The Mechanism Of Inhibitory Effect On Host Antiviral Response Mediated By VSV M Protein

Vesicular stomatitis(VS)is a highly contagious Zoonotic disease in swine,horses,cattle and other mammals.It is caused by vesicular stomatitis virus(VSV)and characterized by widely erosive and vesicular lesions on the surface of the lips,tongue,gums,and teats.Since the outbreak of the disease in the United States in 1821,it has been reported in many countries.The spread of VSV in many countries has led to chaos in the world meat market and to serious economic losses as the production capacity of infected animals(such as pigs and cattle)has decreased.Therefore,the potential harm of the disease to the breeding industry can not be ignored and the disease was listed as a important animal disease by the World Organisation for Animal Health(OIE).Virus is one of the most dangerous pathogenic microbial.Innate immune system plays an important role in protecting against pathogenic microbial infection.Many viruses have evolved with the ability to inhibit or escape host antiviral responses.Many studies have proved that VSV matrix(M)protein can inhibit host transcription,nuclear-cytoplasmic RNA transport and protein translation to block host antiviral response.The mechanism that M protein inhibit host transcription and nuclear-cytoplasmic RNA transport has been extensively reported,but few study has focused on the mechanism of transcription inhibition mediated by M protein.We found that both VSV and M protein inhibited transcription of the luciferase gene in BSR-T7/5 at 4 h post-transfection.To further investigate the underlying mechanism,yeast two-hybrid screen was performed to search for host proteins that interact with the M protein.The subunit of transcription/repair factor TFIIH,p8,was identified as an M binding partner,and the interaction was validated with a GST-pull down assay and laser confocal microscopy.Through a mutagenesis analysis,we found that the p8-M interaction was impaired when I96,E156,R159 and R160 residues on M were replaced with Ala.These mutants reduced the inhibitory effect on transcription of the reporter gene.Furthermore,the transcription inhibition mediated by M was impaired when co-expressed with p8.These results indicate that the p8-M interaction plays an important role in inhibiting transcription of host genes.To further understand the mechanism of translation inhibition mediated by M protein,eukaryotic translation initiation factor 3,subunit i(eIF3i)was identified to be an M-binding partner,and this interaction was validated by GST pull-down and laser confocal assays.Through a mutagenesis analysis,we found that some mutants of M between amino acids 122 and 181 impaired but did not completely abolish the M–eIF3i interaction.To investigate whether eIF3 i was involved in VSV propagation,we have determined the transcription and replication level of VSV after knock down or over-expression of the eIF3 i.The result showed that knockdown of eIF3 i by RNA interference increased viral replication,transcription.But,VSV propagation was not changed after the over-expression of eIF3 i.Finally,we also demonstrated that VSV could inhibit the activity of Akt1 and that the knockdown of eIF3 i inhibited the expression of the ISGs regulated by phospho-Akt1.These results indicated that eIF3 i may affect VSV growth by regulating the host antiviral response in Hela cells.These studies not only elucidate the mechanism exploited by VSV to inhibit the antiviral response,but also provide new target for antiviral therapy.