TY - JOUR
T1 - Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag
AU - Rizvi, Tahir A.
AU - Kenyon, Julia C.
AU - Ali, Jahabar
AU - Aktar, Suriya J.
AU - Phillip, Pretty S.
AU - Ghazawi, Akela
AU - Mustafa, Farah
AU - Lever, Andrew M.L.
N1 - Funding Information:
This research was funded primarily by a grant from the Wellcome Trust (078007/Z/05/Z to T.A.R. and A.M.L.L.) and, in part, by grants from the Terry Fox Fund for Cancer Research, Sheikh Hamdan Award for Medical Sciences, and Research Affairs of the United Arab Emirates University to T.A.R. We express our thanks to Dr Didier Trono (Ecole Polytechnique Fédérale de Lausanne, Switzerland) for providing MD.G. and Dr Ellen Collisson (Texas A&M University, College Station, TX) for providing polyclonal anti-serum from a cat infected with the Petaluma strain of FIV. The FIV molecular clone p34TF10 was obtained from the AIDS Research and Reference Reagent Program of the National Institutes of Health. We thank Ms Noura Salem Al Dhaheri and Ms Soumeya Ali Jaballah (Department of Microbiology & Immunology, FMHS, UAE University) for stimulating discussions and critical reading of the manuscript. We thank Ms Nourah Al Shamsi and Ms Amna Al Neyadi for their assistance in cloning some of the transfer vectors as a part of their curricular research program.
PY - 2010/10/15
Y1 - 2010/10/15
N2 - 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.
AB - 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.
KW - Feline immunodeficiency virus (FIV)
KW - Lentiviral vectors and gene therapy
KW - Long-range interaction
KW - Palindromic sequences (pal)
KW - Retroviral RNA packaging
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U2 - 10.1016/j.jmb.2010.08.019
DO - 10.1016/j.jmb.2010.08.019
M3 - Article
C2 - 20732330
AN - SCOPUS:77957260171
SN - 0022-2836
VL - 403
SP - 103
EP - 119
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -