The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

Abstract

Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5ʹ untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging

“…This assay employs primers and probes in a region common to both the wild type and mutant RNAs (nts 702-770; see Supplemental Table 1 for primers and probe details), ensuring perfect complementarity with all target RNAs [20]. Finally, a commercially-available human β-actin assay was used as an endogenous control, as described previously [20,34,37,38].…”

“…In order to correlate the effects of the introduced mutations in the MPMV packaging signal RNA with their secondary structure, the 5´end of the wild type and mutant transfer vector RNAs (region between R and the first 120 nts of gag) were subjected to hSHAPE chemical probing [64][65][66] following the protocol described previously [15,20,34,38]. Briefly, in vitro transcribed RNAs were folded in a buffer favoring gRNA dimerization (50 mM sodium cacodylate (pH 7.5), 300 mM KCl and 5 mM MgCl 2 ) and modified with benzoyl cyanide (BzCN) in the presence of 2 μg total yeast tRNA (Sigma Aldrich).…”

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

“…Mfold predicts all optimal and suboptimal RNA secondary structures based on energy matrices by taking into account the minimum free energy of the provided RNA sequence. Next, the predicted structure for each mutant was validated by SHAPE ( Merino et al, 2005 ; Mortimer and Weeks, 2007 , 2009 ; Aktar et al, 2013 , 2014 ; Kalloush et al, 2016 , 2019 ; Mustafa et al, 2018 ).…”

“…The wild-type (RCR001) as well as mutant plasmids created for in vitro RNA transcription (FN series of clones; Figure 2A ) were linearized with an Sma I restriction enzyme located at the 3′ end of the MPMV sequence. The linearized DNA fragments were subjected to in vitro transcription using the bacteriophage T7 RNA polymerase (MEGAscript T7 Transcription kit, Thermo Fischer Scientific) as described earlier ( Mustafa et al, 2018 ; Kalloush et al, 2019 ). A portion of the in vitro transcribed RNA was analyzed on 8% acrylamide/8M urea gels to confirm the absence of abortive transcripts, followed by DNase treatment (Turbo DNA, Thermo Fischer Scientific) of the resulting RNA.…”

“…The presence of purine-rich sequences in retroviral packaging signal RNA has been proposed to facilitate gRNA packaging by functioning as a potential Gag binding site in HIV-1 ( Clever et al, 1995 ; Abd El-Wahab et al, 2014 ; Smyth et al, 2015 , 2018 ; Bernacchi et al, 2017 ), human immunodeficiency virus-2 (HIV-2; Damgaard et al, 1998 ; Baig et al, 2009 ), and mouse mammary tumor virus (MMTV; Aktar et al, 2014 ; Mustafa et al, 2018 ). The precise role of ssPurines in MPMV life cycle has not been validated, despite indirect evidence for their role in MPMV gRNA packaging ( Jaballah et al, 2010 ).…”