Role of stem B, loop B, and nucleotides next to the primer binding site and the kissing-loop domain in human immunodeficiency virus type 1 replication and genomic-RNA dimerization

Stem-loop B is a 12-nucleotide [nt] -long completely conserved sequence pos tulated to form a 4-bp stem and a 4-nt internal loop under the kissing-loop hairpin (klh) (nt 248 to 270) of human immunodeficiency virus type 1 (HIV- 1) genomic RNA. We investigated its role in viral replication, genomic RNA dimerization, and dimerization of partial HIV-1 RNA transcripts. The putati ve CUCG246-CGAG277 duplex was replaced by nine alternative complementary se quences, five likely to base pair only in short RNAs and four likely to bas e pair in long (similar to 500-nt) RNAs, as assessed by the algorithm mfold . Among the five former sequences, none preserved genome dimerization and a ll reduced viral replication by 98 to 99.9%. Among the four latter sequence s, three (MB6, -9, and -10) preserved genome dimerization, one (MB7) did no t significantly inhibit it, and two (MB9 and -10) preserved viral replicati on. We conclude that duplex formation by stem B nucleotides is necessary fo r viral infectivity and complete genome dimerization. Deleting the 5' or 3' side of loop B or of stem B had little impact on dimerization of partial R NA transcript and no impact on klh folding (and, for loop B mutations, on s tem B folding), but each deletion inhibited genome dimerization almost as m uch as klh destruction. This suggests that loop B is required for complete genome dimerization and that loop B and stem B stimulate dimerization only in very long RNAs and/or in the presence of unidentified viral and cellular factors. Finally, we asked if nine deletions or nucleotide substitutions w ithin nt 200 to 242 and/or nt 282 to 335 could influence genome dimerizatio n. These mutations had intermediate inhibitory impacts consistent with thei r predicted influence on stem B, loop B, and klh formation. Two exceptions were Delta 200-226 and Delta 236-242 genomic RNAs, which dimerized relative ly poorly despite having neutral or positive influences on stem B, loop B, and klh folding.