Avian Marek's Disease Virus Mediates The DNA-sensing Pathway To Escape Host Innate Immune Response

Herpesviruses are important pathogens associated with a wide range of diseases in humans and animals.In particular,Marek's disease virus(MDV)constitutes a highly pathogenic and oncogenic herpesvirus of chickens.As a disease that affects poultry worldwide with economic implications,Marek's disease(MD)has contributed substantially to our understanding of herpesvirus-associated oncogenicity.MD lymphomas exhibit many biological parallels with the lymphoid neoplasias associated with human herpesviruses such as Epstein-Barr virus(EBV).Despite the success of vaccination in controlling MD over the last 40 years,continuous evolution of virulence among MDV strains remains a major challenge for sustainable control of this disease.Evasion of the host innate immune response is essential for herpesviruses to successfully establish infection,latency,and lifelong persistence in the host.Innate responses are initiated upon the detection of invading pathogens by various host pattern-recognition receptors that recognize conserved pathogen-associated molecular patterns and trigger the production of type I interferons(IFNs)and other antiviral factors.In addition to Toll-like receptors,retinoic acid-inducible gene I-like receptors,and Nod-like receptors,several cytosolic DNA sensors have been recently discovered.Among these DNA sensors,cyclic GMP-AMP(cGAMP)synthase(cGAS)is currently considered the principal sensor of cytosolic DNA in different cell types.Recently,the cGAS-STING DNA-sensing pathway was reported to play an important role in type I IFN responses against human herpesviruses.Moreover,a number of viral proteins that inhibit type I IFN production through modulation of this signaling pathway have been identified.However,how avian DNA viruses target the cGAS-STING axis for immune evasion remains largely unknown.A better understanding of MDV-host interactions is,therefore,important to not only elucidate the events in oncogenesis but also develop more sustainable control strategies to combat infectionIn this study,we found that the cGAS-STING signaling pathway is critical for the production of MDV-induced IFN-?.MDV antagonizes host innate immune responses during the late phases of viral infection.We screened MDV proteins for their ability to inhibit the cGAS-STING pathway and identified five viral proteins that block IFN-? activation through this pathway,including Meq?RLORF4?US3?UL46?VP23.The major oncoprotein of MDV,Meq,targeted STING by preventing its interaction with TANK-binding kinase 1 and interferon regulatory factor 7(IRF7),thereby leading to the inhibition of IRF7 activation and IFN-? induction during viral infection.Meq overexpression markedly reduced antiviral responses stimulated by cytosolic DNA,whereas knockdown of Meq heightened MDV-triggered induction of IFN-? and downstream antiviral genes.Moreover,Meq-deficient MDV induced more IFN-? production in vitro and in vivo than wild-type MDV.The levels of CD8+ T cell responses induced by Meq-deficient MDV were higher than those induced by wild-type MDV.Conversely,wild-type MDV replicated more efficiently and induced higher rates of lymphoma than the Meq-deficient mutant.Taken together,these results revealed that MDV antagonizes STING-mediated DNA sensing for immune evasion,which is essential for viral replication and oncogenesis.These findings improve our understanding of the virus-host interaction in MDV-induced lymphoma.Here,we also report that RLORF4,an MDV-specific protein directly involved in viral attenuation,is an inhibitor of the DNA-sensing pathway.The results showed that ectopically expressed RLORF4 blocked IFN-? promoter activation induced by cyclic GMP-AMP synthase(cGAS)and stimulator of interferon genes(STING).RLORF4 selectively inhibited the activation of NF-?B but not IFN-regulatory factor 7.RLORF4 was found to bind the endogenous NF-?B subunits p65 and p50,and it also bound to the Rel homology domains of these subunits.Furthermore,RLORF4 suppressed the tumor necrosis factor alpha-mediated nuclear translocation of p65 and p50.Finally,deletion of RLORF4 from the MDV genome promoted IFN-? production in vitro and in vivo.In the absence of RLORF4,the host cellular immunity was significantly increased,and reduced viral titers were observed during infection of chickens.Our results suggest that the RLORF4-mediated suppression of the host antiviral innate immunity might play an important role in MDV pathogenesis.In summary,our findings suggest a new role of the MDV oncoprotein Meq and RLORF4 in inhibition of the DNA-sensing mediated IFN-? production.Given the multiple roles played by the cGAS-STING axis in not only the recognition of a variety of pathogens and tumor-derived DNA but also the induction of antitumor immunity and tumor cell-specific apoptosis.This study not only reveals a very important mechanism for MDV to escape the host innate immune response,but also explains why MDV infection induces the host to produce tumor so quickly.