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The Epstein-Barr virus (EBV) SM protein enhances pre-mRNA processing of the EBV DNA polymerase transcript
S C Key1, T Yoshizaki, J S Pagano
1Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Abstract:
The Epstein-Barr virus (EBV) DNA polymerase (pol) mRNA, which contains a noncanonical polyadenylation signal, UAUAAA, is cleaved and polyadenylated inefficiently (S. C. S. Key and J. S. Pagano, Virology 234:147-159, 1997). We postulated that the EBV early proteins SM and M, which appear to act posttranscriptionally and are homologs of herpes simplex virus (HSV) ICP27, might compensate for the inefficient processing of pol pre-mRNA. Here we show that the SM and M proteins interact with each other in vitro. In addition, glutathione S-transferase-SM/M fusion proteins precipitate the heterogeneous ribonucleoprotein (hnRNP) C1 splicing protein. Further, the SM protein is coimmunoprecipitated from SM-expressing cell extracts with an antibody to the hnRNP A1/A2 proteins, which are splicing and nuclear shuttling proteins. Finally, the amount of processed EBV DNA polymerase mRNA was increased three- to fourfold in a HeLa cell line expressing SM; this increase was not due to enhanced transcription. Thus, inefficient processing of EBV pol RNA by cellular cleavage and polyadenylation factors appears to be compensated for and may be regulated by the early EBV protein, SM, perhaps via RNA 3'-end formation.
Insights
The Epstein-Barr virus (EBV) DNA polymerase mRNA is processed inefficiently. The EBV SM protein compensates for this, potentially regulating RNA 3'-end formation.
Area of Science:
- Virology
- Molecular Biology
- RNA Processing
Background:
- Epstein-Barr virus (EBV) DNA polymerase (pol) mRNA processing is inefficient due to a noncanonical polyadenylation signal (UAUAAA).
- EBV early proteins SM and M are homologs of herpes simplex virus (HSV) ICP27 and may posttranscriptionally regulate mRNA processing.
Purpose of the Study:
- To investigate whether EBV early proteins SM and M compensate for inefficient EBV pol mRNA processing.
- To elucidate the mechanism by which SM protein might regulate EBV pol mRNA processing.
Main Methods:
- In vitro protein-protein interaction assays (SM and M proteins).
- Glutathione S-transferase (GST) pull-down assays with GST-SM/M fusion proteins to identify interacting hnRNP proteins.
- Coimmunoprecipitation of SM protein with hnRNP A1/A2 antibodies from cell extracts.
- Quantification of processed EBV DNA polymerase mRNA in a HeLa cell line expressing SM protein.
Main Results:
- SM and M proteins interact in vitro.
- GST-SM/M fusion proteins precipitate hnRNP C1 splicing protein.
- SM protein is coimmunoprecipitated with hnRNP A1/A2 proteins.
- Expression of SM protein in HeLa cells increased processed EBV DNA polymerase mRNA levels three- to fourfold without affecting transcription.
Conclusions:
- The EBV early protein SM compensates for inefficient processing of EBV pol mRNA.
- SM protein may regulate EBV pol RNA 3'-end formation, potentially involving interactions with hnRNP proteins.
- This compensation mechanism highlights a regulatory role for EBV early proteins in viral gene expression.