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

Author:

Krishnan Anjana,Ali Lizna M.,Prabhu Suresha G.,Pillai Vineeta N.,Chameettachal Akhil,Vivet-Boudou Valérie,Bernacchi Serena,Mustafa Farah,Marquet Roland,Rizvi Tahir A.

Abstract

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 mapped the feline immunodeficiency virus (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 using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed that the unpaired U85CUG88stretch 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 (WT) and mutant FIV ψ sequences, with substitutions in the U85CUG88stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U85CUG88sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50Gagon WT and mutant U85CUG88ψ RNAs led to reduced levels of Pr50Gagbinding to mutant U85CUG88ψ RNAs, indicating that the U85CUG88stretch is crucial for ψ RNA–Pr50Gaginteractions. 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 the development of FIV-based vectors for human gene therapy.

Funder

Zayed Bin Sultan Center for Health Sciences

United Arab Emirates University

RetroPack International Research Project from the CNRS

College of Medicine and Health Sciences, United Arab Emirates University

Abu Dhabi Department of Education and Knowledge (ADEK) ASPIRE

College of Graduate Studies of the UAEU

UAE University UPAR

ASPIRE

the technology program management pillar of Abu Dhabi's Advanced Technology Research Council

the ASPIRE Precision Medicine Research Institute, Abu Dhabi

Publisher

Cold Spring Harbor Laboratory

Subject

Molecular Biology

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