NEGATIVE AND POSITIVE mRNA SPLICING ELEMENTS ACT COMPETITIVELY TO REGULATE HIV-1 VIF GENE EXPRESSION

Department of Microbiology, University of Iowa, Iowa City, IA 52242

^* To whom correspondence should be addressed. Email: marty-stoltzfus{at}uiowa.edu.

	Abstract

Over 40 different HIV-1 mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for Gag and Pol gene products. Regulation of splicing at HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tacts and positive or negative regulation occurs through binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A1, which produces single spliced vif mRNA and promotes inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5' splice site (5'ss) downstream of exon 2 (5'ss D2). Here we show that mutations within 5'ss D2 that are predicted to lower or increase the affinity of the 5'ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5'ss D2 was not necessary for the effect of 5'ss D2 on Vif expression. In addition, we have found that mutations of a GGGG motif proximal to 5'ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5'- proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE specifically binds to cellular SR protein SRp75. Our results suggest that the 5' ss D2, proximal GGGG silencer and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA compatible with optimal virus replication.