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1.
Biophys J ; 120(21): 4874-4890, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34529947

RESUMO

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5' untranslated region (5'UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5'UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5'UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5'UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5'UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5'UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation.


Assuntos
HIV-1 , Regiões 5' não Traduzidas , Genômica , HIV-1/genética , Conformação de Ácido Nucleico , Nucleocapsídeo , RNA Viral/genética , Vírion
3.
Viruses ; 12(10)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-33008123

RESUMO

Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems-a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.


Assuntos
Genoma Viral , Espectroscopia de Ressonância Magnética/métodos , RNA Viral/química , Retroviridae/genética , Empacotamento do Genoma Viral , Sequência de Bases , HIV-1/genética , Conformação de Ácido Nucleico , Filogenia , Estrutura Secundária de Proteína , RNA Viral/genética , Montagem de Vírus
4.
Proc Natl Acad Sci U S A ; 117(30): 17737-17746, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32647061

RESUMO

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5'-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5'-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 µM, and that all high-affinity sites (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles' heel to which HIV therapeutics may be targeted.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Nucleocapsídeo/metabolismo , RNA Viral , Sequências Reguladoras de Ácido Ribonucleico , Montagem de Vírus , Sequência de Bases , Sítios de Ligação , Genoma Viral , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/metabolismo , Ligação Proteica
5.
Proc Natl Acad Sci U S A ; 113(47): 13378-13383, 2016 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-27834211

RESUMO

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.


Assuntos
Guanosina/genética , HIV-1/genética , RNA Viral/química , Sítio de Iniciação de Transcrição , Heterogeneidade Genética , Genoma Viral , HIV-1/fisiologia , Estrutura Molecular , Mutação , Polirribossomos/genética , Regiões Promotoras Genéticas , RNA Viral/genética , Transcrição Gênica , Montagem de Vírus , Replicação Viral
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