TY - JOUR
T1 - The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA
AU - Mustafa, Farah
AU - Vivet-Boudou, Valérie
AU - Jabeen, Ayesha
AU - Ali, Lizna M.
AU - Kalloush, Rawan M.
AU - Marquet, Roland
AU - Rizvi, Tahir A.
N1 - Funding Information:
This research was funded by a joint grant from the United Arab Emirates University (UAEU; Zayed Center for Health Sciences) and Terry Fox Funds for Cancer Research grants (fund codes 31R020 and 21M095 respectively) and College of Medicine & Health Sciences grant (NP-14-34) to TAR and in part from and UAEU-National Research Foundation grant (31M101) and UAEU CMHS grant 31M331 to FM .
Funding Information:
This research was funded by a joint grant from the United Arab Emirates University (UAEU; Zayed Center for Health Sciences) and Terry Fox Funds for Cancer Research grants (fund codes 31R020 and 21M095 respectively) and College of Medicine & Health Sciences grant (NP-14-34) to TAR and in part from and UAEU-National Research Foundation grant (31M101) and UAEU CMHS grant 31M331 to FM. Authors would like to thank Ms. Faiza Moureen, Dr. Jaleel Kizhakkayil, and Dr. Suriya Aktar (Department of Microbiology & Immunology, CMHS, UAEU) for their help in some of the cloning and experimental procedures at the initial stages of the project as a part of their training and Dr Fabrice Jossinet for his help in drawing the secondary structure models of Figures 3 and 5 with Assemble. The authors would also like to thank the editorial assistance of Akhil Chameettachal, Fathima Nuzra Nagoor Pitchai, and Vineeta Pillai (Department of Microbiology & Immunology, CMHS, UAEU) for reading the manuscript and providing their inputs.
Publisher Copyright:
© 2018, © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
PY - 2018/8/3
Y1 - 2018/8/3
N2 - 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.
AB - 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.
KW - Mouse mammary tumor virus
KW - RNA packaging and dimerization
KW - RNA secondary structure
KW - Retroviruses
KW - palindrome
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U2 - 10.1080/15476286.2018.1486661
DO - 10.1080/15476286.2018.1486661
M3 - Article
C2 - 29929424
AN - SCOPUS:85049569851
SN - 1547-6286
VL - 15
SP - 1047
EP - 1059
JO - RNA Biology
JF - RNA Biology
IS - 8
ER -