The secondary structure of the 5′ end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem–loop

Abstract

The secondary structure of the 59 end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem-loop JULIA C. KENYON ABSTRACT Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV).Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem-loops and a conserved long-range interaction between heptanucleotide sequences 59-CCCUGUC-39 in R/U5 and 59-GACAGGG-39 in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in speciesspecific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 59 UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.Keywords: FIV; lentivirus; packaging signal; dimerization; RNA INTRODUCTIONFeline immunodeficiency virus (FIV) is a lentivirus related to human immunodeficiency virus (HIV), the causative agent of AIDS (Olmsted et al. 1989;Talbott et al. 1989). Like HIV, FIV causes in its host, the domestic cat, a prolonged disease that is characterized by progressive depletion of CD4 + T cells, ultimately leading to a fatal immunodeficiency (Pedersen et al. 1987;Yamamoto et al. 1988;Siebelink et al. 1990). Many other parallels exist between the two viruses and these render FIV infection in cats a potentially useful small animal model for HIV (Olmsted et al. 1989; and reviewed in Elder et al. 2008). In addition to this, FIV is being developed as a human gene therapy vector as, like all lentiviruses, it is able to transduce nondividing cells while being associated with fewer safety concerns than primate lentiviral vectors (reviewed in Saenz and Poeschla 2004). FIV also poses a serious veterinary concern in its own right, as it infects domestic cats and big cats alike, with a worldwide distribution and a high seroprevalence (Troyer et al. 2005). A better understanding of the mechanisms of FIV replication is needed in order to utilize the virus to its full potential as an HIV model or as a gene therapy vector, and to seek ways to combat its spread in feline species. Examining the structure of functionally important regions of the FIV RNA will not only aid this, but may also enhance our understand...

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

“…These results suggest that LRI-I and LRI-II play an important architectural role in stabilizing the RNA secondary structure of the 5 ′ -UTR sequences required for MPMV gRNA packaging (Schmidt et al 2003;Jaballah et al 2010). Such a scenario could be comparable to that observed in other retroviruses (HIV-1, HIV-2, and FIV) where interactions between the R/U5 regions and the start of gag sequences have been proposed to stabilize the overall RNA secondary structure essential for gRNA dimerization and packaging (Paillart et al 2002;Kenyon et al 2008Kenyon et al , 2011Song et al 2008;Rizvi et al 2010;Lu et al 2011b).…”

“…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.…”

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).…”

“…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.…”