Variant effects of non-native kissing-loop hairpin palindromes on HIV replication and HIV RNA dimerization: Role of stem-loop B in HIV replication and HIV RNA dimerization

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. In vitro synthesized RNAs from human immunodefic iency virus type 1 (HIV-1) can form loose or tight dimers depending on whet her their respective kissing-loop hairpins (nts 248-270 in HIV-1(Lai)) bond via their hexameric autocomplementary sequences (ACS), also called palindr omes, or via the ACS and stem sequences [Laughrea, M., and Jette, L. (1996) Biochemistry 35, 1589-1598]. To understand the role of the ACS in HIV-1 re plication and in the formation and stability of HIV-1 RNA dimers, we replac ed the central CGCG261(or tetramer) of the HIV-1(Lai) ACS by two other HIV- 1 tetramers (UGCA/ UGCG), four non-HIV-1 tetramers [GUAC, UUAA (respectivel y found in HIV-2(Rod) and SIVmnd), GGCC and AGCU (absent from HIV and SIV v iruses)], or GGCG, a nonpalindromic tetramer. The infectivity of GGCC, GUAC , and UGCA viruses was unchanged or insignificantly decreased; the infectiv ity of AGCU and UGCG viruses was decreased by 80%; the infectivity of UUAA and GGCG viruses was decreased by 92-98%. Thus, the four non-HIV-1 palindro mes yielded phenotypes ranging from wild-type to as defective as a virus be aring a nonpalindrome. Studies of in vitro synthesized HIV-1 RNAs were gene rally consistent with in vivo results, specifically: (i) loose dimerization of GGCC and GUAC RNAs, but not of UUAA and AGCU RNAs, was influenced by th e 3' DLS (a sequence located downstream of the 5' splice junction) in a way expected for a wild-type ACS; (ii) the 3' DLS strongly reduced tight dimer ization of UUAA and AGCU RNAs, but not of GGCC and GUAC RNAs. We conclude t hat HIV-1 is sensitive to the ACS sequence without discriminating against a ll nonnative ACS: GGCC/GUAC, but not AGCU/ UUAA, are good substitutes for t he prevalent CGCG/UGCA native tetramers and better substitutes than the ver y rare UGCG native tetramer. The correlation between in vivo and in vitro r esults suggests that in vitro assays measure parameters of in vivo relevanc e. Deletion of CUCGG247 (the 5' strand of stem-loop B) decreased the replic ative capacity by more than 99.9% and metamorphosed the 3' DLS into an inhi bitor of the loose dimerization of HIV-1 RNA.