A CONSERVED HAIRPIN MOTIF IN THE R-U5 REGION OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RNA GENOME IS ESSENTIAL FOR REPLICATION

The untranslated leader region of the human immunodeficiency virus (HI V) RNA genome contains multiple hairpin motifs. The repeat region of t he leader, which is reiterated at the 3' end of the RNA molecule, enco des the well-known TAR hairpin and a second hairpin structure with the polyadenylation signal AAUAAA in the single-stranded loop [the poly(A ) hairpin]. The fact that this poly(A) stem-loop structure and its the rmodynamic stability are well conserved among HIV and simian immunodef iciency virus isolates, despite considerable divergence in sequence, s uggests a biological function for this RNA motif in viral replication. Consistent with this idea, we demonstrate that mutations that alter t he stability of the stem region or delete the upper part of the hairpi n do severely inhibit replication of HIV type 1. Whereas destabilizing mutations in either the left- or right-hand side of the base-paired s tem interfere with virus replication, the double mutant, which allows the formation of new base pairs, replicates more rapidly than the two individual virus mutants. Upon prolonged culturing of viruses with an altered hairpin stability, revertant viruses were obtained with additi onal mutations that restore the thermodynamic stability of the poly(A) hairpin. Transient transfection experiments demonstrated that transcr iption of the proviral genomes, translation of the viral mRNAs, and re verse transcription of the genomic RNAs are not affected by mutation o f the 5' poly(A) hairpin. We show that the genomic RNA content of the virions is reduced by destabilization of this poly(A) hairpin but not by stabilization or truncation of this structure, These results sugges t that the formation of the poly(A) hairpin structure at the 5' end of the genomic RNA molecule is necessary for packaging of viral genomes into virions and/or stability of the virion RNA.