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

Abstract

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)

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

“…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.…”

“…hSHAPE experiments [64–66] were performed on in vitro transcribed RNAs corresponding to nucleotides 1 to 713 using benzoyl cyanide (BzCN) and following the protocol described previously [25,33,57]. Briefly, RNAs were modified in a buffer favoring gRNA dimerization (50 mM sodium cacodylate (pH 7.5), 300 mM KCl and 5 mM MgCl 2 ) in the presence of 2 µg total yeast tRNA (Sigma Aldrich).…”

The Life-Cycle of the HIV-1 Gag–RNA Complex

“…Evidence for this is provided by the observation that the duplication of the HIV-1 packaging site in one full length RNA leads to the packaging of the monomer with an intramolecular interaction [ 109 ]. Indeed, the dimerization and encapsidation of HIV-1 gRNA seem to be strongly interrelated processes [ 76 , 84 , 97 , 110 , 111 , 112 , 113 , 114 ], and this feature is observed in all retroviruses studied to date [ 9 , 18 , 115 , 116 , 117 ]. This has given rise to several models where conformational changes in the gRNA regulate Gag binding through dimerization.…”

Cross- and Co-Packaging of Retroviral RNAs and Their Consequences

“…For example, the poly(A) stem loop of HIV-1 is involved in long-range interactions (LRIs) with sequences in the matrix (MA), forming a pseudoknot [71]. Similarly, the U5 stem loop sequences are involved in long-range interactions (LRI) with the stem loop containing the Gag AUG in many retroviruses, including HIV-1 and 2, SIV, FIV, MMTV, and MPMV [69,70,71,72,73,74,75,76,77,78,79], as well as other plus-strand RNA viruses with icosahedral capsids [80]. Mutations that destabilize these interactions affect several important steps in the retroviral replication cycle, including RNA packaging and dimerization [10,48,49,71,72,79,81,82].…”

“…Detailed RNA structural determinants of two important beta retroviruses, MMTV and MPMV, as well as lentiviruses such as HIV-1 and FIV have recently been reported as employing selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) followed by validation of predicted structural motifs for RNA packaging and dimerization using mutational analysis in a biologically relevant replication assay [69,70,74,76,79,82,83]. Emerging themes from these and other studies (reviewed in [3,8,33]) on RNA packaging can be summarized as follows:…”