Overview:

The long term vision of the HMP Unit is to discover novel fundamental knowledge concerning herpesvirus molecular biology, in particular mechanisms of host immune system evasion or subversion, and apply this knowledge to the development of novel prophylactic, therapeutic and/or diagnostic procedures against herpesvirus disease. The Unit utilises a natural herpesvirus/host system, murine cytomegalovirus (MCMV) infection of mice, as a model to investigate molecular mechanisms underlying pathogenesis which are evolutionarily conserved with human herpesviruses, such as human cytomegalovirus (HCMV). This will continue to be a primary research area and in addition we will look to exploit our model systems and know-how through collaboration with research and commercial groups interested in vaccine and drug development.

HCMV is a highly prevalent betaherpesvirus that, in the absence of effective immune control, may cause serious disease. HCMV is recognised as one of the major causes of congenital disease in many countries worldwide; within Australia, approximately 200-600 children born each year are affected by congenital CMV disease, with many requiring lifelong support for the resulting conditions. Moreover, HCMV is a major cause of morbidity and mortality in transplant recipients and patients with AIDS. An effective commercial vaccine aginst HCMV has yet to be developed. Moreover, current antiviral chemotherapy suffers from problems associated with toxicity and rapid acquisition of drug resistance.

HCMV and related viruses are notable for encoding a large number of genes which are involved in virus-host interactions, in particular the modulation of host immune responses. All herpesviruses that infect circulating blood cells, i.e. betaherpesviruses such as HCMV and gammaherpesviruses such as Epstein-Barr virus (glandular fever, Burkitt's lymphoma) or HHV-8 (Kaposi's sarcoma), have been found to encode homologues of G protein-coupled receptors (GPCR). The GPCR are a large and diverse family of proteins which act as 'sensory organs' for cells, by binding ligands at the cell surface and transmitting signals to the interior of the cell. A sub-class of GPCR, the chemokine receptors (CKR), are critical components of the host immune system, directing the tissue specific migration and activation of immune effector cells in response to chemokines released at the site of infection or within lymphoid organs. Interestingly, most of the herpesvirus GPCRs are most closely related to CKR and several have been found to transmit signals constitutively, without requiring binding of extracellular ligand. In this way, they may function by activating pathways within the cell that result in migration of virus-infected cells to preferred sites of replication/latency or otherwise enhance virus replication. The viral CKR may also act by interfering with normal cellular CKR-mediated signalling, thereby interfering with immune responses.

Research Projects

CORE PROJECT AREAS

CMV G protein-coupled receptors: from function to therapy
Collaborators: Dr. Rhonda Cardin (Cincinnati Children's Hospital, USA), Prof. Mette Rosenkilde (University of Copenhagen, Denmark), Dr. Thomas Kledal (INAGEN, Roskilde, Denmark), Dr. Barry Slobedman (Millenium Institute, Sydney), Dr. Stacey Efstathiou (University of Cambridge, U.K.)
Funding: NHMRC Project grant 631401

Characterisation of the MCMV G protein-coupled receptor homologue (GPCR), M78
Many viral GPCRs exhibit rapid, constitutive endocytosis, which may contribute to their biological functions. M78 is the mouse CMV member of the 'UL78 family' of betaherpesvirus specific GPCRs, which have been shown to contribute to replication in tissue culture and in vivo for mouse and rat CMV. We previously demonstrated that M78 was rapidly and constitutively endocytosed and have undertaken mutagenic analysis to determine key motifs directing endocytosis. The importance of the C-terminus of M78 has been demonstrated, including a canonical di-leucine motif characteristic of clathrin mediated endocytosis. Deletion analysis demonstrated that M78 endocytosis could occur in the absence of the di-leucine motif and suggests novel positive and negative regulators of endocytosis (Figure 1). Incorporation of M78 with deletion of most of the C-tail, which has substantially reduced endocytosis, was found to enhance replication in certain cell-types, but attenuate replication in macrophages. These results indicate novel activities of M78 which will be the subject of future studies.

