Structural And Functional Insights Into Polymerase Of Influenza D Virus

The influenza virus is one of the important human pathogens,and it has a significant impact on public health and economic development.The influenza virus belongs to the Orthomyxoviridae family and is a type of single-stranded,negative-sense,segmented RNA virus.The influenza virus polymerase is responsible for the replication and transcription of the viral genome and is highly conserved among different influenza strains.The polymerase and is also closely related to host adaptability.In the past few decades,the structure of the influenza virus polymerase has been studied relentlessly,but only partial domain information of the polymerase has been obtained[5].Since we do not have a high-resolution structure of the intact influenza virus polymerase,it severely hinders our understanding of the transcription and replication processes of influenza viruses at the molecular level.It is difficult for the influenza virus polymerase to be structurally analyzed mainly because of its excessive molecular weight and,low expression level in the recombinant system[6].Until recently,the expression and purification techniques of macromolecular complexes have advanced rapidly,and the crystal structures of intact bat influenza A virus polymerase,influenza B virus polymerase and influenza C virus polymerase have been resolved[7-8].The subject of this study is the influenza D virus polymerase.Of the Orthomyxoviridae family of viruses,influenza A,B and C viruses are well known and all can cause disease in humans.Recently,a new influenza D virus(IDV)was isolated from pigs and cattle.The genome of newly-identified IDV virus showed approximately 50%overall amino acid homology to ICVs and was unable to reassort with human ICVs.So far,more IDV strains or viral genomic segments were identified in United States,France,China and other areas,implicating the wide geographic distribution of IDV.IDV has a broader cell tropism than human ICV and is capable of infecting ferrets,pigs and guinea pigs.The influenza virus polymerase plays a central role not only in the replication of the virus,but also in the interspecies transmission of the virus[9].Currently,the main measures to deal with influenza virus infection are vaccination and medical treatment.It is difficult to effectively immunize high-risk groups such as the old man and the children,and also the vaccination can not provide broad and longlasting immune protection due to the continuous quick mutation of influenza virus strains.Therefore,medical treatment with drugs remains the alternative stragety for controlling influenza.Influenza polymerase plays a pivotal role in viral infection and has conservation in different strains,so it is an promising target for the design of broadly-reactive drugs.In this study,by using the baculovirus expression system to express three polymerase subunits(P3,PB1 and PB2)in tandem,a uniformly stable influenza D virus polymerase(FluDPol)protein was successfully obtained and in vitro enzyme activity was obtained.The system verified that FluDPol has transcription,replication and endonuclease activities,and found that enzyme activity is different from the other three types of FluPol.The FluDPol could efficiently produce full-length(FL)cRNA product using the vRNA promoter,with a small fraction of short product(SP)that might result from aberrant termination of the elongation process.In contrast,most of the vRNA product synthesized via cRNA template was SP,possibly a result of aberrant termination or internal initiation,with very low efficiency for producing FL vRNA product.The unique enzymatic behavior of FluDPol indicates that vRNA synthesis process is probably much more dependent on other viral or cellular factors in the host cells as compared to the other three types of FluPols.In this study,the three-dimensional structure of the influenza A virus polymerase was successfully analyzed by means of cryo-EM.The results show that FluDPol has a very similar overall structure compared to the other three types of FluPol,most similar to FluCPol.In the cryoelectron structure of FluDPol,the C-terminal domain of the PB2 subunit contains a cap binding domain.Because its conformation is very flexible,it is impossible to obtain a clear electron density map and resolve its three-dimensional structure,while the P3 subunit The endonuclease domain and the NLS domain of the PB2 subunit have multiple flexible conformations.This result indicates that the state of FluDPol in solution is not a static unique conformation,it can be in a state of dynamic equilibrium between a variety of different conformations,including closed and open conformations.These findings have given us new insights into the regulatory mechanisms of transcriptional replication of influenza virus polymerases and provide a valuable scientific reference for us to better understand the replication mechanisms of influenza viruses.