Identification Of Host Protein Interactions For Species A Rotavirus VP4 And Study Of Their Role And Mechanism In Viral Infection

Rotavirus gastroenteritis is an acute and highly contagious zoonotic disease caused by rotavirus,with severe symptoms including diarrhea,vomiting,and even death in young animals and infants.Rotavirus spreads widely among various mammals and birds,including pigs,cows,sheep,dogs,cats,horses,rabbits,mice,deer,camels,donkeys,bats,chickens,ducks,and pigeons,significantly affecting animal husbandry,companion animal breeding,and wildlife disease control in multiple veterinary medicine-related fields.Rotavirus belongs to the family Reoviridae,and is a non-envelope and doublestranded RNA(ds RNA)virus with 11 segments,and species A is the most epidemic.Rotavirus possesses 6 structural proteins and 5 non-structural proteins,comprising triple layers of capsid structure.The outer layer consists of VP4 and VP7 proteins,with VP4 primarily mediating rotavirus entry,playing a crucial role in determining the rotavirus tropism.Although several carbohydrates or proteins such as Histo-blood group antigens,sialic acid,integrins,and heat shock protein family A(Hsp70)member8(HSPA8,also known as HSC70)have been reported to be involved in rotavirus entry,the discovery of molecules related to rotavirus entry,particularly proteins,remains limited compared to other significant zoonotic viruses.Furthermore,research on VP4 interacting proteins is insufficient,with only one systematic identification of VP4 interacting proteins currently reported.Therefore,this study aims to identify interactions between VP4 and host proteins to discover key host proteins associated with species A rotavirus infection and conduct functional,mechanistic,and drug studies.This study applied Turbo ID proximity labeling to identify VP4 interacting proteins.This method,reported in 2018,enables biotinylation of neighboring proteins within 10 n M,facilitating identification of interacting proteins through mass spectrometry.Through this method,174 high-confidence VP4 interacting host proteins were identified.Bioinformatics analysis indicates that these proteins are not only associated with mediating virus entry into host cells but also with RNA binding and ds RNA binding functions,suggesting that VP4 interacting proteins may be involved not only in rotavirus entry but also in innate immunity and virus replication functions.Consequently,this study focuses on the selection and functional validation of VP4 interacting proteins from two aspects: the primary biological function of VP4,which is mediating rotavirus entry,to discover proteins associated with virus entry,and analysis of proteins related to innate immunity and replication.Proteins involved in virus entry should be located in the extracellular region,plasma membrane,and cytoskeleton.Subcellular localization analysis revealed 88 VP4 interacting proteins located in these regions.Further bioinformatics analysis and experimental validation identified Vimentin(VIM)and Actin Related Protein 2(ACTR2)as promoting rotavirus infection in intestinal cells.Regarding the analysis of proteins related to innate immunity and virus replication,6 members of the DEx D/Hbox Helicase family were identified as VP4 interacting proteins.Among them,DEADBox Helicase 3 X-Linked(DDX3X)showed the highest biotinylation abundance and was located in the interferon activation domain,suggesting its potential crucial role in rotavirus infection.Therefore,VIM,ACTR2,and DDX3 X were selected for further research.Further confirmation of the roles of VIM and ACTR2 in rotavirus infection showed that both proteins promote rotavirus infection and interact with VP4.Experiments involving gene knockdown,antibody blocking,and recombinant protein incubation demonstrated that VIM and ACTR2 facilitate rotavirus binding to HIEC-6 cells,thus promoting rotavirus infection.Non-histone protein acetylation modification plays a significant role in protein interactions.Another study by our group identified the most significant decrease in acetylation modification at the K439 site of VIM after rotavirus infection.Point mutation experiments revealed that simulating deacetylation modification K439 R mutation reduced the interaction ability between VIM and VP4,and subsequent infection experiments showed that the K439 R mutation reduced VIM’s ability to promote rotavirus infection.These findings suggest that deacetylation modification at the VIM K439 site reduces virus infection by lowering its interaction ability with VP4,demonstrating the host’s antagonistic role against virus infection.Investigation of upstream regulators of VIM revealed that its expression can be regulated by thrombospondin 1(THBS1),which promotes VIM expression and rotavirus infection.THBS1 abundance significantly decreased after MYAS33 strain rotavirus infection of HIEC-6 cells,also reflecting the host’s antagonistic mechanism against rotavirus infection.Study on DDX3 X showed that it promotes rotavirus infection and assists rotavirus in suppressing the production of interferon lambda 1(IFNL1).Further studies revealed that VP4 binds to the interferon activation domain of DDX3 X,inhibiting DDX3X’s IFNL1 induction effect.Additionally,the enzymatic activity region of DDX3 X plays a crucial role in promoting rotavirus infection,with the inhibitor RK-33 exerting inhibitory effects on rotavirus infection.Based on the completion of the above phenotypic identification and mechanistic studies,as VIM,ACTR2,and DDX3 X all play facilitating roles in the process of rotavirus infection,this study conducted experiments with relevant inhibitors to explore their potential as antiviral agents against rotavirus infection.The results demonstrated that the VIM inhibitor Fi Ve 1 and the DDX3 X inhibitor RK-33 exhibited anti-rotaviral effects both in vitro and in mice,while the ACTR2-related inhibitors CK-636,CK-666,and Benproperine phosphate showed anti-rotavirus activity in vitro.In summary,using Turbo ID proximity labeling technology,this study identified a total of 174 host proteins interacting with rotavirus VP4.Through bioinformatics analysis and preliminary experimental results,three molecules,VIM,ACTR2,and DDX3 X,not previously reported in rotavirus infection,were selected for investigation.The findings indicate that all three molecules can facilitate rotavirus infection,elucidating their respective mechanisms and identifying potential candidates for antiviral therapy against rotavirus infection.This research not only expands our understanding of rotavirus-host interactions,providing a theoretical basis for the development of antiviral drugs against rotavirus,but also opens new directions and perspectives for the discovery of new drug targets in the interactions between other viruses and hosts.