INEFFICIENT SPLICEOSOME ASSEMBLY AND ABNORMAL BRANCH SITE SELECTION IN SPLICING OF AN HIV-1 TRANSCRIPT IN-VITRO

Continuous replication of human immunodeficiency virus type I (HIV-1) requires balanced expression of spliced and nonspliced mRNAs in the cy toplasm. This process is regulated post-transcriptionally by the viral -encoded Rev protein. An important prerequisite for Rev responsiveness is the presence of weak splice sites in the viral mRNA, We have inves tigated the splicing of the second intron of the HIV-1 Tat/Rev transcr ipt in vitro and show that the S'-splice site region is responsible fo r the inefficient splicing of the HIV-1 transcript. In contrast, the H IV-1 5'-splice site is highly functional in combination with a heterol ogous S' splice site. Incubation of the HIV-1 transcript in nuclear ex tract leads to a rapid accumulation of 50 S nonproductive pre-spliceos ome complexes. These complexes contain mainly U1 and U2 small nuclear ribonucleoproteins and are formed independently of the presence of the downstream 3'-splice site. The HIV-1 transcripts, which do proceed th rough the first splicing step, utilize primarily a uridine as the bran ch acceptor nucleotide. Sequence comparison with other HIV-1 introns s uggests that nucleotides other than adenosines are commonly used as br anch points in these viruses.