Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag

Tahir A. Rizvi, Julia C. Kenyon, Jahabar Ali, Suriya J. Aktar, Pretty S. Phillip, Akela Ghazawi, Farah Mustafa, Andrew M.L. Lever

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)103-119
Number of pages17
JournalJournal of Molecular Biology
Volume403
Issue number1
DOIs
Publication statusPublished - Oct 15 2010

Keywords

  • Feline immunodeficiency virus (FIV)
  • Lentiviral vectors and gene therapy
  • Long-range interaction
  • Palindromic sequences (pal)
  • Retroviral RNA packaging

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Molecular Biology

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