The Molecular Mechanism Of Ectromelia Virus Protein PAPL Inhibiting The Antiviral Activity Of Host GBP2

Since the report of human infection with monkeypox virus in the UK in May 2022,concerns regarding poxvirus epidemics have resurfaced.This has emphasized the urgent requirement for the development of novel vaccines and antiviral drugs to effectively combat monkeypox virus and other orthopoxvirus infections.However,the study of monkeypox virus pathogenic mechanisms is somewhat limited due to the requirement for biosafety level three laboratories.Ectromelia virus（ECTV）is a nucleoplasmic large DNA virus,closely related to monkeypox virus and variola virus,which belongs to the Orthopoxvirus genus of the Chordopoxvirinae subfamily,exhibits similarities in genome structure,genetic characteristics,and disease characteristics.This makes it a valuable model virus for investigating the pathogenesis of orthopoxviruses and their interaction with hosts.Guanylate-binding protein（GBP）,as a member of the interferon-induced GTPase superfamily,plays a significant role in the host’s defense against various pathogens,including viruses,bacteria,and parasites.While the antiviral innate immunity function of GBP has been extensively studied,particularly for RNA viruses,there is limited research on DNA viruses,specifically poxviruses.This study aims to utilize ECTV as a model virus to elucidate the role of GBP in resisting ECTV infection.Through transcriptome and q PCR analysis,it was observed that ECTV infection significantly increased the expression of GBP^chr3 both in vivo and in vitro,particularly GBP2.Consequently,our study aims to investigate the antiviral effect of GBP2 on ECTV.Our findings demonstrate that overexpression of GBP2 has a dose-dependent inhibitory effect on ECTV replication.Furthermore,when the endogenous expression levels of GBP2 were knockdown,ECTV replication significantly increased,suggesting the crucial role of GBP2 in host defense against ECTV infection.Notably,GBP2primarily restricts ECTV replication through its N-terminal GTPase domain.Mutations in the key GTP binding site K51 of GBP2 impair its inhibitory effect on viral replication.However,studies have indicated that despite the high transcriptional expression levels of GBP2,its antiviral effect is not very pronounced.This raises the question of whether the antagonism of virus-encoded proteins is responsible,considering the abundance of such molecules in the poxvirus genome.Through IP/MS and Co-IP technology,it was found that the poly（A）polymerase catalytic subunit（PAPL）protein of ECTV is a viral regulatory molecule that interacts with GBP2.PAPL promotes the degradation of GBP2 by the ubiquitin-proteasome pathway,thereby counteracting the antiviral activity of GBP2.Knocking out the PAPL gene of ECTV with CRISPR/Cas9 system,significantly diminish the replication ability of the virus and enhance the antiviral activity of GBP2,indicating the indispensable role of PAPL in the replication process of ECTV.Overall,this study is the first to explore the antiviral effect of GBP2 on ECTV.It also identified the viral protein that interacts with GBP2,clarified the molecular pathway of their interaction,and revealed a new mechanism by which the ECTV-encoded protein PAPL antagonizes the host immune response.These findings lay the groundwork for further exploration of the interaction mechanism between poxviruses and their hosts and also provide a theoretical foundation and technical knowledge for the development of safe and effective vaccines and antiviral drugs.