Expression of Kaposi’s sarcoma herpesvirus vFLIP, a potent activator of NFkB signaling, promotes latency. Inhibition of NFkB signaling promotes lytic reactivation. We previously reported that lytic inducer, RTA, inhibits vFLIP induced NFkB signaling by inducing the degradation of vFLIP via the proteasome. Here we report that the cellular ubiquitin ligase, Itch, is required for RTA induced degradation of vFLIP. Expression of either Itch targeting shRNA or a dominant negative mutant of the ubiquitin ligase both increased the stability of vFLIP in the presence of RTA. Itch potently ubiquitinated vFLIP in vivo and in vitro. We provide evidence for interaction between RTA, vFLIP and Itch and we identified an RTA resistant mutant of vFLIP that is unable to interact with Itch. These observations contribute to our understanding of how RTA counteracts the activities of vFLIP.

Pertussis is an infectious disease caused by the bacterial pathogen Bordetella pertussis. In recent years, there has been a re-emergence of pertussis disease. Pertussis morbidity is extensive and spans across all age groups, but a higher incidence of severe disease and mortality is exhibited in young infants. Mortality is associated with severe respiratory infection and other systemic secondary co-morbidities. The infant immune system has reduced capacity to generate proinflammatory/T-helper (Th) 1 cell polarizing responses. Secretion of Interferon gamma (IFN-γ), a Th1 polarizing cytokine, by immune cells plays a critical role in macrophage activation and microbicidal activity in the lung. Furthermore, B. pertussis respiratory challenge of adult IFN-γ receptor deficient mice results in systemic dissemination and lethality. We hypothesize that the dissemination and lethality observed in our neonatal mouse model is a result of inadequate IFN-γ signaling or production, which contribute to insufficient control of the infection. We found that B. pertussis infection results in a significantly lower level of transcription of IFN-γ in the lungs and increased bacterial dissemination in neonatal mice when compared to adult mice. Additionally, infected neonatal mice deficient in IL-10, a potent regulator of IFN-γ, showed reduced dissemination and significantly increased IFN-γ and other pro-inflammatory cytokines as compared to age-matched wild type mice. Future studies include characterization of IFN-γ secreting immune cells during infection. Results generated by this work will contribute to a better understanding of severe disease in infants, addressing the limitations of infant immunity and treatment options for pertussis.

KSHV vFLIP is a potent activator of NFκB signaling and an inhibitor of apoptosis and autophagy. Inhibition of vFLIP function and NFκB signaling promotes viral reactivation. Here we provide evidence for a novel function of vFLIP in promoting NFߢB signaling through inhibition of the DUB activity of the negative regulator, A20. We demonstrate interaction of vFLIP with the Itch/A20 ubiquitin editing complex. We have identified a SUMO interaction motif in vFLIP that is required for NFκB activation. We observed a decrease in vFLIP induced NFκB when the SIM in vFLIP was mutated. Small molecule inhibition of SUMOylation resulted in a dose dependent increase lytic reactivation and virus production. Our results suggest a role for SUMO in mediating vFLIP function and provide evidence for vFLIP modulation of the negative regulation of NFκB signaling by A20. Our results provide further insight into the function of vFLIP and SUMO in the regulation of NFκB signaling and the latent lytic transition.