Herpesvirus Research Group

The herpesvirus research unit at the CHRC has a focus on cytomegalovirus disease.  The long term vision of our group is to apply our fundamental studies of herpesvirus molecular biology to the development of new ways of preventing the disease, or new ways to diagnose and treat herpesvirus infection.

Why study Human Cytomegalovirus (HCMV)?

Human CMV is a highly prevalent betaherpesvirus that is recognised as one of the major causes of congenital disease worldwide. It is estimated that 200-600 children born each year in Australia are affected by congenital HCMV disease, with many requiring lifelong support for the resulting conditions. The virus can cross the placenta to infect the unborn child, which may result in foetal death or congenital disease of varying severity. 

HCMV may also cause disease in immunosuppressed people, for example transplant recipients.  Moreover, CMV is a major cause of morbidity and mortality in transplant recipients and AIDS patients. An effective commercial vaccine against CMV has yet to be developed. Moreover, current antiviral chemotherapy suffers from problems associated with toxicity and rapid acquisition of drug resistance.

Our Research Aims

Our research is seeking to understand how HCMV causes disease,  HCMV deploys various ‘weapons’ to counteract or hijack the immune system.  We have been investigating how one class of such ‘weapons’, which are important in delivering messages to cells that affect how they behave in response to infection, act during CMV infection, using human cells in tissue culture and mouse CMV in animals.  This work has identified specific functions of the virus which may be suitable targets for future drugs to counter CMV disease. 

The Unit utilises a natural herpesvirus/host system, mouse CMV (MCMV) infection of mice, as a model to investigate molecular mechanisms underlying pathogeneses which are evolutionarily conserved with human herpesviruses, such as human CMV (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 design.

Current Research Studies

Characterisation of novel targets for antivirals directed against CMV

HCMV and related viruses are notable for encoding multiple genes that are not essential for virus replication in vitro, but are critical for the pathogenesis of the virus in vivo.  These “non-essential” gene products are attractive antiviral targets against HCMV.  Candidates in this group include the viral homologues of G protein-coupled receptors (GPCRs).  All herpesviruses that infect circulating blood cells, such as the betaherpesviruses (e.g. HCMV) and gammaherpesviruses (e.g Epstein-Barr Virus that causes glandular fever or HHV-8 that causes Kaposi’s sarcoma), have been found to encode homologues of GPCRs.  Significantly, these comprise distinct gene families: beta 27/28, beta 33, beta 78 (betaherpesviruses); gamma 74, gamma BILF1 (gammaherpesviruses).  Work by our group and others has demonstrated the potential of the vGPCRs to contribute to various aspects of viral pathogenesis.

Identification of biological function and key signalling activities for CMV vGPCRs

We previously established the importance of beta 33 for virus tropism to the salivary gland and persistence/reactivation from latency, via studies of mouse CMV infection.  Furthermore, we demonstrated the importance of signalling activated by M33 of mouse CMV (beta 33 family) for the biological functions.  In addition, we demonstrated that vGPCR from human CMV (US28 and UL33) are able to at least partially rescue salivary gland and reactivation phenotypes for mouse CMV lacking M33, thus generating an in vivo model to probe the mechanism of action of the HCMV GPCRs (Farrell et al., 2011). By construction of human CMV US28 mutants with defined effects upon signalling (determined in transiently transfected and virus infected cells), we have delineated key signalling pathways for the function of US28 in our mouse model (Farrell et al, 2013).  Collectively, these studies have:

  1. demonstrated the importance of CMV vGPCRs to pathogenesis, thereby supporting their potential as antiviral drug targets
  2. identified key signalling pathways responsible for biological function, thereby providing readouts for pre-clinical assessment of future antivirals

Current studies are determining the activities of vGPCRs in specific cell types relevant to CMV disease.  In macrophages, M33 is required for efficient replication in tissue culture and this function is complemented by US28, but G protein-coupled signalling is not required, suggesting an alternative vGPCR activity is responsible for this phenotype (Farrell et al 2013 and unpublished data).  We have also demonstrated activities of the vGPCR in human trophoblasts, an important cell type of the placenta, which may be relevant to HCMV disease/transmission to the foetus during pregnancy (Figure 2).  HCMV UL33 and (to a lesser degree) US28 were both found to induce migration of trophoblasts toward a serum source.  This activity is associated with high constitutive signalling including activation of CREB (unpublished data).

