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Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

ABSTRACT: G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4-binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step.

SUBMITTER: Amrane S

PROVIDER: S-EPMC9757044 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

Amrane Samir S Jaubert Chloé C Bedrat Amina A Rundstadler Tiffany T Recordon-Pinson Patricia P Aknin Cindy C Guédin Aurore A De Rache Aurore A Bartolucci Laura L Diene Ibra I Lemoine Frédéric F Gascuel Olivier O Pratviel Geneviève G Mergny Jean-Louis JL Andreola Marie-Line ML

Nucleic acids research 20221101 21

G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during inf ...[more]

PMID: 36453997

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Project description:BackgroundAlternative splicing is a key step in Human Immunodeficiency Virus type 1 (HIV-1) replication that is tightly regulated both temporally and spatially. More than 50 different transcripts can be generated from a single HIV-1 unspliced pre-messenger RNA (pre-mRNA) and a balanced proportion of unspliced and spliced transcripts is critical for the production of infectious virions. Understanding the mechanisms involved in the regulation of viral RNA is therefore of potential therapeutic interest. However, monitoring the regulation of alternative splicing events at a transcriptome-wide level during cell infection is challenging. Here we used the long-read cDNA sequencing developed by Oxford Nanopore Technologies (ONT) to explore in a quantitative manner the complexity of the HIV-1 transcriptome regulation in infected primary CD4+ T cells.ResultsONT reads mapping to the viral genome proved sufficiently long to span all possible splice junctions, even distant ones, and to be assigned to a total of 150 exon combinations. Fifty-three viral RNA isoforms, including 14 new ones were further considered for quantification. Relative levels of viral RNAs determined by ONT sequencing showed a high degree of reproducibility, compared favourably to those produced in previous reports and highly correlated with quantitative PCR (qPCR) data. To get further insights into alternative splicing regulation, we then compiled quantifications of splice site (SS) usage and transcript levels to build "splice trees", a quantitative representation of the cascade of events leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2 and its impact on viral isoform levels. Furthermore, we produced the first dynamic picture of the cascade of events occurring between 12 and 24 h of viral infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation.ConclusionONT sequencing is a convenient and reliable strategy that enabled us to grasp the dynamic of the early splicing events modulating the viral RNA landscape in HIV-1 infected cells.

Project description:Fluorescence microscopy imaging of individual HIV-1 viruses necessitates a specific labeling of viral structures that minimally perturbs the infection process. Herein, we used HIV-1 pseudoviruses containing NCp7 fused to a tetracystein (TC) tag, labeled by a biarsenical fluorescein derivative (FlAsH) to quantitatively monitor the NCp7 protein concentration in the viral cores during the early stages of infection. Single particle imaging of individual pseudoviruses with defined ratios of TC-tagged to non tagged NCp7 proteins, together with theoretical modeling of energy transfer between FlAsH dyes, showed that the high packaging of TC-tagged proteins in the viral cores causes a strong fluorescence quenching of FlAsH and that the fluorescence intensity of individual viral complexes is an appropriate parameter to monitor changes in the amount of NCp7 molecules within the viral particles during infection. Interestingly, we observed a dramatic fluorescence increase of individual FlAsH-labeled pseudoviruses containing 100% TC-tagged NCp7 proteins in infected cells at 8 and 16 h post-infection. This effect was significantly lower for pseudoviruses expressing TC-tagged integrase. Therefore, this fluorescence increase is likely related to the cytoplasmic viral transformation and the release of NCp7 molecules from the viral complexes. This loss of quenching effect is largely reduced when reverse transcriptase is inhibited, showing that NCp7 release is connected to viral DNA synthesis. A spatial analysis further revealed that NCp7-TC release is more pronounced in the perinuclear space, where capsid disassembly is thought to be completed. Quantification of NCp7-TC content based on fluorescence quenching presented in this study evidences for the first time the cytoplasmic release of NCp7 during the remodeling of HIV-1 viral particles on their journey toward the nucleus. The developed approach can be applied to quantify dye concentrations in a wide range of nano-objects by fluorescence microscopy techniques.

Dataset Information

Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

Publications

Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

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