SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization

Abstract

Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem–loops, extensive long-range interactions (LRIs) and a small, palindromic stem–loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem–loops are static structures, the 5′ and 3′ regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem–loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs.

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

“…Thus, packaging signals may act as ‘structural switches’ that favor either translation or dimerization/packaging of the gRNA, and also help the virus differentiate between spliced and unspliced viral RNAs. Several riboswitch models have been proposed for HIV-1 [50–53], and in the case of FIV, the existence of dual structures has been validated by SHAPE and RNA dimerization assays [54]. Flexibility in retroviral gRNA is created via long-range interactions that have now been observed in several retroviruses, from HIV-1, FIV, MLV, to MPMV and MMTV [6,25,26,33,55–59].…”

“…Flexibility in retroviral gRNA is created via long-range interactions that have now been observed in several retroviruses, from HIV-1, FIV, MLV, to MPMV and MMTV [6,25,26,33,55–59]. The DIS in one conformation is occluded by base pairing and not available for dimerization, thus earmarking this RNA for translation, while the DIS in the other conformation is available as an apical loop on a stem loop, thus allowing dimerization and packaging of the dimeric RNA [54]. In a similar manner, we propose that SL4 may be part of an RNA structural switch that may facilitate the ability of MMTV to convert its genomic RNA from a form that favors translation to a dimeric form that may act as a substrate for RNA packaging.…”

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

“…1; Jaballah et al 2010;Aktar et al 2013). LRIs have been found to be conserved in several retroviruses, including HIV-1 and 2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and mouse mammary tumor virus (MMTV) (Paillart et al 2002(Paillart et al , 2004bAbbink and Berkhout 2003;Kenyon et al 2008Kenyon et al , 2011Aktar et al 2014;Siegfried et al 2014;Keane et al 2015;Smyth et al 2015;Tran et al 2015). Despite the fact that there are substantial sequence heterogeneities among human, simian, and feline lentiviruses, the preservation of such LRIs in these lentiviruses suggest they play important function(s) in the retroviral life cycle (Paillart et al 2002;Kenyon et al 2008Kenyon et al , 2011.…”

“…LRIs have been found to be conserved in several retroviruses, including HIV-1 and 2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and mouse mammary tumor virus (MMTV) (Paillart et al 2002(Paillart et al , 2004bAbbink and Berkhout 2003;Kenyon et al 2008Kenyon et al , 2011Aktar et al 2014;Siegfried et al 2014;Keane et al 2015;Smyth et al 2015;Tran et al 2015). Despite the fact that there are substantial sequence heterogeneities among human, simian, and feline lentiviruses, the preservation of such LRIs in these lentiviruses suggest they play important function(s) in the retroviral life cycle (Paillart et al 2002;Kenyon et al 2008Kenyon et al , 2011. A pivotal role of LRIs is further evidenced by the fact that mutations that destabilize these interactions affect several important steps in the retroviral life cycle, including RNA packaging and dimerization (Paillart et al 2002;Abbink and Berkhout 2003;Song et al 2008;Rizvi et al 2010;Lu et al 2011).…”

SHAPE analysis of the 5′ end of the Mason-Pfizer monkey virus (MPMV) genomic RNA reveals structural elements required for genome dimerization

“…The ssPurine-rich region (or its repeat in region "B" when predicted to refold as the ssPurine-rich region) has been shown to be essential for RNA packaging, possibly functioning as a potential NC binding site. Two LRIs between U5 and gag sequences could potentially play a role in MPMV RNA packaging by maintaining the overall RNA secondary structure as has recently been shown in the case of FIV (Kenyon et al 2008(Kenyon et al , 2011Rizvi et al 2010). Even though the deletion analysis of 5 ′ end of MPMV gRNA provides a plausible explanation for the discontinuous nature of MPMV packaging signals, the existence of the different structural motifs of the predicted RNA secondary structure of this region has not been validated experimentally.…”