Overlapping Cis sites used for splicing of HIV-1 env/nef and rev mRNAs

Alternative splicing is used to generate more than 30 human immunodeficienc y virus type 1 (HIV-1) spliced and unspliced mRNAs from a single primary tr anscript. The abundance of HIV-1 mRNAs is determined by the efficiencies wi th which its different 5' and 3' splice sites are used. Three splice sites (A4c, A4a, and A4b) are upstream of the rev initiator AUG. RNAs spliced at A4c, A4a, and A4b are used as mRNAs for Rev. Another 3' splice site (A5) is immediately downstream of the rev initiator. RNAs spliced at A5 are used a s mRNAs for Env and Nef, In this report, primer extension analysis of splic ing intermediates was used to show that there are eight branch points in th is region, all of which map to adenosine residues. In addition, cis element s recognized by the cellular splicing machinery overlap; the two most 3' br anch points overlap with the AG dinucleotides at rev 3' splice sites A4a an d A4b, Competition of the overlapping cis sites for different splicing fact ors may play a role in maintaining the appropriate balance of mRNAs in HIV- 1-infected cells. In support of this possibility, mutations at rev 3' splic e site A4b AG dinucleotide dramatically increased splicing of the env/nef 3 ' splice site A5, This correlated with increased usage of the four most 3' branch points, which include those within the rev 3' splice site AG dinucle otides. Consistent with these results, analysis of a mutant in which three of the four env/nef branch points were inactivated indicated that use of sp lice site A5 was inhibited and splicing was shifted predominantly to the mo st 5' rev 3' splice site A4c with preferential use of the two most 5' branc h points. Our results suggest that spliceosomes formed at rev A4a-4b, rev A 4c, and env/nef A5 3' splice sites each recognize different subsets of the eight branch point sequences.