We have used systematic deletion analysis of the 5′ untranslated region (UTR) of the feline immunodeficiency virus (FIV) genome, both in the presence and absence of various amounts of gag, to define the cis-acting sequences responsible for efficient RNA packaging. Our analyses revealed that the primary FIV packaging signal consists of two essential core elements located within the first 90–120 bp of the 5′UTR and the first 90 bp of the gag gene. Interestingly, the region between the major splice donor (SD) and gag, including ∼130–160 bp upstream of the SD, is dispensable for encapsidation. Finally, other determinants of packaging were found to be present in the viral LTR and/or within the 3′ end of the viral genome. Taken together, our results suggest that the primary packaging determinants of FIV are multipartite and discontinuous, composed of two elements within the 5′UTR and gag gene.
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