MUTATIONS IN THE KISSING-LOOP HAIRPIN OF HUMAN-IMMUNODEFICIENCY-VIRUSTYPE-1 REDUCE VIRAL INFECTIVITY AS WELL AS GENOMIC RNA PACKAGING AND DIMERIZATION

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Bec ause it plays a key role in the in vitro dimerization of short HIV-1 R NA transcripts (M. Laughrea and L. Jette, Biochemistry 35:1589-1598, 1 996, and references therein; M. Laughrea and L. Jette, Biochemistry 35 :9366-9374, 1996, and references therein) and because dimeric RNAs may be preferably encapsidated into the HIV-1 virus, alterations of the k issing-loop hairpin might affect the in vivo dimerization and encapsid ation processes. Accordingly, substitution and deletion mutations were introduced into the kissing-loop hairpin of an infectious HIV-1 molec ular clone in order to produce viruses by transfection methods. The in fectivity of the resulting viruses was decreased by at least 99%, the amount of genomic RNA packaged per virus was decreased by 50 to 75%, a nd the proportion of dimeric genomic RNA was reduced from >80 to 40 to 50%, but the dissociation temperature of the genomic RNA was unchange d, There is evidence suggesting that the deletion mutations moderately inhibited CAp24 production but had no significant effect on RNA splic ing. These results are consistent with the kissing-loop model of HIV-1 RNA dimerization. In fact, because intracellular viral RNAs are proba bly more concentrated in transfected cells than in cells infected by o ne virus and because the dimerization and encapsidation processes are concentration dependent, it is likely that much larger dimerization an d encapsidation defects would have been manifested within cells infect ed by no more than one virus.