Cre-lox switch’ viruses to identify vGPCR effects upon infection of specific cell types

We are employing a ‘Cre-lox switch’ approach to identify the cell types important for dissemination to/replication in different organs and the contribution of vGPCRs to cell-type specific functional effects.  The ‘Cre-lox switch’ approach uses a system that directs efficient rearrangements of DNA sequences containing lox sites in cells which express the Cre recombinase.  We have engineered mouse CMV with a ‘colour-switch’ cassette, where the virus changes from production of a green to a red fluorescent protein in response to Cre.  Using transgenic mice expressing Cre in specific cell types, the ability of virus to infect and subsequently spread from selected cell-types may be determined. By combining the ‘colour-switch’ with our panel of mouse CMV vGPR mutants, we can establish the contribution of the vGPCR to infection and dissemination involving specific cell types such as macrophages and endothelial cells (Figure 3).  We have also engineered mouse CMV with ‘gene-switch’ cassettes, where the expression of M33 will be either switched on or switched off in the presence of Cre.  These viruses will enable us to determine whether M33 is required for efficient infection of, or dissemination from, particular cell types.

CMV latency model in mice

The question of whether human and mouse CMV are similar with regard to sites/cell-types important for latency has been a matter of debate. Given the importance of latency/reactivation for CMV disease and transmission, determination of similarities/differences between human and mouse CMV is a key issue when considering the application of mouse CMV as a model for therapies designed to prevent establishment of, or repress reactivation from, human CMV latency. Monocyte precursors are believed to be an important reservoir for latent CMV in humans. Although hCMV DNA is detected at a low frequency in such cells, reactivation has been demonstrated upon stimulation of the cells to mature.  Recently, in collaboration with Rhonda Cardin (Cincinnati), we have been investigating the contribution of specific cell types as a reservoir for latent CMV in mice.  These studies suggest that mouse CMV is similar to human CMV with regard to low level infection of myeloid cells, including monocyte precursors, during latency.  In addition, stromal cells of the spleen and bone marrow are implicated as a site for latency – consistent with recent studies of human CMV. 

Auxiliary project areas

1. Tagged HSV-1 recombinants as neuronal tracing agents

The urge to cough is a ‘regulated reflex’ involving both brainstem and higher level control centres.  Dr. Mazzone’s group is interested in defining the anatomy, physiology and pharmacology of neural pathways regulating airway defensive reflexes.  By determining how sensory-related information arising from the airways is integrated into the central pathways regulating smooth muscle tone and cough reflex, Dr. Mazzone aims to describe how the nervous system contributes to symptoms of respiratory disease, thereby identifying novel targets for therapeutic intervention.  Mapping the sensory neuronal pathways from the airways to different areas of the brain will identify regions where airway sensory information is processed.

In collaboration with Dr. Mazzone and Miss Alice McGovern (UQ), we have utilised an HSV-1 strain (H129) that selectively transmits between neurons via the anterograde route (from cell body to axon tip) as a tool to map sensory neuron pathways. The H129 strain has been engineered to express fluorescent markers, including a ‘colour-switch’ version which will indicate whether the virus has infected specific regions of the brain before passing on to other areas.  These novel reagents enable tracing of neuronal pathways relevant to the respiratory tract and determination of differences between eg. tracheal versus lung sensory networks, relevant to responses to aggravating stimuli or infection.

Rats were infected with HSV-1 either in the trachea or lung and monitored for appearance of virus in specific regions of the brain.  The image shows three different brain regions, where either the tracheal and lung sensory pathways overlap (A’, B) or are mostly focussed in different areas (A’’).  The image shows neurons originating from virus inoculated either into the trachea (red) or lung (green) (McGovern et al, 2014, Brain Structure and Function (in press)).

2. Novel delivery of antiviral agents

In collaboration with Dr. Coombes (UQ, Pharmacy) and PhD student Ms Nhung Thi Tuyet Dhang, 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 have investigated, using HSV-2 as a model target virus (Asvadi et al, 2103) and extended to anti-HIV drug delivery (Dang et al 2014).  A new collaboration with Dr. Ben Ross (UQ, Pharmacy) and RHD student Mr. Eddy Lee will be investigating silica nanoparticles as a novel platform for blocking virus infection, via surface displayed virus-binding molecules.

 

Collaborations

In collaboration with the Virus Immunity research unit of Philip Stevenson (UQ, SCMB), we have been investigating the very earliest stage of CMV infection using the mouse model.  Through development of viruses that carry markers that enable virus-infected cells to be readily identified, we are determining the critical steps in virus infection and how the immune system responds to the virus.  Ultimately, these studies are seeking to enable development of effective vaccines against HCMV and other herpesviruses.

HCMV has considerable genetic variability.  In collaboration with David Warrilow (Public Health Laboratory Service), Lutz Krause (UQ, Diamantina Institute) and William Rawlinson (SEALS and Uni NSW), we have initiated a pilot project investigating HCMV sequence variability.  Ultimately this work is seeking to determine whether there are genetic markers for viruses which have either high, or low risk of causing congenital disease.

Our Staff and Students

This group is led by Associate Professor Nick Davis-Poynter.

Meet the rest of our team