Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

Coey, Aaron; Larsen, Kevin; Puglisi, Joseph D.; Puglisi, Elisabetta Viani

doi:10.1261/rna.056804.116

Cited by 13 publications

(26 citation statements)

References 42 publications

(66 reference statements)

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“…The RNA fold in the RTIC likely sequesters important sequences for vRNA-tRNA interactions, such as the PAS/anti-PAS and A-rich loop/anticodon sequence interactions 9,11–18 , which may subsequently form as RNAs rearrange in response to RT extension during initiation. The RNA tertiary conformation within the RTIC is clearly dynamic, as shown by prior single-molecule data 19,27,30 and suggested by our cryo-EM data. We observe several conformations with variable orientations and density for the extended tRNA helix and three-way junction of H1, H2 and PBS (Extended Data Figure 7).…”

supporting

confidence: 81%

“…Accordingly, we propose that the 5′ end of tRNA (nts 1–54) refolds to form a secondary structure with a contiguous helix (Figure 2c). Specifically, the D and anticodon stems from nucleotides 10–41 rearrange to form a continuous helical structure, which fits the observed density far better than the three-way junction 27,28 observed in the free initiation complex (Extended Data Figure 6). …”

supporting

confidence: 55%

“…HIV-1 vRNA constructs were prepared by in vitro transcription with T7 RNA polymerase as previously described 27,28 . Transcripts were denatured in 8 M urea and purified on a sequencing PAGE gel.…”

Section: Methodsmentioning

confidence: 99%

“…Single-molecule FRET experiments were performed using a prism-based total internal reflection instrument with a diode-pumped solid-state 532 nm laser as previously described 27,33–38 . This includes the use of an oxygen scavenging system (protocatechuate 3,4-dioxygenase (PCD) and β-carboxy-cis,cis-muconic acid (PCA)) and a triplet state relaxer (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)) to reduce aberrant dye behaviors.…”

Section: Methodsmentioning

confidence: 99%

“…No additional interactions between the vRNA and tRNA occur beyond the extended PBS helix at the +1 stage of initiation (Figure 2b), consistent with biochemical results on similar HIV-1 subtype-B sequences 13,14 . Notably absent is any PAS/anti-PAS interaction between HIV-1 nts 123–131 and tRNA nts 48–55 (Figure 1a), which has been implicated in RT initiation and shown to form dynamically in the absence of RT 11,12,27,30 . The formation and positions of vRNA H1 and H2 are consistent with their proposed function as barriers during initiation 13,14,19 .…”

mentioning

confidence: 99%

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Architecture of an HIV-1 reverse transcriptase initiation complex

Larsen

Mathiharan

Kappel

et al. 2018

Nature

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Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in infection1 and a common target of antiretrovirals2. The reaction is catalyzed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion along with 2 copies of dimeric viral genomic RNA5 and host tRNALys3, which acts as a primer for initiation of reverse transcription6,7. Upon viral entry, initiation is slow and non-processive compared to elongation8,9. Despite extensive efforts, the structural basis for RT function during initiation has remained a mystery. Here we apply cryo-electron microscopy (cryo-EM) to determine a three-dimensional structure of the HIV-1 RT initiation complex. RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer-template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5′ end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.

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supporting

confidence: 81%

supporting

confidence: 55%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Architecture of an HIV-1 reverse transcriptase initiation complex

Larsen

Mathiharan

Kappel

et al. 2018

Nature

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Illuminating the virus life cycle with single-molecule FRET imaging

Mothes

2019

Advances in Virus Research

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Single-molecule Förster resonance energy transfer (smFRET) imaging has emerged as a powerful tool to probe conformational dynamics of viral proteins, identify novel structural intermediates that are hiding in averaging population-based measurements, permit access to the energetics of transitions and as such to the precise molecular mechanisms of viral replication. One strength of smFRET is the capability of characterizing biological molecules in their fully hydrated/native state, which are not necessarily available to other structural methods. Elegant experimental design for physiologically relevant conditions, such as intact virions, has permitted the detection of previously unknown conformational states of viral glycoproteins, revealed asymmetric intermediates, and allowed access to the real-time imaging of conformational changes during viral fusion. As more laboratories are applying smFRET, our understanding of the molecular mechanisms and the dynamic nature of viral proteins throughout the virus life cycle are predicted to improve and assist the development of novel antiviral therapies and vaccine design.

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Stability and conformation of the dimeric HIV-1 genomic RNA 5′UTR

Blakemore

Burnett²,

Swanson³

et al. 2021

Biophysical Journal

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During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5 0 untranslated region (5 0 UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5 0 UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5 0 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 0 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 Fo ¨rster resonance energy transfer imaging to demonstrate that 5 0 UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5 0 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.

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Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

Cited by 13 publications

References 42 publications

Architecture of an HIV-1 reverse transcriptase initiation complex

Architecture of an HIV-1 reverse transcriptase initiation complex

Illuminating the virus life cycle with single-molecule FRET imaging

Stability and conformation of the dimeric HIV-1 genomic RNA 5′UTR

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