A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag.

Akhil Chameettachal,Vineeta N Pillai,Anjana Krishnan,Farah Mustafa,Roland Marquet,Tahir A Rizvi,Valérie Vivet-Boudou,Lizna Mohamed Ali,Fathima Nuzra Nagoor Pitchai,Serena Bernacchi

doi:10.1093/nar/gkab223

Abstract

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem–loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.

Highlights

Viruses consist of a protein shell that encloses a genome that can either be DNA or RNA
dynamic light scattering (DLS) experiments revealed that the mean hydrodynamic radius (Rh) based on number distribution was estimated to be ∼6.00 nm, which corresponds to Pr77Gag trimers (Supplementary Figure S2)
Most reported studies on gRNA packaging in the literature have been performed on human immunodeficiency virus type 1 (HIV-1), where purinerich apical or internal loops in hairpins structures have been proposed to govern gRNA packaging by functioning as Gag binding sites

Summary

Introduction

Viral structural proteins have the ability to recognize their genome and ‘package’ it into the assembling virus particles. They must incorporate their genomes into the virus particles with high specificity to ensure continuity of their life cycle. Retroviruses belong to a special class of viruses that use RNA as their genome (full length unspliced gRNA), which harbors cis-acting packaging sequences (called psi, ), that facilitate specific encapsidation of the gRNA [1,2,3,4,5,6,7,8]

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