Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.
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.
Department of Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.
Rawan M
RM
Kalloush
Architecture et Réactivité de l'ARN, CNRS, IBMC, Université de Strasbourg, 67084 Strasbourg cedex, France.
Valérie
V
Vivet-Boudou
Department of Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.
Lizna M
LM
Ali
Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.
Farah
F
Mustafa
Architecture et Réactivité de l'ARN, CNRS, IBMC, Université de Strasbourg, 67084 Strasbourg cedex, France.
Roland
R
Marquet
Department of Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.
Tahir A
TA
Rizvi
D015750:Genes, gag; D016093:Mason-Pfizer monkey virus / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D019065:Virus Assembly
D012367:RNA, Viral
Mason-Pfizer monkey virus (MPMV); RNA packaging and dimerization; RNA secondary structure; SHAPE (selective 2′hydroxyl acylation analyzed by primer extension); long-range interactions (LRI); retroviruses