A NATURALLY ARISING MUTATION OF A POTENTIAL SILENCER OF EXON SPLICINGIN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INDUCES DOMINANT ABERRANT SPLICING AND ARRESTS VIRUS PRODUCTION

We have isolated a naturally arising human immunodeficiency type 1 (HI V-1) mutant containing a point mutation within the env gene. The point mutation resulted in complete loss of balanced splicing, with dominan t production of aberrant mRNAs. The aberrant RNAs arose via activation of normally cryptic splice sites flanking the mutation within the env terminal exon to create exon 6D, which was subsequently incorporated in aberrant env, tat, rev, and nef mRNAs. Aberrant multiply spliced me ssages contributed to reduced virus replication as a result of a reduc tion in wild-type Rev protein. The point mutation within exon 6D activ ated exon 6D inclusion when the exon and its flanking splice sites wer e transferred to a heterologous minigene, Introduction of the point mu tation into an otherwise wild-type HIV-1 proviral clone resulted in vi rus that was severely inhibited for replication in T cells and display ed elevated usage of exon 6D, Exon 6D contains a bipartite element sim ilar to that seen in tat exon 3 of HIV-1, consisting of a potential ex on splicing silencer (ESS) juxtaposed to a purine-rich sequence simila r to known exon splicing enhancers, In the absence of a flanking 5' sp lice site, the point mutation within the exon 6D ESS-like element stro ngly activated env splicing, suggesting that the putative ESS plays a natural role in limiting the level of env splicing, We propose, theref ore, that exon silencers may be a common element in the HIV-1 genome u sed to create balanced splicing of multiple products from a single pre cursor RNA.