Co-expression of a trans-dominant negative mutant of the human immunodeficiency virus type 1 (HIV-1) Rev protein affects the Rev-dependent splicing pattern and expression of HIV-1 RNAs

Trans-dominant negative mutants of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev inhibit the function of wild-type Rev in a d ose-dependent manner. This was previously shown to be caused by nuclear ret ention of the wild-type protein. In the present work, further analysis of t he trans-dominant negative effect was performed using cotransfection experi ments with different constructs encoding HIV-1 Rev and viral structural pro teins together with a plasmid encoding a trans-dominant negative Rev mutant . Thus, one species of pre-mRNA was transcribed from the reporter plasmids, This pre-mRNA was then either spliced or exported by Rev as unspliced RNA for translation of the HIV structural proteins. An immunofluorescence assay and Western blot analysis were used for analysis of protein expression. In situ hybridization was applied for labelling of unspliced mRNA in transfec ted cells, and RNase protection analysis was used to determine the relative amount of unspliced versus spliced mRNAs. The experiments confirmed that t he transdominant negative mutant inhibited nuclear export of unspliced mRNA . It was, in addition, demonstrated for the first time that the trans-domin ant negative mutant also affected a Rev-dependent regulatory step connected with viral pre-mRNA splicing. As a consequence, proteins expressed from un spliced and singly spliced HIV mRNAs decreased while there was an increase in protein products encoded by spliced and alternatively spliced mRNAs.