Molecular Mechanisms Of Antagonizing Antiviral Activity Of Host Proteins IRF1 And CH25H By Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV)is an important pathogen that causes economically huge losses to the pig industry worldwide.As well known,PRRSV has the abilities of rapid variation and recombination and antagonism of host innate immunity,resulting in the difficulty in the prevention and control of PRRSV infection and cinical disease in practice.Thus,to explore the anti-PRRSV activity of innate antiviral molecule and the related antagonism action of PRRSV is of important theoretically and practically significance for screening the anti-PRRSV molecule and understanding the mechanism of escaping the innate immunity of PRRSV.In the present study,the antiviral effects and mechanisms of IRF1,CH25H and 25HC against PRRSV were investigated;and the antagonism mechanisms of PRRSV to these molecules were also analyzed,in order to provide evidence for elucidating the molecular mechanisms of escaping the innate immunity of PRRSV and new strategies in PRRS prevention and control.Over-expression of IRF1 gene using lentiviral expression system in MARC-145 cells inhibited PRRSV replication.Moreover,the knockdown of IRF1 gene using the siRNA was beneficial for the viral replication.To further analyze the effect of PRRSV infection on IRF1 expression,MARC-145 cells and PAMs were infected with PRRSV and western blot was used to test the level of IRF1,the result showed that the levels of endogenous IRF1 were up-regulated in the early stage of infection and were down-regulated in the late stage.The expressing plasmid of PRRSV proteins were transfected into HEK293FT cells expressing IRF1,respectively.Western blot analysis showed that the nsp1?,nsp10 and nspll down-regulated IRF1 expression.The expressing plasmid of nsp1?/nsp11 mutants were co-transfected into HEK293FT cells together with the IRF1-expressing plasmid and the results showed that His-159 of nsplp and His-129,His-144,Lys-173 of nsp11 were key amino acid sites involved in down-regulation of IRF1.Degradation assays showed that nsp1? could degrade IRF1 via the lysosomal pathway and nsp10/nsp11 induce the degradation of IRF1 via the lysosomal and proteasomal pathways.Over-expression of CH25H gene using lentiviral expression system in MARC-145 cells inhibited PRRSV replication.To determine the effect of 25 HC on PRRSV replication,the levels of PRRSV growth in 25HC treated cells were tested.The results showed that 25HC exhibited significant inhibitory effect on the titer of PRRSV,suggesting that 25HC shared the anti-PRRSV activity.Viral attachment assay,penetration assay and release assay were conducted to investigate the antiviral mechanisms of 25HC on PRRSV.Real-time RT-PCR assay was used to analyze the effect of 25HC on the genome synthesis of PRRSV.The results indicated that 25HC could impair the attachment and entry of PRRSV in vitro,but not affect viral genome synthesis,virion release and infectivity.To further analyze the effect of PRRSV infection on the expression of CH25H,PAMs were infected with PRRSV and western blot was used to examine the level of CH25H,the result showed that the level of endogenous CH25H was obviously reduced.The expressing plasmids of PRRSV proteins were transfected into HEK293FT cells expressing CH25H,respectively.Western blot analysis showed that nspl(3 and nspll down-regulated CH25H expression.The expressing plasmids of nsp1?/nsp11 mutants were co-transfected into HEK293FT cells together with the CH25H-expressing plasmid and the results showed that His-159 of nsp1? and His-129,His-144,Lys-173 of nspll were key amino acids involved in down-regulation of CH25H.Degradation assays showed that nsp1? and nspll could degrade CH25H via the lysosomal pathway.The anti-PRRSV activity of CH25H was also antagonized by the nsp1? and nsp11.In summary,our results suggest that:(i)IRF1 and CH25H have anti-PRRSV activites,25HC exerts antiviral effect against PRRSV infection via impairing the attachment and entry of PRRSV;(ii)PRRSV nsp1? induces the degradation of IRF1 via the lysosomal pathway and nsp10/nsp11 induce the degradation of IRF1 via the lysosomal and proteasomal pathways;(iii)PRRSV nsp1? and nsp11 induce the the degradation of CH25H via the lysosomal pathway.These findings provide scientific evidence for elucidating the molecular mechanisms of escaping the innate immunity of PRRSV and screening the anti-PRRSV molecules and are helpful for understanding the multiple functions of PRRSV nsp1? and nsp?.