POSITION DEPENDENCE OF FUNCTIONAL HAIRPINS IMPORTANT FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RNA ENCAPSIDATION IN-VIVO

At least two hairpins in the 5' untranslated leader region, stem-loops 1 and 3 (SL1 and SL3), contribute to human immunodeficiency virus typ e 1 RNA encapsidation in vivo. We used a competitive assay, which meas ures the relative encapsidation efficiency of mutant viral RNA in the presence of competing wild-type RNA, to compare the contributions of S L1, SL3, and two adjacent secondary structures, SL2 and SL4, to encaps idation. SL2 is not required for RNA encapsidation, while SL1, SL3, an d SL4 all contribute approximately equally to encapsidation, To determ ine whether these hairpins function in a position dependent manner, we interchanged the positions of two of these stem-loop structures. This resulted in substantial diminution of encapsidation, indicating that the secondary structures that comprise E, the encapsidation signal, fu nction only in their correct contests. Mutation of nucleotides flankin g SL1 and SL3 had little effect on encapsidation, me also showed that SL1, while present on both genomic and subgenomic viral RNAs, nonethel ess contributes to selective encapsidation of genomic RNA. Taken toget her, these data are consistent with the formation of a higher-order RN A structure, partially composed of SL1, SL3, and SL4, that functions t o effect concurrent encapsidation of full-length RNA and exclusion of subgenomic RNA, Finally, it has been reported that E is required for e fficient translation of Gag mRNA in vivo, However, we have found that a variety of mutants, including a mutant lacking the entire region enc ompassing SL1, SL2, and SL3, still produce RNAs that are efficiently t ranslated. These data indicate that E is unlikely to contribute to eff icient Gag mRNA translation in vivo.