Functional complementation studies of murine cytomegalovirus encoded G protein-coupled receptor homologue M33

Abraham, Alexander Mathews (2012). Functional complementation studies of murine cytomegalovirus encoded G protein-coupled receptor homologue M33 MPhil Thesis, School of Chemistry & Molecular Bioscience, The University of Queensland.

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Author Abraham, Alexander Mathews
Thesis Title Functional complementation studies of murine cytomegalovirus encoded G protein-coupled receptor homologue M33
School, Centre or Institute School of Chemistry & Molecular Bioscience
Institution The University of Queensland
Publication date 2012
Thesis type MPhil Thesis
Supervisor Nicholas Davis-Poynter
Helen Farrell
Total pages 103
Total colour pages 17
Total black and white pages 86
Language eng
Subjects 0605 Microbiology
060506 Virology
Formatted abstract
Human Cytomegalovirus (HCMV) is a species specific member of the betaherpesvirus family which causes serious disease in the absence of effective immune control. However, infection in an immunocompetent individual is generally asymptomatic. In common with other herpesviruses, HCMV establishes lifelong latency where it maintains its carriage in specific sites of the host in the absence of productive infection. Periodically, reactivation from latency results in acute infection, especially in immunocompromised individuals.

HCMV and related viruses encode numerous genes involved in modulation of host immune response, several of which are sequence homologues to host cellular GPCR. This indicates that these genes may have been captured by the virus during its coevolution with the host. Viral GPCR homologues may function to enable the replicative success of the virus and thereby contribute to viral pathogenesis.

GPCRs are 7 transmembrane receptors that function to regulate various cellular processes by activating diverse intracellular signalling pathways. In the case of classical, G protein-coupled signalling, a predominant factor determining the major signalling pathways activated by the receptor is coupling to one or more of the four families of mammalian G protein alpha-subunits, namely Gαs, Gαi, Gαq or Gα12/13. Typically, the GPCR is in an inactive state until engaged by an extracellular ligand, which promotes a conformational change of the transmembrane domains, resulting in activation of the associated G protein complex and hence triggering of second messenger pathways.

All beta and gammaherpesviruses encode GPCR homologues. Viral GPCR homologues namely US28 and UL33 (encoded by HCMV) and M33 (UL33 counterpart encoded b MCMV) were shown to trigger signalling pathways either constitutively, in the absence of exogenous ligand (all three) and/or in response to stimulation by ligands (US28 only). While UL33 constitutively signalled promiscuously via Gαi, Gαq and Gαs, M33 was more similar to US28 wherein these receptors coupled specifically to Gαq to signal constitutively.

Previous studies in our laboratory demonstrated that M33 is required for in vivo replication of MCMV in salivary glands and was also required for efficient reactivation of the virus from latency from spleen and lung explants. Subsequently it was shown that these phenotypes were dependent on M33 mediated G protein- coupled constitutive signalling of M33.

The first part of this study describes completion of work to investigate whether the human CMV GPCR homologues UL33 and US28 were able to functionally complement the activity of M33. These studies demonstrated partial complementation of M33 function (salivary gland replication and reactivation from latency) by both HCMV GPCR. Given the importance of signalling for the in vivo M33 phenotype, we speculated that functional complementation of US28/UL33 was due to conserved signalling pathways shared by these receptors. Based on these findings, we proposed that other GPCRs (cellular) would be able to rescue M33 dependent phenotypes, consistent with their signalling ability.

The second part of this study determined the ability of a selection of cellular GPCR to rescue the M33 dependent phenotype of salivary gland replication, namely EBI2, GPR18, GPR39 and Ghrelin receptor, which have been reported to constitutively signal via interaction with either Gαi (EBI2 and GPR18) or Gαq (GPR39 and Ghrelin receptor) G protein subtypes. Each receptor was cloned with an epitope tag and expression confirmed in transiently transfected cells. Each of the receptors trafficked to the cell surface, but they showed marked differences in constitutive endocytosis properties. Constitutive activation of Gαq coupled signalling by GPR39 and Ghrelin receptor and of Gαi signalling by EBI2 was confirmed, whereas GPR18 signalling via Gαi was not detected.

Recombinant MCMV expressing each of the cellular GPCRs were generated and confirmed to replicate similarly to wild type in tissue culture. The in vivo replication characteristics of these recombinant viruses for salivary gland replication in infected mice were determined. EBI2 and GPR18 partially rescued salivary gland replication while GPR39 and Ghrelin receptor were defective for replication. Thus, cellular receptors coupling to Gαi rather than Gαq were able to partially complement the function of M33. These results suggest that whereas ablation of Gαq signalling by M33 is associated with loss of function (salivary gland replication), constitutive activation of Gαq by the cellular GPCR was neither necessary nor sufficient for functional complementation. Our results suggest that functional complementation may be determined by activation of downstream effectors, which may occur via either convergence of different G protein trigged pathways or via non-classical (G protein-independent) signalling. The recombinant viruses expressing cellular GPCRs generated in this study could be used as a tool to help dissect the signalling pathways and downstream effectors activated by M33 that are required for its biological function.
Keyword Herpesvirus
G protein - coupled receptor homologue
Mouse cytomegalovirus model
Constitutive G protein-coupled signalling
Recombinant virus

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Created: Thu, 27 Sep 2012, 06:04:36 EST by Alexander Mathews Abraham on behalf of Scholarly Communication and Digitisation Service