A G–C-Rich Palindromic Structural Motif and a Stretch of Single-Stranded Purines Are Required for Optimal Packaging of Mason–Pfizer Monkey Virus (MPMV) Genomic RNA

Jaballah, Soumeya Ali; Aktar, Suriya J.; Ali, Jahabar; Phillip, Pretty Susan; Dhaheri, Noura Salem Al; Jabeen, Aayesha; Rizvi, Tahir A.

doi:10.1016/j.jmb.2010.06.043

Cited by 20 publications

(78 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The specificity for gRNA during packaging, thus, has been ascribed to high-affinity interactions between the NC domain of Gag and the packaging signal [5–13]. We have made a similar observation in the case of MPMV, where genetic and structure-prediction analyses have suggested that either ssPurines (Region A) or its partially repeated but base-paired sequence (Region B) plays a crucial role in gRNA packaging, possibly by functioning as a NC binding site [32,33]. …”

Section: Discussionmentioning

confidence: 70%

“…Following transfection, cultured cells were fractionated into nuclear and cytoplasmic fractions, as described previously [24,25,32,57,59,62,63]. Briefly, packaged viral RNA from the pelleted viral particles and cellular RNAs from the cytoplasmic fractions were isolated using Trizol and Trizol LS reagents (Invitrogen), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The presence of a stretch of purines in the packaging sequences on retroviral gRNA has been proposed to facilitate RNA packaging by functioning as a potential NC binding site [26–31]. It is also interesting to note that the positioning of the purine-rich loop in MMTV adjacent to the pal II loop is reminiscent of the situation found in Mason-Pfizer monkey virus (MPMV) [32,33]. In the case of MPMV, genetic and structure-prediction analyses have suggested that either ssPurines or its partially-repeated base-paired sequence in an adjacent region plays a crucial role in gRNA packaging, possibly by functioning as a NC binding site [32,33].…”

Section: Introductionmentioning

confidence: 99%

“…It is also interesting to note that the positioning of the purine-rich loop in MMTV adjacent to the pal II loop is reminiscent of the situation found in Mason-Pfizer monkey virus (MPMV) [32,33]. In the case of MPMV, genetic and structure-prediction analyses have suggested that either ssPurines or its partially-repeated base-paired sequence in an adjacent region plays a crucial role in gRNA packaging, possibly by functioning as a NC binding site [32,33]. Therefore, it is reasonable to propose that the bifurcated SL4 structure in MMTV functions as a major dimerization and packaging determinant during MMTV gRNA packaging process with pal II initiating the process of dimerization and ssPurines facilitating the encapsidation of the dimerized RNA into the virus particles by functioning as a potential Gag binding site.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 2018

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 70%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, the significance of these structural motifs to gRNA packaging remains largely unclear. One such characteristic feature of retroviral gRNA packaging signals is the long-range interaction (LRI) involving sequences in R/U5 that are complementary to a sequence found downstream in the gag gene (Paillart et al 2002(Paillart et al , 2004bAbbink and Berkhout 2003;Kenyon et al 2008;Song et al 2008;Jaballah et al 2010;Aktar et al 2013Aktar et al , 2014.…”

Section: Introductionmentioning

confidence: 99%

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

Kalloush¹,

Vivet-Boudou²,

Ali³

et al. 2016

RNA

Self Cite

View full text Add to dashboard Cite

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2 ′ hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5 ′ region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. Keywords: retroviruses; Mason-Pfizer monkey virus (MPMV); RNA secondary structure; RNA packaging and dimerization; long-range interactions (LRI); SHAPE (selective 2 ′ hydroxyl acylation analyzed by primer extension)

show abstract

References

2022

Structures and Functions of Retroviral RNAs

View full text Add to dashboard Cite

A G–C-Rich Palindromic Structural Motif and a Stretch of Single-Stranded Purines Are Required for Optimal Packaging of Mason–Pfizer Monkey Virus (MPMV) Genomic RNA

Cited by 20 publications

References 56 publications

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

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

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

References

Contact Info

Product

Resources

About