Final Report Summary - MCV IMMUNOREGULATION (Identification of novel immunomodulators in the human poxvirus molluscum contagiosum virus) Infectious diseases kill 16 million people world-wide each year. Understanding how the immune system functions to sense and fight pathogens is essential for the development of novel therapeutics. Poxviruses have evolved a wide-spectrum of open-reading frames (ORFs) encoding immunoregulators which bind host proteins and disrupt the antiviral immune response. These ORFs are acquired from the host and are incorporated into the terminal regions of the viral genome. The acquisition of this extensive ‘toolbox’ of immunoregulatory sequences over long periods of host-virus co-evolution make them highly incisive tools for revealing novel facets of host immune system under the continual selective pressure of pathogens. Most research on poxviral immunoregulation has been conducted using the genome of Vaccinia virus (VV), a virus that is not adapted to human infection. Molluscum Contagiosum virus (MCV), is a human poxvirus that causes a long-term infection with a relatively weak immune response from the host. It has a highly diverse genome with largely unique terminal ORFs, only 10% of which currently have a known function. MCV is remarkable in poxvirus biology for several reasons. Firstly, it is the only known extant, natural human poxvirus and appears to be specifically adapted to human infection. Secondly, it displays an unparalleled ability to inhibit human anti-viral immunity and inflammation at sites of infection, since it often persists in skin lesions for months to years mostly without widespread inflammation and clearance. Thirdly, due to the ability of MCV to efficiently evade human immunity, an understanding of the mechanisms the virus uses to actualize this may present valuable avenues for therapeutic intervention of human inflammatory diseases. However there are no animal or cell culture based MCV infection models.In this project, we used biochemical and cell biology approaches to analyse unique ORFs of MCV and identify sequences that modulate human anti-viral immunity. Viral immunoregulators from MCV were identified and initially characterised with a view to the long term development of immunotherapeutics. In particular we report the discovery of four novel inhibitors of NFκB encoded by the MCV genome. NFκB is a critical host transcription factor in driving inflammation, and as such is an important therapeutic target. The four new MCV inhibitors identified were capable of inhibiting both pattern recognition receptor- and proinflammatory cytokine-stimulated NFκB activation, and also acted combinatorially. Host target proteins for the four MCV inhibitors were characterised, and in particular we elucidated the precise mechanism of one inhibitor, MC132, which has convergently evolved to target NFκB subunit p65 for degradation via cullin-5, in a similar fashion to the gammaherpesvirus Kaposi Sarcoma Herpesvirus.Two MCV proteins were also found to inhibit interferon regulatory factor (IRF) activation after cell stimulation. IRFs are critical anti-viral transcription factors, and further analysis of the mechanism of action of these MCV proteins is revealing insights into how such IRFs operate to mobilise an anti-viral response. Overall, the discovery of these novel MCV inhibitors of human immune transcription factors profoundly extends our knowledge of how this fascinating and mysterious poxvirus is likely able to evade human immunity to persist in skin. Further study of the MCV inhibitors will reveal new ways to manipulate human innate immunity.