Repair of a Rev-minus human immunodeficiency virus type 1 mutant by activation of a cryptic splice site

We isolated a revertant virus after prolonged culturing of a replication-im paired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initi ation codon. Sequencing of the tat-rev region of this revertant virus ident ified a second-site mutation in tat that restored virus replication in the mutant background. This mutation activated a cryptic 5' splice site (ss) th at, when used in conjunction with the regular HN 3' ss #5, fuses the tat an d rev reading frames to encode a novel T-Rev fusion protein that rescues Re v function. We also demonstrate an alternative route to indirectly activate this cryptic 5' ss by mutational inactivation of an adjacent exon splicing silencer element.