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Journal of Virology, July 2001, p. 6033-6041, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.6033-6041.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Epstein-Barr Virus SM Protein Interacts with mRNA In Vivo and Mediates a Gene-Specific Increase in Cytoplasmic mRNA

Vivian Ruvolo,1 Ashish K. Gupta,1 and Sankar Swaminathan1,2,*

University of Florida Shands Cancer Center1 and Department of Medicine and Department of Molecular Genetics & Microbiology,2 University of Florida, Gainesville, Florida 32610

Received 16 January 2001/Accepted 3 April 2001

SM is an Epstein-Barr virus (EBV) gene expressed during early lytic replication of EBV. SM encodes a nuclear phosphoprotein that functions as a posttranscriptional regulator of gene expression. SM has been implicated in several aspects of gene regulation, including nuclear mRNA stabilization, posttranscriptional processing, and nuclear mRNA export. Activation by SM is promoter independent but gene specific. The mechanism by which SM selectively activates some EBV target genes or heterologous reporter genes remains to be determined. SM binds RNA in vitro, suggesting that sequence- or structure-specific mRNA interactions might mediate SM specificity. We have further analyzed RNA binding by SM and demonstrated that proteolytic cleavage of SM and consequent exposure of an arginine-rich region are necessary to allow RNA binding in vitro. However, SM mutants with deletions of this arginine-rich region localized normally in the nucleus and were fully functional in gene activation. We therefore developed an assay to study in vivo interactions of SM with target mRNAs based on immunoprecipitation of SM from cell lysates followed by RNase protection analysis. Using this assay, we demonstrated that SM forms complexes with specific mRNAs in vivo. SM binds mRNAs from both SM-responsive as well as nonresponsive intronless genes and increases the nuclear accumulation of both types of mRNAs. In addition, SM preferentially associates with newly transcribed mRNAs. These data indicate that SM forms complexes with mRNAs in the nucleus and enhances their nuclear accumulation. However, SM does not enhance cytoplasmic accumulation of all transcripts that it binds to the same degree, suggesting that additional mRNA-specific characteristics, such as nuclear retention motifs or binding sites for cellular proteins, also determine responsiveness to SM.

* Corresponding author. Mailing address: University of Florida Shands Cancer Center, Box 100232, University of Florida, 1600 SW Archer Rd., Gainesville, FL 32610-0232. Phone: (352) 392-9302. Fax: (352) 392-5802. E-mail: sswamina{at}ufl.edu.

Journal of Virology, July 2001, p. 6033-6041, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.6033-6041.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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