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

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

doi:10.3389/fmicb.2020.595410

Abstract

A distinguishing feature of the Mason-Pfizer monkey virus (MPMV) packaging signal RNA secondary structure is a single-stranded purine-rich sequence (ssPurines) in close vicinity to a palindromic stem loop (Pal SL) that functions as MPMV dimerization initiation site (DIS). However, unlike other retroviruses, MPMV contains a partially base-paired repeat sequence of ssPurines (bpPurines) in the adjacent region. Both purine-rich sequences have earlier been proposed to act as potentially redundant Gag binding sites to initiate the process of MPMV genomic RNA (gRNA) packaging. The objective of this study was to investigate the biological significance of ssPurines and bpPurines in MPMV gRNA packaging by systematic mutational and biochemical probing analyses. Deletion of either ssPurines or bpPurines individually had no significant effect on MPMV gRNA packaging, but it was severely compromised when both sequences were deleted simultaneously. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) analysis of the mutant RNAs revealed only mild effects on structure by deletion of either ssPurines or bpPurines, while the structure was dramatically affected by the two simultaneous deletions. This suggests that ssPurines and bpPurines play a redundant role in MPMV gRNA packaging, probably as Gag binding sites to facilitate gRNA capture and encapsidation. Interestingly, the deletion of bpPurines revealed an additional severe defect on RNA propagation that was independent of the presence or absence of ssPurines or the gRNA structure of the region. These findings further suggest that the bpPurines play an additional role in the early steps of MPMV replication cycle that is yet to be identified.

Highlights

Retroviruses are versatile mobile genetic elements that have historically been used to study the regulation of eukaryotic gene expression (Pachori et al, 2001; Gong et al, 2007; Ly et al, 2007, 2008; Lyon et al, 2008; Yang et al, 2010)
The current study was undertaken to establish the role of ssPurines and its base-paired partial repeat, bpPurines, in Mason-Pfizer monkey virus (MPMV) genomic RNA (gRNA) packaging and propagation
The SHAPE-validated structure of MPMV packaging signal RNA revealed a stretch of ssPurines, as well as a partial repeat in the form of bpPurines (Aktar et al, 2013)

Summary

Introduction

Retroviruses are versatile mobile genetic elements that have historically been used to study the regulation of eukaryotic gene expression (Pachori et al, 2001; Gong et al, 2007; Ly et al, 2007, 2008; Lyon et al, 2008; Yang et al, 2010). Retroviral packaging signal RNAs (known as psi-ψ) assume higher-order structures and are confined to the first ∼100 to ∼400 nucleotides (nt) of the gRNA that invariably extend into the gag gene (Berkhout and van Wamel, 1996; Das et al, 1997; Jewell and Mansky, 2000; Clever et al, 2002; Browning et al, 2003; D’Souza and Summers, 2005; Mustafa et al, 2005; Lever, 2007; Kenyon et al, 2008, 2011; Johnson and Telesnitsky, 2010; Rizvi et al, 2010; Jouvenet et al, 2011; Ali et al, 2016; Comas-Garcia et al, 2016; Kaddis Maldonado and Parent, 2016; Mailler et al, 2016; Dubois et al, 2018). Based on the cross- and co-packaging studies among distinct retroviruses, it has been proposed that structural motifs (rather than their primary sequence) are crucial during gRNA packaging (Embretson and Temin, 1987; Rizvi and Panganiban, 1993; Yang and Temin, 1994; Das et al, 1997; Yin and Hu, 1997; White et al, 1999; Browning et al, 2001; Balvay et al, 2007; Al Dhaheri et al, 2009, Al Shamsi et al, 2011)

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