The impact of viral gene expression and cellular signalling upon the control of HCMV latency and reactivation
Lead Research Organisation:
University College London
Department Name: Virology
Abstract
Human Cytomegalovirus is a herpes virus that co-exists within the human host for life. Usually benign in healthy adults, in certain patients whose body defence mechanism (immune system) against pathogens is switched off (i.e. transplant, cancer and AIDS patients) the virus spreads uncontrollably throughout the body. This results in severe disease and, if not brought back under control, can lead to death.
The ultimate aim is to understand how the virus remains dormant in humans and what host signals tell the virus to awaken (‘reactivate‘) from its‘ dormant state. By understanding what the signals are which reactivate the virus we can then design therapies to intercept that signal and prevent it from telling the virus to reactivate until the patients‘ immune system has recovered sufficiently to regain control over the virus. Furthermore, if we can understand how the virus hijacks the cells it resides in during its‘ dormant phase we can hopefully, by determining what the virus needs to survive, design novel therapies that could allow us to understand what the virus needs for long term survival in the host. Such an approach may kill the virus and result in the eradication of this pathogen from humans.
The ultimate aim is to understand how the virus remains dormant in humans and what host signals tell the virus to awaken (‘reactivate‘) from its‘ dormant state. By understanding what the signals are which reactivate the virus we can then design therapies to intercept that signal and prevent it from telling the virus to reactivate until the patients‘ immune system has recovered sufficiently to regain control over the virus. Furthermore, if we can understand how the virus hijacks the cells it resides in during its‘ dormant phase we can hopefully, by determining what the virus needs to survive, design novel therapies that could allow us to understand what the virus needs for long term survival in the host. Such an approach may kill the virus and result in the eradication of this pathogen from humans.
Technical Summary
Although primary infection with human Cytomegalovirus (HCMV) is usually asymptomatic in healthy individuals, infection and reactivation of immuno-suppressed AIDS, cancer and transplant patients, causes severe morbidity and mortality. Additionally, infection in utero occurs in approximately 1-2% of all live births and is associated with severe long-term consequences including blindness, deafness and mental retardation.
Previous studies on HCMV latency and reactivation suggest that the myeloid lineage is important. HCMV establishes latency in bone-marrow progenitor cells and reactivation is linked with the differentiation state of myeloid cells whereby myeloid cell maturation induces chromatin remodelling of the major lytic (‘reactivation‘) promoter into an active form concomitant with reactivation. However, the apparent contradiction between efficiency of HCMV reactivation in vivo compared with studies in vitro suggests that further, yet undefined, factors augment reactivation in vivo.
Firstly, I propose to study the impact of chromatin remodelling of the MIEP during differentiation in the context of inflammatory signalling induced by myeloid cell maturation. Specifically, I intend to identify and characterise signalling pathways involved in reactivation. Broad spectrum chemical inhibitors will be used to identify potential pathways involved and then more detailed analyses using adenoviral vectors to introduce mutant forms of signalling proteins, siRNA technology and more specific inhibitors of points in the pathway will be carried out. These analyses will be performed in an in vitro model that has been used in previously to analyse the molecular mechanisms of HCMV reactivation.
Secondly, I propose the further study of a recently identified transcript, UL81-82ast (LUNA), expressed during both lytic and latent infection. Preliminary data suggests that LUNA can disrupt ND10 bodies which maybe due to a potential de-sumoylase activity of the protein. To assess the role of the de-sumoylase function a mutant virus within the putative active site will be characterised for its ability to establish latency and reactivate using the in vitro system. Furthermore, expression of a SUMO protease would allow HCMV to target a number of diverse processes, thus I will perform a yeast 2 hybrid assay and then characterise positive interactions to give further insight into the precise role of this protein during latency, reactivation and lytic infection.
It is hoped that these studies will increase our understanding of how HCMV manipulates the host cell environment to so successfully establish both lytic and latent infections, and ultimately lead to the identification of key signalling pathways that promote reactivation providing a target for therapeutic intervention.
Previous studies on HCMV latency and reactivation suggest that the myeloid lineage is important. HCMV establishes latency in bone-marrow progenitor cells and reactivation is linked with the differentiation state of myeloid cells whereby myeloid cell maturation induces chromatin remodelling of the major lytic (‘reactivation‘) promoter into an active form concomitant with reactivation. However, the apparent contradiction between efficiency of HCMV reactivation in vivo compared with studies in vitro suggests that further, yet undefined, factors augment reactivation in vivo.
Firstly, I propose to study the impact of chromatin remodelling of the MIEP during differentiation in the context of inflammatory signalling induced by myeloid cell maturation. Specifically, I intend to identify and characterise signalling pathways involved in reactivation. Broad spectrum chemical inhibitors will be used to identify potential pathways involved and then more detailed analyses using adenoviral vectors to introduce mutant forms of signalling proteins, siRNA technology and more specific inhibitors of points in the pathway will be carried out. These analyses will be performed in an in vitro model that has been used in previously to analyse the molecular mechanisms of HCMV reactivation.
Secondly, I propose the further study of a recently identified transcript, UL81-82ast (LUNA), expressed during both lytic and latent infection. Preliminary data suggests that LUNA can disrupt ND10 bodies which maybe due to a potential de-sumoylase activity of the protein. To assess the role of the de-sumoylase function a mutant virus within the putative active site will be characterised for its ability to establish latency and reactivate using the in vitro system. Furthermore, expression of a SUMO protease would allow HCMV to target a number of diverse processes, thus I will perform a yeast 2 hybrid assay and then characterise positive interactions to give further insight into the precise role of this protein during latency, reactivation and lytic infection.
It is hoped that these studies will increase our understanding of how HCMV manipulates the host cell environment to so successfully establish both lytic and latent infections, and ultimately lead to the identification of key signalling pathways that promote reactivation providing a target for therapeutic intervention.
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| Matthew Reeves (Principal Investigator / Fellow) |