Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation

Ali, Lizna Mohamed; Pitchai, Fathima Nuzra Nagoor; Vivet-Boudou, Valérie; Chameettachal, Akhil; Jabeen, Ayesha; Pillai, Vineeta; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A.

doi:10.3389/fmicb.2020.595410

Cited by 5 publications

(4 citation statements)

References 86 publications

(173 reference statements)

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“…Several recent studies have suggested that unpaired purines or purine-rich sequences play an important role in retroviral gRNA packaging ( Damgaard et al 1998 ; Baig et al 2009 ; Jaballah et al 2010 ; Abd El-Wahab et al 2014 ; Bernacchi et al 2017 ; Mustafa et al 2018 ; Wu et al 2018 ; Ali et al 2020 ; Nikolaitchik et al 2020 ; Chameettachal et al 2021 ; Pillai et al 2021 ; Pitchai et al 2021 ; Umunnakwe et al 2021 ). Therefore, to understand the role of purines in the packaging of FIV gRNA, we introduced substitution mutations in several stretches of purine-rich regions of the FIV packaging signal RNA whose secondary structure was previously published ( Kenyon et al 2011 ).…”

Section: Resultsmentioning

confidence: 99%

“…This is because many of these nucleotides may act synergistically, and mutating a single or a short stretch of unpaired Gs may not significantly affect RNA packaging. Furthermore, in the case of MPMV, it has been demonstrated that two stretches of purines act redundantly in RNA packaging ( Ali et al 2020 ; Pitchai et al 2021 ). Consequently, mutating one motif may not lead to a loss of packaging due to compensation by the other region.…”

Section: Discussionmentioning

confidence: 99%

“…The cytoplasmic and viral RNAs were treated with DNase (Turbo DNase, Thermo Fisher Scientific) and tested for the presence of DNA using FIV-specific primers (OTR 660 and OTR 662, Supplemental Table S1) to ensure that the RNA preparations were not contaminated with any residual plasmid DNA from the process of transfection. Next, the DNased-RNAs were reverse transcribed using random hexamers (5 ′ NNNNNN 3 ′ ) and M-MLV reverse transcriptase (Promega), as previously described (Ali et al 2020;Chameettachal et al 2021;Pillai et al 2021). In order to ensure proper fractionation and the quality of the cDNAs prepared, multiplex RT-PCR was performed using unspliced β-actin primers FIV genomic RNA packaging www.rnajournal.org 81 (Tan et al 1995; Supplemental Table S1) as well as primers for 18S rRNA (18S Quantum competimer control, Ambion), as described previously (Mustafa et al 2005(Mustafa et al , 2012Ghazawi et al 2006;Rizvi et al 2010).…”

Section: Nucleocytoplasmic Fractionation Isolation Of Rna and Cdna Pr...mentioning

confidence: 99%

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Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging

Krishnan,

Ali,

Prabhu

et al. 2023

RNA

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The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we have mapped the FIV ψ to two discontinuous regions within the 5’ end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences employing genetic, biochemical, and structure-function relationship approaches. Our mutational analysis revealed that the unpaired U85CUG88 stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput Selective 2' Hydroxyl Acylation analyzed by Primer Extension (hSHAPE) performed on wild type and mutant FIV ψ sequences with substitutions in the U85CUG88 stretch revealed that these mutations had limited structural impact and maintained nucleotides 80 to 92 unpaired, as in the wild type structure. Since these mutations dramatically affected packaging, our data suggests that the single-stranded U85CUG88 sequence is important during FIV RNA packaging. Filter binding assays performed using purified FIV Pr50Gag on wild type and mutant U85CUG88 ψ RNAs led to reduced levels of Pr50Gag binding to mutant U85CUG88 ψ RNAs, indicating that the U85CUG88 stretch is crucial for ψ RNA-Pr50Gag interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as development of FIV-based vectors for human gene therapy.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Nucleocytoplasmic Fractionation Isolation Of Rna and Cdna Pr...mentioning

confidence: 99%

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Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging

Krishnan,

Ali,

Prabhu

et al. 2023

RNA

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“…Packaging signals are commonly located at the 5 end of retroviral gRNAs. They are formed by several stem-loops including the dimerization initiation site (DIS) that mediates base-pairing between two copies of gRNA, and display stretches of unpaired purines that might be potential binding sites for NC [87][88][89]. Moreover, genomic dimerization is thought to be a general prerequisite for retroviral gRNA packaging and, accordingly, aberrant gRNA dimerization was found to impair viral assembly (reviewed in [90,91]).…”

Section: Retroviral Packaging Signals and Grna Dimerizationmentioning

confidence: 99%

Visualization of Retroviral Gag-Genomic RNA Cellular Interactions Leading to Genome Encapsidation and Viral Assembly: An Overview

Bernacchi

2022

Viruses

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Retroviruses must selectively recognize their unspliced RNA genome (gRNA) among abundant cellular and spliced viral RNAs to assemble into newly formed viral particles. Retroviral gRNA packaging is governed by Gag precursors that also orchestrate all the aspects of viral assembly. Retroviral life cycles, and especially the HIV-1 one, have been previously extensively analyzed by several methods, most of them based on molecular biology and biochemistry approaches. Despite these efforts, the spatio-temporal mechanisms leading to gRNA packaging and viral assembly are only partially understood. Nevertheless, in these last decades, progress in novel bioimaging microscopic approaches (as FFS, FRAP, TIRF, and wide-field microscopy) have allowed for the tracking of retroviral Gag and gRNA in living cells, thus providing important insights at high spatial and temporal resolution of the events regulating the late phases of the retroviral life cycle. Here, the implementation of these recent bioimaging tools based on highly performing strategies to label fluorescent macromolecules is described. This report also summarizes recent gains in the current understanding of the mechanisms employed by retroviral Gag polyproteins to regulate molecular mechanisms enabling gRNA packaging and the formation of retroviral particles, highlighting variations and similarities among the different retroviruses.

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Identification of Pr78Gag Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants

Pitchai

Chameettachal

Vivet-Boudou

et al. 2021

Journal of Molecular Biology

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Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation

Cited by 5 publications

References 86 publications

Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging

Identification of a putative Gag binding site critical for feline immunodeficiency virus genomic RNA packaging

Visualization of Retroviral Gag-Genomic RNA Cellular Interactions Leading to Genome Encapsidation and Viral Assembly: An Overview

Identification of Pr78Gag Binding Sites on the Mason-Pfizer Monkey Virus Genomic RNA Packaging Determinants

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