ
CES to enable cotranscriptional shedding of the 5′ cap promoted Ψ
CES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5′ cap and exposure of NC binding sites that reside fully within the Ψ
CES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5′-capped RNA genomes.
