Optimal Packaging of FIV Genomic RNA Depends upon a Conserved Long-range Interaction and a Palindromic Sequence within gag

Abstract

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5′ 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5′ and 3′ sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem–loop (SL2) and a small palindromic stem–loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8–5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5–3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.

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

“…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]. 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].…”

“…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.…”

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

“…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). Similarly, an earlier study on MPMV has shown the importance of gag sequences (involved in maintaining U5-gag LRIs) in RNA packaging, further arguing in favor of the existence of LRIs in vivo and their functional role in the MPMV life cycle (Schmidt et al 2003).…”

“…Nucleocytoplasmic fractionation, virion and RNA isolation, and cDNA preparation Transfected cells were fractionated into cytoplasmic and nuclear fractions and RNA extracted as described earlier (Mustafa et al 2005;Ghazawi et al 2006;Jaballah et al 2010;Rizvi et al 2010). Packaged viral RNAs were prepared from clarified supernatants from transfected cultures after filtration through 0.2 µm filters as described previously (Mustafa et al 2005;Ghazawi et al 2006;Jaballah et al 2010;Rizvi et al 2010).…”

“…Packaged viral RNAs were prepared from clarified supernatants from transfected cultures after filtration through 0.2 µm filters as described previously (Mustafa et al 2005;Ghazawi et al 2006;Jaballah et al 2010;Rizvi et al 2010). RNA preparations were DNase-treated and subjected to 30-cycle PCR to test for the presence of any residual contaminating plasmid DNA using MPMV specific primer pair: OTR 1161 (S; 5 ′ -GATCAGAACACTGTCTTGTC-3 ′ ) and OTR 1163 (AS; 5 ′ -CTT TCTTATCTATCAATTCTTTAA-3 ′ ) as described previously (Jaballah et al 2010).…”