Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome.
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 orCap3G) 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 ofCap1G,Cap2G, andCap3G 5'-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which theCap1G 5'-leader RNA adopts a dimeric structure, theCap2G andCap3G 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.
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109.
Siarhei
S
Kharytonchyk
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250.
Sarah
S
Monti
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109.
Philip J
PJ
Smaldino
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250.
Verna
V
Van
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250.
Nicholas C
NC
Bolden
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250.
Joshua D
JD
Brown
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250.
Emily
E
Russo
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250.
Canessa
C
Swanson
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250.
Alex
A
Shuey
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; summers@hhmi.umbc.edu ateles@umich.edu.
Alice
A
Telesnitsky
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250; summers@hhmi.umbc.edu ateles@umich.edu.|Howard Hughes Medical Institute, University of Maryland Baltimore County, Baltimore, MD 21250.
Michael F
MF
Summers
D018740:Genetic Heterogeneity; D016679:Genome, Viral; D006151:Guanosine / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D015394:Molecular Structure; D009154:Mutation; D011132:Polyribosomes / Q000235:genetics; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D019065:Virus Assembly; D014779:Virus Replication
D012367:RNA, Viral; D006151:Guanosine
5′-leader; HIV-1; RNA; structure; transcription