Identification Of Interaction Sites Between Chicken CARD11 Protein With Newcastle Disease Virus Phosphoprotein And Influence On Virus Properties

Virus replication and pathogenicity are determined by both the virus itself and the host.Newcastle disease virus（NDV）is a virus that causes virulent disease in birds and its replication is strictly dependent on the ribonucleoprotein（RNP）complex,which is composed of polymerase large protein（L）,phosphoprotein（P）,nucleoprotein（NP）and genomic RNA,and is the basic unit of NDV replication and transcription.The host factors also influence the function of the RNP complex.In our laboratory,a host caspase recruitment domain family protein 11（CARD11）,which inhibits NDV replication,was identified as a scaffolding protein that controls key signals for antigen-induced lymphocyte activation in the adaptive immune response.The CC1 domain of chicken CARD11（CARD11-CC1）inhibits viral replication by competing with NDV L proteins to bind the X domain（P-X）of NDV phosphoprotein to block P-L protein interactions and reduce viral RNA polymerase activity.However,the precise site of CARD11-CC1 binding to P-X is not known.In this study,the following results were obtained by using protein bioinformatics,immunoprecipitation（co-IP）,microgenomic,and reverse genetics techniques.1.Analysis of CARD11-CC1 interaction amino acid sitesUsing bioinformatics to simulate the protein structure of CARD11-CC1,a series of truncated bodies were constructed and the reciprocal region was identified at amino acids255 to 264 by co-IP with phosphoprotein;then a point mutant of CARD11-CC1(²⁵⁵LKNDIENRPK²⁶⁴)was constructed,and the key amino acid site was shortened to256KNDIENR262 by co-IP with phosphoprotein.Detection by dual luciferase reporter minigenome system revealed that K256A,D258A,E260A,N261A and P263A lost their inhibitory effect on viral RNA polymerase activity compared with the control.2.Analysis of phosphoprotein interaction amino acid sitesAfter simulating the protein structure of P-X,we constructed a series of phosphoprotein truncates and then performed co-IP with CARD11-CC1 to identify the interacting region at amino acids 385 to 388;then we constructed a P(³⁸⁵IRKI³⁸⁸)point mutant and found that all four mutations affected the reciprocal interaction of P-CC1 by co-IP,and detection by dual luciferase reporter minigenome system revealed that all four loci significantly reduced viral RNA polymerase activity after single mutation.3.Effect of P protein R386 mutation on virus characteristics and antiviral role of CARD11Full-length genomic plasmids with P protein reciprocal site mutations I385A,R386A,K387A,and I388A were constructed using the infectious clone of the strong F48E9 strain as the backbone,and cotransfected with the helper plasmids p CMV-NP-P-L and p CAGGS-T7in BHK-21 cells to rescue the recombinant mutant virus r F48E9/P（R386A）.Other recombinant mutant viruses failed to be rescued successfully,and it is possible that mutations at these amino acid sites are lethal to the rescued virus.After infection of BHK-21cells with recombinant virus at 0.01 MOI,Western blot and viral proliferation results showed no significant change in P protein expression and viral proliferation ability of r F48E9/P（R386A）compared to r F48E9.However,the r F48E9/P（R386A）syncytium formation ability was reduced and the viral plaque size became smaller.The results of pathogenicity index MDT and ICPI indicated that the virulence of r F48E9/P（R386A）was weakened to medium to strong virulence.The recombinant virus was infected with CARD11overexpressing and knockdown DF-1 cells at 0.01 MOI,respectively,and it was found that the expression of CARD11 significantly inhibited the proliferation of r F48E9 but not r F48E9/P（R386A）.After the recombinant virus infected 4-week-old chicks with 105PFU/each,the mortality of the control r F48E9 was 100%,and the clinical symptoms and pathological changes were significant,but r F48E9/P（R386A）did not cause significant clinical symptoms and pathological changes,and no lethality.The above results suggest that the P protein R386 locus plays a key role in virulence,pathogenicity and antiviral of CARD11.In summary,this study identified the key amino acid sites of CC1-X interactions as³⁸⁵IRKI³⁸⁸of P and ²⁵⁶KNDIENR²⁶²of CARD11,all of which affected viral RNA polymerase activity.Meanwhile,the mutant virus r F48E9/P（R386A）was successfully rescued,and R386is more critical for virus virulence and pathogenicity.In this study,the precise mechanism of NDV P protein antagonizing NDV by interacting with CARD11 amino acid sites was elucidated,which laid the foundation for further understanding of NDV-host interactions and enriched the molecular virology of NDV,while providing new strategies for the development of antiviral drugs.