Downloaded from www.microbiologyresearch.org by IP: 54.157.140.51 On: Sat, 27 Feb 2016 21:48:01 Analysis of the subcellular trafficking properties of murine cytomegalovirus M78, a 7 transmembrane receptor homologue E. L. Sharp, 1 N. J. Davis-Poynter 1,2 and H. E. Farrell 1,2 Correspondence H. E. Farrell h.farrell1@uq.edu.au 1 Infectious Diseases, Animal Health Trust, Newmarket, Suffolk CB8 7UU, UK 2 The University of Queensland, Clinical Medical Virology Centre and Royal Children’s Hospital, Sir Albert Sakzewski Virus Research Centre, QLD 4072, Australia Received 12 June 2008 Accepted 10 September 2008 Murine cytomegalovirus (MCMV) M78 is a member of the betaherpesvirus ‘UL78 family’ of seven transmembrane receptor (7TMR) genes. Previous studies of M78 and its counterpart in rat cytomegalovirus (RCMV) have suggested that these genes are required for efficient cell–cell spread of their respective viruses in tissue culture and demonstrated that gene knockout viruses are significantly attenuated for replication in vivo. However, in comparison with other CMV 7TMRs, relatively little is known about the basic biochemical properties and subcellular trafficking of the UL78 family members. We have characterized MCMV M78 in both transiently transfected and MCMV-infected cells to determine whether M78 exhibits features in common with cellular 7TMR. We obtained preliminary evidence that M78 formed dimers, a property that has been reported for several cellular 7TMR. M78 traffics to the cell surface, but was rapidly and constitutively endocytosed. Antibody feeding experiments demonstrated co-localization of M78 with markers for both the clathrin-dependent and lipid raft/caveolae-mediated internalization pathways. In MCMV-infected cells, the subcellular localization of M78 was modified during the course of infection, which may be related to the incorporation of M78 into the virion envelope during the course of virion maturation. INTRODUCTION The cytomegaloviruses (CMVs) are characterized by slow replication cycles, with periods of persistent virus shedding and latency. Virus dissemination is highly cell-associated, principally via infected leukocytes (Sinclair & Sissons, 2006). In order to maintain a stable relationship with their host, the CMVs have evolved various mechanisms to manipulate normal cellular responses, in particular innate and adaptive immune responses (reviewed by Mocarski, 2002). Among the viral gene products that are predicted to be linked to CMV dissemination are seven transmembrane receptors (7TMR) that signal via coupling to G proteins. Leukocyte trafficking is regulated by the concerted responses of multiple cellular 7TMR, both in response to infection and during normal cellular homeostasis (Le et al., 2004) and thus, the CMV 7TMR homologues have been implicated in usurping this role during infection to promote virus dissemination. Additional roles for CMV 7TMR include chemokine sequestration, membrane fusion and production of an intracellular environment favourable for virus replication (Vischer et al., 2006). Human cytomegalovirus (HCMV) encodes four 7TMR: UL33, UL78, US27 and US28. Of these, US28 has been the most extensively characterized, both pharmacologically and with respect to its intracellular trafficking properties. US28 has been shown to bind both CC and CX3C chemokines with high affinity, resulting in the activation of multiple intracellular activation pathways (Kuhn et al., 1995). In addition to agonist-induced signalling, US28 exhibits constitutive signalling and endocytosis, which appear to be modulated by the binding of fractalkine (Casarosa et al., 2001). Unlike most cellular chemokine receptors, US28 is located predominantly in late and recycling endosomes, rather than at the cell surface, which support the suggestion that it plays a role in sequestration of cellular chemokines (Bodaghi et al., 1998; Fraile-Ramos et al., 2001). While constitutive endocytosis of US28 has been shown to occur independently of b-arrestin proteins, it is nevertheless internalized, at least in part, via a clathrin-mediated pathway (Fraile-Ramos et al., 2003). Studies with UL33 and US27 have demonstrated that, like US28, they co-localize with endocytic vesicles. Notably, the localization of UL33 and US27 in endocytic vesicles in HCMV-infected cells has been shown to overlap with intracellular membranes containing HCMV glycoproteins important for virion maturation, consistent with the incorporation of these viral 7TMR in the virion envelope (Fraile-Ramos et al., 2002). It has been suggested that dimerization/oligomerization of 7TMR influences their intracellular trafficking. Homo- and Journal of General Virology (2009), 90, 59–68 DOI 10.1099/vir.0.004853-0 004853 G 2009 SGM Printed in Great Britain 59