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Journal of Virology, August 2009, p. 8141-8152, Vol. 83, No. 16
0022-538X/09/$08.00+0     doi:10.1128/JVI.02116-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Activation of Intracellular Signaling Pathways by the Murine Cytomegalovirus G Protein-Coupled Receptor M33 Occurs via PLC-β/PKC-Dependent and -Independent Mechanisms{triangledown} ,{dagger}

Joseph D. Sherrill, Melissa P. Stropes, Olivia D. Schneider, Diana E. Koch, Fabiola M. Bittencourt, Jeanette L. C. Miller, and William E. Miller*

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524

Received 7 October 2008/ Accepted 22 May 2009

The presence of numerous G protein-coupled receptor (GPCR) homologs within the herpesvirus genomes suggests an essential role for these genes in viral replication in the infected host. Such is the case for murine cytomegalovirus (MCMV), where deletion of the M33 GPCR or replacement of M33 with a signaling defective mutant has been shown to severely attenuate replication in vivo. In the present study we utilized a genetically altered version of M33 (termed R131A) in combination with pharmacological inhibitors to further characterize the mechanisms by which M33 activates downstream signaling pathways. This R131A mutant of M33 fails to support salivary gland replication in vivo and, as such, is an important tool that can be used to examine the signaling activities of M33. We show that M33 stimulates the transcription factor CREB via heterotrimeric Gq/11 proteins and not through promiscuous coupling of M33 to the Gs pathway. Using inhibitors of signaling molecules downstream of Gq/11, we demonstrate that M33 stimulates CREB transcriptional activity in a phospholipase C-β and protein kinase C (PKC)-dependent manner. Finally, utilizing wild-type and R131A versions of M33, we show that M33-mediated activation of other signaling nodes, including the mitogen-activated protein kinase family member p38{alpha} and transcription factor NF-{kappa}B, occurs in the absence of Gq/11 and PKC signaling. The results from the present study indicate that M33 utilizes multiple mechanisms to modulate intracellular signaling cascades and suggest that signaling through PLC-β and PKC plays a central role in MCMV pathogenesis in vivo.

* Corresponding author. Mailing address: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524. Phone: (513) 558-0866. Fax: (513) 558-8474. E-mail: william.miller{at}uc.edu

{triangledown} Published ahead of print on 3 June 2009.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, August 2009, p. 8141-8152, Vol. 83, No. 16
0022-538X/09/$08.00+0     doi:10.1128/JVI.02116-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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