The Study Of Structure And Function Of M1 Protein Of Infectious Salmon Anemia Virus

Many enveloped viruses encode matrix proteins,including orthomyxoviruses,paramyxoviruses,retroviruses and filoviruses.Viral matrix proteins are called virus assembly ?adaptors? because they interact with both the viral envelope and the viral ribonucleoprotein(RNP)complexes,thus facilitating the incorporation of the viral genome into newly assembled viruses as well as virus budding.All members of the Orthomyxoviridae family,including the influenza viruses A,B,and C,thogotovirus,and isavirus,encode a matrix protein called M1.In the influenza A virus,the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses.The influenza virus M1 is also known to mediate nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particlesas well as virus budding.Despite extensive studies on the influenza virus M1,only crystal structures of its N-terminal domainare available.Infectious salmon anemia virus(ISAV)is an important orthomyxovirus that causes high mortality in farmed Atlantic salmon.ISAV and influenza share many similarities in terms of virus structure,genomic composition,life cycle,and the functions of the various virally encoded proteins.Therefore,ISAV can be used as an alternative model to study influenza viruses,especially for structural and functional studies of the viral proteins.To better understand the structure and function of the orthomyxovirus matrix protein,here we determined the crystal structure of the full-length M1 from ISAV(ISAV-M1)to 2.6 ? resolution.The crystal structure of ISAV-M1 has an elbow shape with a N-domain and a C-domain.The structure of the N-domain is comparable to that of the influenza A virus M1 despite the low sequence similarity.The C-domain,which is connected to N-domain through an extended linker loop,folds into an ?-helical bundle with four ?-helices.In the crystal,ISAV-M1 monomers assemble into an infinite 2-D lattice with structural features resembling the membrane-associated protein matrix of the influenza A virus.Using liposome floatation experiments,we showed that ISAV-M1 binds membrane through electrostatic interactions and mapped the key membrane association site to the N-domain(i.e.K63 K,K66,and K68).An RNA binding activity has also been demonstrated for the ISAV-M1.Cryo-electron tomography of intact ISA viron showed that ISAV-M1 forms a protein matrix underneath the viral envelope.Fitting the ISAV-M1 2-D assembly into the averaged tomogram indicated perfect agreement with the thickness of the matrix protein density,suggesting the 2-D M1 lattice closely mimics the authentic matrix protein layer found in infectious particles.NS1 and M2 of ISAV have been well known as antagonist of type I interferon.However,little is known about the characterization of M1 or NEP.In addition,heat shock cognate 70(Hsc70)has been reported to interact with M1 and NEP of influenza viruses for the export of viral ribonucleoprotein(vRNP)via vRNP-M1-NEP complex,the goal of this study therefore was to characterize the subcellular localization and interactions of ISAV M1 and NEP as well as cellular Hsc70.When M1,NEP,and Hsc70 were individually expressed in the stripped snakehead(SSN-1)cells,we found that M1 protein was localized in both cytosol and nucleus of the cells,NEP was localized only in the cytosol and accumulated adjacent to the nucleus,while Hsc70 was localized throughout the cytosol,but not in the nucleus.However,when two of them were co-expressed,we found that both M1 and Hsc70 were co-localized with NEP in the cytosol and accumulated adjacent to the nucleus,while M1 and Hsc70 were still localized as they were expressed individually.Furthermore,pull-down assay was performed and showed that NEP could interact with both M1 and Hsc70,and M1-Hsc70 interaction was also observed although the interaction was weaker than that of NEP-Hsc70.Our study characterized the subcellular localization and interactions of three proteins including M1 and NEP of ISAV,and Hsc70.These data will help towards a better understanding of the life cycle of ISAV,especially the process of vRNP export.