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Journal of Virology, September 2001, p. 8487-8497, Vol. 75, No. 18
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.18.8487-8497.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

RNA Splicing at Human Immunodeficiency Virus Type 1 3' Splice Site A2 Is Regulated by Binding of hnRNP A/B Proteins to an Exonic Splicing Silencer Element

Patricia S. Bilodeau,1 Jeffrey K. Domsic,2 Akila Mayeda,3 Adrian R. Krainer,4 and C. Martin Stoltzfus1,2,*

Department of Microbiology1 and Program in Molecular Biology,2 University of Iowa, Iowa City, Iowa 52242; Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 331363; and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-22084

Received 22 February 2001/Accepted 8 June 2001

The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1B, A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.

* Corresponding author. Mailing address: Department of Microbiology and Program in Molecular Biology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7793. Fax: (319) 335-9006. E-mail: marty-stoltzfus{at}uiowa.edu.

Journal of Virology, September 2001, p. 8487-8497, Vol. 75, No. 18
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.18.8487-8497.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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