Effects Of Host PKM Protein On CSFV Proliferation And Interaction With NS5B

Classical swine fever virus(CSFV)is the pathogen of classical swine fever(CSF),which is a highly infectious and fatal disease around the world.Although in a few regions CSF has been eradicated,it is still occasionally observed in China and thus hinder the normal development of Chinses pig industry.China needs to find more effective method to prevent the occurrence of CSF.CSFV is similar to other animal viruses,and would utilize host factors during infection.Therefore,the research on the interaction between host factors and CSFV is of great scientific value.Pyruvate kinase is an essential enzyme in glycolysis.It has been reported that pyruvate kinase would interact with the RNA-dependent RNA polymerase(Rd Rp)of hepatitis virus C(HCV)and influenza virus.In addition,our laboratory has previously found that M type pyruvate kinase(PKM)was the same complex with CSFV C using co-immunoprecipitation-mass spectrometry(co-IP-MS),while C would co-localize with NS5B,the Rd Rp of CSFV,during infection.Therefore,this research is to explore the influence of PKM on CSFV proliferation and the interaction between PKM with NS5B.The results obtained are as follows:(1)PKM knockdown restrains CSFV propagation,while its overexpression propels CSFV propagation.After knockdown and overexpression of PKM;CSFV proliferation was examined by Western blot and real-time quantitative PCR(RT-q PCR).The results showed that the overexpression of PKM significantly increases CSFV proliferation(P<0.05 or P<0.001),the knockdown of PKM highly significantly decreases CSFV proliferation(P<0.01 or P<0.001).(2)CSFV infection upregulates PKM expression.Using Western blot and RT-q PCR,the PKM level in CSFV-infected cells was found to highly significantly increase(P<0.01 or P<0.01);the diseased regions of CSFV-infected piglets were found to exhibit significantly higher PKM level using immunohistochemistry(IHC)(P<0.05,P<0.01 or P<0.001).(3)PKM binds to CSFV NS5B and not C.It was found through co-immunoprecipitation(co-IP)and confocal microscopy that NS5B co-located with PKM in CSFV-infected and non-infected cells,while C only co-located with PKM in CSFV-infected cells;using glutathione S-transferase(GST)pull-down,PKM was found to bind to NS5B and not C.(4)PKM propel the replication stage of CSFV.Using IFA,we found that CSFV-infected cells released progeny virions around 10 hpi;the results of RT-q PCR showed that before release of progeny virions,PKM overexpression restrains the replication of CSFV genome(P<0.05 or P<0.01)and PKM knockdown propels the replication of CSFV genome(P<0.05,P<0.01 or P<0.001).(5)PKM enhances NS5B activity.The reporter plasmid pGL4.21-CSFV-IRES was constructed.The results of luciferase assay show that PKM overexpression highly significantly enhanced NS5B activity(P < 0.001)while PKM knockdown highly significantly restrained NS5B activity(P<0.01).In summary,PKM increases CSFV proliferation through enhancing its NS5B activity.