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

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

doi:10.1016/j.jmb.2021.166923

Abstract

How retroviral Gag proteins recognize the packaging signals (Psi) on their genomic RNA (gRNA) is a key question that we addressed here using Mason-Pfizer monkey virus (MPMV) as a model system by combining band-shift assays and footprinting experiments. Our data show that Pr78Gag selects gRNA against spliced viral RNA by simultaneously binding to two single stranded loops on the MPMV Psi RNA: (1) a large purine loop (ssPurines), and (2) a loop which partially overlaps with a mostly base-paired purine repeat (bpPurines) and extends into a GU-rich binding motif. Importantly, this second Gag binding site is located immediately downstream of the major splice donor (mSD) and is thus absent from the spliced viral RNAs. Identifying elements crucial for MPMV gRNA packaging should help in understanding not only the mechanism of virion assembly by retroviruses, but also facilitate construction of safer retroviral vectors for human gene therapy.

Highlights

Specific selection of the retroviral genome is central to the process of virion assembly, during which a dimeric form of retroviral genomic RNA is selectively packaged into the nascently forming virions [1,2,3,4,5,6]
Our results indicate that the Mason-Pfizer monkey virus (MPMV) polyprotein, Pr78Gag binds to two loops: 1) the ssPurines loop (U191UAAAAGUGAAAGUAA206) and 2) a second loop (A252AGUGU257) corresponding to the last two purines of the bpPurines and extending into a GU-rich region (Figure 1B)
To determine whether the two purine-rich regions important for MPMV genomic RNA (gRNA) packaging, act as binding sites for the Gag precursor polyprotein, large scale expression and purification of Pr78Gag was performed and the protein characterized for its biological function, revealing that it could assemble in vitro to form virus-like particles (VLPs), and form VLPs in bacteria, and that the VLPs produced in eukaryotic cells could encapsidate MPMV RNA containing the packaging signals (Psi) region [62]

Summary

Introduction

Specific selection of the retroviral genome is central to the process of virion assembly, during which a dimeric form of retroviral genomic RNA (gRNA) is selectively packaged into the nascently forming virions [1,2,3,4,5,6]. Despite the fact that the viral gRNA constitutes only ~1% of the total RNA in the cell milieu it is still selected from a vast array of spliced viral and cellular RNAs [7,8,9,10,11,12,13] This highly controlled and selective process is dependent on two important factors: (1) the presence of specific sequences or structures within the gRNA, and (2) the retroviral precursor polyprotein Gag and its ability to identify and bind to these unique sequences or structures [7,9,10,11,12,13,14,15]. These structural motifs are involved in the specific selection of gRNA independent of their primary sequence [15,20,23,24,25,26,27]

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Conclusion