Viral manipulation of DBC1: a novel strategy to promote cell survival and suppress inflammation

Human and animal cells have very sophisticated networks to communicate and respond to stress. Infection by a virus is a stress that can lead to the death of the cell. Accordingly cells respond vigorously to viral infection with the aim of blocking viral multiplication and alerting the body's immune system to the ongoing infection. Only viruses that have the capacity to avoid this hostile cell response survive. How viruses achieve this is not always clear. However, if and when one of these viral strategies is discovered, an opportunity for the development of antiviral interventions emerges. This project concerns the discovery of one of those strategies employed by a group of viruses named poxviruses, a member of which was responsible for the devastating disease smallpox. The smallpox virus killed more people in recorded history than all other infectious diseases combined, but fortunately was eradicated thanks to a worldwide vaccination campaign that used vaccinia virus (VACV), another member of the poxvirus family. VACV is currently being studied as a vaccine against another smallpox-like disease known as monkeypox as well as other several important human and animal diseases such as tuberculosis, AIDS, or rabies.
This project will study how poxviruses manipulate the activity of a cellular protein termed DBC1. DBC1 belongs to one of those communication networks that cells use to respond to external stress. Its main role is to block the action of another protein known as SIRT1. Both DBC1 and SIRT1 were only discovered ~10 years ago, so our understanding of how they work is still in its infancy. However, it is now clear that both DBC1 and SIRT1 are very important in the process of ageing and in age-related diseases such as cancer and chronic inflammation.
This project demonstrates that poxviruses specifically bind and relocalise DBC1, the negative regulator of SIRT1, in a part of the cell where SIRT1 is not normally present. This suggests that poxviruses break the DBC1-SIRT1 connection and benefit from this in a number of ways that are not fully understood yet. This project will therefore determine 1) how poxviruses sequester DBC1 away from SIRT1, 2) what advantage this has for the virus, and 3) what consequences this viral action has in disease and vaccination. To address how, detailed molecular biology and protein localisation studies will be developed. To address why, we will conduct a series of functional tests in cells previously modified to lack DBC1, SIRT1 or both genes. The response of these cells to infection will be studied and compared to that of normal cells. The conclusions from these studies will establish for the first time the role of the DBC1-SIRT1 axis during infection with viruses, and will provide valuable information not only for emerging poxviruses such as the monkeypox virus, but perhaps also for other viruses with similar biology such the African Swine Fever virus - an emerging, economically important pig pathogen that might also sequester DBC1.
Finally, given the importance of VACV as a vaccine, the project will study the impact of the DBC1-SIRT1 axis in vaccination using viruses modified in the laboratory to relocate DBC1 or not. The response to vaccination is known to be complex and to change with age. If the modified virus that does not break the DBC1-SIRT1 connection triggers a better immune response, this modification can be introduced into VACV-based vaccines that are currently being developed. Therefore this project has the potential to impact on the design of vaccines and to improve our understanding of ageing and its biology.

The mechanisms by which viruses manipulate cell homeostasis and divert cellular signalling not only provide new therapeutic avenues for antiviral strategies, but also identify key cellular factors and hubs that may be aberrant or non-functional in other diseases. We have recently discovered a new strategy exploited by viruses to hijack a host nuclear protein termed Deleted in Breast Cancer (DBC)-1, an inhibitor of the family of universally conserved histone deacetylases known as Sirtuins (SIRTs). Using state-of-the-art proteomics and microscopy imaging we have identified a poxvirus protein that sequesters DBC1 in the cytosol forming aggregates with mitochondria. We hypothesise that poxvirus relocation of DBC1 into the mitochondrion serves multiple purposes such as inhibition of cell death and inflammatory signalling as well as the de-repression of SIRTs in the nucleus.
This project aims to understand how exactly poxviruses manipulate DBC1, and the contribution of this viral strategy to virulence and pathogenesis. To study mechanism, we will conduct extended mutagenesis and cellular assays on DBC1 and its viral antagonist. To determine purpose, we will study apoptotic and inflammatory responses in the presence of the viral antagonist and mutants thereof as well as during viral infection in cells edited for DBC1 or SIRT1. Finally, we will establish the impact of targeting of DBC1 in virulence and pathogenesis as well as in immunisation.
In summary, this project uncovers the first-in-class DBC1 antagonist encoded by a pathogen and reveals an exciting and unexpected connection between viruses and the DBC1-SIRT1 axis. This work will provide unprecedented insights into host-pathogen interactions relevant not only to poxviruses but potentially other animal and human viruses. In addition, given the importance of SIRTs in age-related diseases our work will impact our understanding of the regulatory mechanisms of SIRT1 in disease.

This study will have a global benefit on public health, as well as direct benefits for the private sector in the following ways -
Anti-viral strategies. Between the 8 and 26 September 2018 3 cases of monkeypox were diagnosed in the UK for the first time ever. The patients are believed to have acquired the infection in Nigeria, where a large-scale, sustained epidemic of monkeypox virus (MPXV) is ongoing since September 2017. The risk of MPXV adds to the long-standing concern for the use of variola virus as a bioterrorism agent. There are no vaccination programs in place against poxviruses after the eradication of smallpox and the vast majority of the population has become immunologically naïve. An outbreak of MPXV and/or VARV in the UK could have catastrophic consequences given the high mortality rates of these viruses. Our work with vaccinia virus as well as ectromelia virus, the most appropriate model for human smallpox and monkeypox, will contribute to our understanding of poxvirus pathogenesis and host range and the emergence of zoonotic outbreaks. Furthermore, our studies with SIRTs can be expanded to other human and animal viruses, some of which such as the African Swine Fever virus share very similar biology with poxviruses, and so they have the potential to illuminate new anti-viral interventions.
Immunomodulation. The global market for immunomodulators was worth $131.7 billion in 2015 and is expected to reach $233.7 billion by 2025. Immunosuppressants, immunostimulants, vaccines and antibodies will be the four product segments to lead, in this order. The increasing incidence of autoimmune and inflammatory disorders is thought to be the key factor responsible for this huge increase. Chronic diseases including asthma, cancer, allergic conditions, and multiple sclerosis are observing a significant growth. In addition, there is increasing drugs resistance and a larger number of transplantation procedures and the need to prevent organ rejection. Taken together, the pressure to meet these clinical needs is rising and the generation of new immunomodulators with lower adverse effects and targeted therapy mechanisms is pressing. This project will uncover how viruses manipulate cellular inflammatory responses via DBC1 and SIRTs, thereby generating original and exciting knowledge that can lead to novel immunomodulatory strategies.
Cellular biology and cancer. Our discovery that viruses have evolved to target DBC1 indicates that this protein controls important functions in the cell. Indeed our data suggest that targeting of DBC1 enhances the survival of the cell and inhibits the responses that lead to cell death. These are properties typically associated with cancer cells. Our work deciphering how poxviruses manipulate DBC1 and DBC1-controlled responses will highlight novel connections and regulatory mechanisms that govern cell viability and might be exploited by viruses and cancer cells alike.
Ageing. Since their discovery SIRTs have been tightly linked to cell proliferation, cell division and ageing. SIRTs control multiple metabolic responses in the cell including mitochondrial biogenesis, energy consumption and genotoxic stress. Therefore SIRTs and their regulatory mechanisms are master regulators of cell homeostasis. Despite the overarching role of SIRTs in ageing and inflammation the role of SIRTs in viral infections has not been appreciated so far. Our studies using viruses as pathogens as well as vaccine vectors have the potential to reveal new interesting features about SIRTs and the biological mechanisms underpinning the biology of ageing.