Molecular Mechanisms Of The Interaction Between Host Restrictive Factors And RNA Viruses

Caprine arthritis encephalitis is a chronic disease caused by Caprine arthritis encephalitis virus(CAEV).The main feature of the disease in adult goats is chronic arthritis,and there may be complications such as interstitial pneumonia and interstitial mastitis;while lambs aged 2 to6 months show ascending paralytic encephalomyelitis symptom.Since 1985,the disease has been found in Gansu,Guizhou,Sichuan,Shaanxi,Shandong and Xinjiang provinces.After infecting ewes,lambs born from ewes are also infected with the disease,with an annual incidence of 16% to 19% and a fatality rate of 100%.At present,there is no vaccine or specific treatment for this disease on the market,so it is of great practical significance to study the mechanism of the interaction between the protein of the pathogen and the host restriction factor,then explore and develop specific drugs or treatment for this disease.CAEV is an enveloped RNA virus.Similar to other lentiviruses such as human immunodeficiency virus(HIV),CAEV also developed a series of interactions between viral proteins and host restriction factors during its long-term coevolution with its host.Goat oaA3Z2-Z3 protein is a homologous protein of the human apolipoprotein B m RNA editing enzyme catalytic subunit 3G(APOBEC3G,A3G).As a human host restriction factor,A3 G can induce the replacement of cytosine on HIV DNA with uracil,thereby inhibiting the normal replication of the virus,but it will be degraded by the vif of the HIV after the host is infected,thereby allowing the virus to escape from the A3 G protein restriction effect.According to previous literature review,we known that oaA3Z2-Z3 protein will also be degraded by CAEV Vif protein,but what is the specific mechanism? And whether the mechanism is similar to that of Medy-Vesna virus(MVV),which also belongs to small ruminant lentivirus(SRLV)subgroup is not clear.This study aimed at solving the above problems.Vif protein degrades host restriction factor by hijacking host protein and assembling into E3 ubiquitin ligase complex.The complexes recruited by the vif protein of different species of viruses will be different.In this study,we identified each protein required by CAEV-Vif to form the E3 ubiquitin ligase complex and found that,unlike the complex recruited by HIV-1-Vif(CUL5,ELOB/C and CBF-?),the complex hijacked by CAEV-Vif is composed of CUL5,ELOB/C and CYPA.In addition,this study revealed and identified the regions of interaction between CAEV-Vif and proteins recruited by CAEV-Vif through sequence alignment,prediction,truncation,and mutant construction.After verification,the binding area of CAEVVif and ELOB/C,namely BC box,is 170SLE172;the binding area with CUL5 is I141 and R142;the key area for binding with CYPA is P21;C132-C134-C154-C157 is used as zinc finger area,it is also involved in the binding of CAEV-Vif to CUL5.The binding sites of oaA3Z2-Z3 are Y39 and L44.Moreover,we determined that CAEV Vif assembled E3 ubiquitin ligase stepwise via its SLE motif(170SLE172)to recruit Elongin B/C,the P21 site and the zinc finger motif(C132-C134-C154-C157)to recruit CYPA,as well as the hydrophobic domain(141IR142)to recruit Cullin5.And finally forms the E3 ubiquitin ligase complex that degrades oaA3Z2-Z3.In addition to the above studies,the interaction between enterovirus and host restriction factor SAMHD1 is also studied in depth.According to the previous results of our laboratory,we found that SAMHD1 could inhibit enterovirus EVA71,but its antiviral mechanism remains unclear.In this study,the molecular mechanism of SAMHD1 inhibiting enterovirus EVA71 was explored in depth.Enteroviruses of the picornaviridae family contain 12 species,including 4 enteroviruses(EV-A to EV-D)and 3 rhinoviruses as human pathogens.Enterovirus type A,which is highly infectious,is the main pathogen causing the outbreak of infectious diseases and poses a great threat to children worldwide.Among them,enterovirus A71(EVA71),Coxsackie virus A6(CVA6)and Coxsackie virus A16(CVA16)are often associated with hand-food-mouth disease(HFMD)and Aseptic Meningitis(AM).Although the vast majority of enterovirus infections have no obvious clinical manifestation and can be cured on their own,the age of onset in the pandemic is only a few years and accounts for 76% of the total annual cases.In these cases,severe viral infections can cause brainstem encephalitis,central pulmonary edema and heart failure,and ultimately lead to death.In addition to poliovirus(PV),non-poliomyelitis enteroviruses such as Coxsackie virus,ECHO virus and several enteroviruses(such as EVA71 and EVD68)can also cause a variety of diseases,including myocarditis,HFMD and acute flaccid paralysis(AFP).In China,the main EV pathogens that cause HEMD are EVA71 and CVA16 subtypes.HFMD causes morbidity and even death in many children every year,so it is urgent to study all aspects of enterovirus.In this study,we explored the restriction mechanism of the host restriction factor SAMHD1 on enteroviruses,hoping to provide a theoretical basis and potential targets for the development of new drugs for HFMD.SAMHD1 has a variety of biological activities,such as virus restriction,innate immune regulation and autoimmunity.The most widely known function is that it can reduce the d NTP concentration in the cytoplasm to make the level of d NTP in the cell lower than the level required for virus replication,thereby inhibiting the replication of HIV and other viruses.Preliminary studies of our group have shown that the host restriction factor SAMHD1 has an inhibitory effect on EVA71,while the host factor TRIM21(an E3 ubiquitin ligase)induced by interferon(IFN)can specifically interact with and degrade SAMHD1 through the proteasome pathway.However,whether SAMHD1 can inhibit other enteroviruses and the molecular mechanism by which SAMHD1 inhibits these viruses remains unclear.In this study,we observed that SAMHD1 can also restrict a variety of enteroviruses,including CVA16 and EVD68,but not CVA6.In this study,we found that SAMHD1 can competitively bind the VP1 protein of EVA71 and EVD68 to the VP2 protein binding region,thus interfering with the interaction between VP1 and VP2 and thus interfering with viral assembly.In addition,we found that the SAMHD1 T592 A mutant can also exercise its inhibitory effect on EVA71 by weakening the interaction between VP1 and VP2,but the T592 D mutant cannot inhibit the replication of EVA71.We also proved that SAMHD1 cannot inhibit CVA6 because the binding site of SAMHD1 and VP1 does not completely overlap with the binding site of CVA6 VP1 and VP2.In conclusion,in this study,we identified that SAMHD1 had broad-spectrum antiviral activity against CVA16 and EVD68,but not against CVA6.And it was revealed for the first time that SAMHD1 affects the assembly of viral particle capsids by affecting the binding of VP1 protein and VP2.This discovery broadens our understanding of the molecular mechanism of host restriction factor SAMHD1 inhibiting enterovirus.The mechanism of interaction between SAMHD1 and enterovirus will help the development of drugs for HFMD.