Figure 1: Effects of M78 mutations upon endocytosis. Endoytosis of a panel of M78 mutants was analysed in transfected cells. The graph shows the ratio of total/surface M78 detected by immunofluorescence following a period of antibody feeding: the higher the ratio, the more efficiently M78 has been endocytosed. Wild type (wt: non-mutated) M78, shown in red, is efficiently endocytosed. Mutated M78 that are endocytosed similarly to wt M78 shown in orange, mutants with significantly reduced endocytosis shown in green or blue.

Demonstration of functions for the MCMV GPCR M33 during latency/reactivation

M33 is the mouse CMV member of the 'UL33 family' of homologous genes conserved throughout the betaherpesvirus family. We had previously demonstrated that constitutive signalling induced by M33 was critical for virus replication in salivary glands, a major site of virus persistence and shedding (Case et al, 2008). More recently, we demonstrated that an M33 null recombinant was defective for reactivation from latency in ex vivo spleen and lung explant cultures (Cardin et al., 2009). We have now extended these studies to demonstrate that although G protein-coupled signalling, mediated by M33, is not important for replication during the acute phase of infection in most organs (apart from salivary glands), it is required for wild-type reactivation efficiency (Farrell et al, 2011). Given the key role of latency/reactivation to the lifecycle of cytomegaloviruses, these findings have important implications for potential novel therapies against CMV disease.

Functional complementation between M33 and other GPCRs.

Recombinant MCMV, where the M33 open reading frame was replaced by either UL33 or US28, were generated and evaluated for their in vivo phenotypes. Both human CMV GPCRs were able to partially recover salivary gland replication and restore explant reactivation efficiency to similar levels to those of wild type virus (Farrell et al., 2011) These data are consistent with the signalling properties of UL33 and US28 enabling functional complementation for M33. Current studies are determining the functional impact of specific mutations of US28 upon biological activity in the MCMV system. Furthermore, we have developed a novel model system for evaluating the effects of prototype drugs targeting the human CMV GPCRs during infection of mice.

We have extended these studies to investigate the ability of cellular GPCRs to complement the function of M33. Recombinant MCMV expressing four different cellular GPCRs, previously demonstrated to exhibit constitutive signalling, have been generated and characterised for salivary gland replication. Two of the cellular GPCRs were found to partially complement the function of M33 for salivary gland replication. Intriguingly, both these GPCRs are naturally expressed in lymphocyte subsets, suggesting that GPCR functions specific to lymphocytes may be important for the activity of the CMV GPCR homologues.

Investigation of our panel of MCMV recombinants (deleted, mutated or substituted) in a variety of cell types has, for the first time, demonstrated cell-type specific replication defects. Future studies will be directed at determining the key pathways (eg. signalling) contributing to these cell-type specific effects and relating these differences to biological function in vivo, thereby determining the mechanisms whereby the CMV GPCRs affect viral pathogenesis.

AUXILARY PROJECT AREAS

1: HCMV immune responses and vaccine development

In collaboration with Dr. Khanna (QIMR), we have developed a recombinant MCMV model for assessment of novel HCMV vaccines directed against defined antigens using the mouse model.

2: Tagged HSV-1 recombinants as neuronal tracing agents

In collaboration with Dr. Mazzone and Miss Alice McGovern (UQ), we have generated a fluorescent protein tagged recombinant of an HSV-1 strain that selectively transmits between neurons via the anterograde route. This recombinant will provide a novel reagent for the tracing of neuronal pathways relevant to the respiratory tract, such as those involved in the cough response.

3: Intravaginal delivery of antiviral agents

In collaboration with Dr. Coombes (UQ, Pharmacy), we have been investigating a novel matrix formulation for medium term, in situ delivery of antiviral agents to the female genital tract, via an intravaginal ring device. The incorporation, stability and release of different classes of anti-viral agent will be investigated, using HSV-2 as a model target virus.