Abstract

Equine infectious anemia virus (EIAV), with the simplest lentiviral genome, is a key model for studying fundamental lentiviral biology. Infectious viral particles are produced only when the Gag protein selectively encapsidates full-length genomic RNA via the packaging signal (Psi), yet the structural and functional features of EIAV Psi remain poorly characterized. Using computational prediction and dimethyl sulfate probing, we identified four stem-loops (SLs) within a ~120 nt region in the 5′ leader of the genome, spanning from downstream of the primer binding site through 20 nt into thegagcoding sequence. In vitro dimerization assays demonstrated that a palindromic sequence (5′-CUGGCCAG-3′) within SL3 acts as a critical determinant of RNA dimerization. Functional screening using both an EIAV pseudovirus packaging system and the infectious clone EIAVuk revealed that deletion or mutation of the stem-loops significantly impairs viral packaging and replication, with SL2 deletion or its stem disruption causing the most severe defects. RNA-seq analysis of RNAs bound by wild-type Gag versus a zinc-finger mutant (H391K/H410K) identified two candidate Gag-associated sites: the SL2 stem and the SL2-SL3 junction. Targeting these regions with phosphorothioate-modified antisense oligonucleotides potently inhibited pseudovirus production and the replication of infectious EIAVuk. Our findings defined the secondary structure and functional organization of the EIAV core packaging region and established the SL2 stem and SL2-SL3 junction as candidate packaging determinants and promising targets for RNA-based antiviral intervention.