Cell-type specific activities of cytomegalovirus GPCR homologues

Abstract

The human cytomegalovirus (HCMV) is the main agent of congenital infection worldwide. Although primary infections or reactivations do not represent a threat to most individuals, they impose a lifethreating event to a developing foetus or immunocompromised individuals. Although we have learnt much about sites of viral replication and cells involved in the end-stage disease, the specific pathway and essential cell types that the virus must go through to later establish a successful infection and latency is still not clear. HCMV encodes four GPCR homologues (vGPCR): US27, US28, UL33 and UL78. vGPCR have been implicated in viral pathogenesis due to their ability to activate signalling pathways and, thus, alter physiological processes to favour infection. US28 and UL33 have been shown to activate several kinases and transcriptional factors. A key component of both US28 and UL33 sequence is the DRY (Asp/Arg/Tyr) motif, a region directly involved with G protein binding.Because Gα protein content is cell type-dependent, signalling activation by vGPCR can differ from cell to cell. Furthermore, these receptors share the particular characteristic of being constitutively Functional studies demonstrated migration induction of HTR-8 following transfection by either US28 or UL33. In order to investigate potential mechanisms, signalling pathways were probed via detection of activated kinases and the secretion of matrix metalloproteinases (MMP) and chemokines (CK) via luminex assays. Although differences in the level of kinases could not be measured by western blot, induction of MMP and CK were detected by Luminex assays, with contrasting results for US28 and UL33 in transfected cells (HTR-8 and HUVEC). An HCMV recombinant disrupted for US28 signalling (DQY) was generated and compared with wild type HCMV and a US28 null mutant for induction of MMP and CK in virus-infected cells. US28-dependent effects were identified for infected human foreskin fibroblasts, but results for HUVEC were equivocal.iii To determine potential bottlenecks to viral dissemination that may require M33 function(s), mice were infected with M33 knockout (M33) viruses by different routes: intranasal, -mimicking the most natural route, footpad -a more compartmentalized route, and intraperitoneal, the most direct route to major organs. To help track viral dissemination, a luciferase tagged M33 was generated and infection was compared to control virus (M78luc). M33 intranasal infection did not result in attenuation for colonisation of the nose, draining (superficial cervical) lymph nodes (dLN) or the lungs. Via footpad route, M33 colonised the footpad and dLN to levels equivalent to control virus, but was significantly attenuated in spread to the spleen and, subsequently, the salivary glands.Following intraperitoneal infection, the virus is readily delivered to major organs due to direct access to the bloodstream, there was no difference in the colonisation of primary organs (spleen, liver) between M33 and control MCMV, but M33 was still unable ...