Distinct Conformational States Underlie Pausing during Initiation of HIV-1 Reverse Transcription.
HIV-1
RNA
cryo-EM
reverse transcriptase
reverse transcription initiation
single-molecule FRET
tRNA
Journal
Journal of molecular biology
ISSN: 1089-8638
Titre abrégé: J Mol Biol
Pays: Netherlands
ID NLM: 2985088R
Informations de publication
Date de publication:
24 07 2020
24 07 2020
Historique:
received:
26
03
2020
revised:
21
05
2020
accepted:
01
06
2020
pubmed:
9
6
2020
medline:
26
1
2021
entrez:
9
6
2020
Statut:
ppublish
Résumé
A hallmark of the initiation step of HIV-1 reverse transcription, in which viral RNA genome is converted into double-stranded DNA, is that it is slow and non-processive. Biochemical studies have identified specific sites along the viral RNA genomic template in which reverse transcriptase (RT) stalls. These stalling points, which occur after the addition of three and five template dNTPs, may serve as checkpoints to regulate the precise timing of HIV-1 reverse transcription following viral entry. Structural studies of reverse transcriptase initiation complexes (RTICs) have revealed unique conformations that may explain the slow rate of incorporation; however, questions remain about the temporal evolution of the complex and features that contribute to strong pausing during initiation. Here we present cryo-electron microscopy and single-molecule characterization of an RTIC after three rounds of dNTP incorporation (+3), the first major pausing point during reverse transcription initiation. Cryo-electron microscopy structures of a +3 extended RTIC reveal conformational heterogeneity within the RTIC core. Three distinct conformations were identified, two of which adopt unique, likely off-pathway, intermediates in the canonical polymerization cycle. Single-molecule Förster resonance energy transfer experiments confirm that the +3 RTIC is more structurally dynamic than earlier-stage RTICs. These alternative conformations were selectively disrupted through structure-guided point mutations to shift single-molecule Förster resonance energy transfer populations back toward the on-pathway conformation. Our results support the hypothesis that conformational heterogeneity within the HIV-1 RTIC during pausing serves as an additional means of regulating HIV-1 replication.
Identifiants
pubmed: 32512005
pii: S0022-2836(20)30390-9
doi: 10.1016/j.jmb.2020.06.003
pmc: PMC7387199
mid: NIHMS1604038
pii:
doi:
Substances chimiques
DNA, Viral
0
HIV Reverse Transcriptase
EC 2.7.7.49
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
4499-4522Subventions
Organisme : NIAID NIH HHS
ID : P50 AI150464
Pays : United States
Organisme : NIGMS NIH HHS
ID : P50 GM082545
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM008294
Pays : United States
Informations de copyright
Copyright © 2020 Elsevier Ltd. All rights reserved.
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