The Nonstructural Protein P7 Of Classical Swine Fever Virus And Its Role In Production Of Infectious Virus

Classical swine fever(CSF)is a highly contagious and lethal infectious disease of swine caused by the classical swine fever virus(CSFV).In China,an attenuated live vaccine was prepared from highly virulent CSFV Shimen strain after serial passages in rabbits in the 1950s.The outbreak and prevalence of acute CSF is effectively controlled by the attenuated CSFV vaccination.However,atypical CSF still exists in China,resulting in huge harmful for pig industry.Therefore,understanding of the structure and function of viral proteins are important for development of the novel techniques of controlling CSF.CSFV p7 is an ion channel forming protein and involved in viral virulence.Homologous protein has been identified in the Hepacivirus genus,not in Flavivirus.HCV p7 modulates virion assembly by interacting with NS2 and regulating the subcellular localization of NS2 and Core.However,the structure and function of CSFV p7 and its role in production of infectious virus remain unknown.We constructed antigenically tagged E2,p7 and NS2 eukaryotic expression plasmids to investigate the viral protein interaction.Data showed that the physical interactions between p7 and NS2,p7 and E2,E2 and NS2 or p7 and p7 are observed.A formation of E2,p7 and NS2 complex in transfected cells was identified.To map the interaction regions between p7 and NS2,a series truncated mutants of p7 and NS2 were constructed,respectively.The Co-IP and Confocal data revealed that the first transmembrane domains of both p7 and NS2 are crucial interaction regions of two proteins.Data also indicated that E2p7 exhibits significantly enhanced interaction with NS2 or p7NS2 compared to E2.To investigate the role of E2p7 on infectious virus production,an internal ribosome entry site(IRES)from encephalomyocarditis virus(EMCV)was inserted between the E2 and p7 genes to generate a CSFV cDNA clone,pSM/E2/IRES.Data showed that a more than 10-fold reduction in virus titer is obtained from the pSIM/E2/IRES compared to pSM.When the E2p7 cleavage was inactivated or partial deletion of p7,no infectious CSFV was rescued from the mutated cDNA clone pSM/E2ASG/NSR or pSM/?p715-51.Our data suggested that the mature E2 and p7 are essential for infectious CSFV production and the presence of E2p7 significantly increase the infectious virus production.Nine amino acid residues(1-9aa)of p7 N-terminus and representive conserved residues of p7 TM1 were analyzed by site-directed mutagenesis.All amino acid mutations resulted in significantly reduced virus titer compared to wt CSFV,but the mutant p7YFY24/26/30AAA abolished infectious virus production.The results indicated amino acids 1 to 9 in p7 N-terminus and the representative amino acids in p7 TM1 regulate the infectious virus production.Mutation of p7 N-terminus(1-9aa)led to different infectious virus production from monocistronic or bicistronic genome.Rescued virus from cDNA clone pSM/E2/IRES exhibited significantly increased titer compared to that from pSM,but the virus titer from mutants p7L3A and p7V8A remained unchanged.Data suggested that the N-terminus of p7 regulate the infectious virus production mostly by mediating E2p7 processing.We constructed the p7 mutants pP7TDI18/19/2AAA,pP7EVV21/22/23AAA and pP7YFY25/26/30AAA based on eukaryotic expression plasmid pP7Myc.Co-IP data showed that the interaction between the mutant protein p7TD118/19/20AAA,p7EVV21/22/23AAA or p7YFY2526/30AAA and NS2 significantly decreased,suggesting that infectious CSFV production is regulated by the interaction between the p7 and NS2.To explore the effect of p7 on viral RNA replication,genomic RNA copies of representative p7 mutants(p7Q5A,p7V8A,p7V9A,p7Y30A,p7TDI18/19/20AAA,p7EVV212223AAA)were investigated based on pSM.Data showed that similar viral RNA level is observed from the mutants,suggesting that p7 regulates the infectious virus production during the precursor processing and morphogenesis after viral RNA replication.Our works contribute to understanding of the structure and function of CSFV genome and pathogenicity.