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：2016
卷号：113
期号：47
页码：13378-13383
DOI：10.1073/pnas.1616627113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceCurrent dogma states that the integrated HIV type 1 provirus encodes a single RNA transcript that serves as both mRNA for generating viral proteins and as genomic RNA that is packaged and used for reverse transcription. We now show that multiple transcripts with different functions are generated in infected cells, a consequence of heterogeneous transcriptional start site usage. Transcripts that begin with a single capped guanosine are specifically selected for packaging, whereas those that begin with two or three capped guanosines are enriched on polysomes and used for translation. In vitro studies with recombinant 5'-leader transcripts reveal a mechanism by which the incorporation of a single 5' guanosine dramatically alters the structure, function, and fate of the viral RNA. The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3-R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5' capped guanosine (Cap1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015) Sci Rep 5:17680]. In addition, we now show that transcripts that begin with two or three capped guanosines (Cap2G or Cap3G) are enriched on polysomes, indicating that RNAs synthesized from different transcription start sites have different functions in viral replication. Because genomes are selected for packaging as dimers, we examined the in vitro monomer-dimer equilibrium properties of Cap1G, Cap2G, and Cap3G 5'-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which the Cap1G 5'-leader RNA adopts a dimeric structure, the Cap2G and Cap3G 5'-leader RNAs exist predominantly as monomers. Mutagenesis studies designed to probe for base-pairing interactions suggest that the additional guanosines of the 2G and 3G RNAs remodel the base of the PolyA hairpin, resulting in enhanced sequestration of dimer-promoting residues and stabilization of the monomer. Our studies suggest a mechanism through which the structure, function, and fate of the viral genome can be modulated by the transcriptionally controlled presence or absence of a single 5' guanosine.
