The Effect And Mechanism Of Host Factor EEF1A1 On Influenza Virus Propagation

As an important zoonosis,influenza have a great impact on breeding industry and public health all the time,and the frequent interspecies transmission of influenza virus made it attracted much attention in recent years.Influenza virus can utilize host proteins for its replication,nevertheless host protien can play a role in the interspecies transmission of influenza virus as a restriction factor.This study selected human e EF1A1(eukaryotic elongation factor 1-alpha 1)protien to explore its effect on the proliferation of avian influenza strain A/duck/Hubei/hangmei01/2006(HM)on A549 cells.We showed that eEF1A1 reduced the proliferation of HM virus on A549 cells,and it can inhibit the activity of Rd Rp by affecting the assembly of vRNP complex.The results showed that eEF1A1 may act as a key factor in limiting the infection of avian influenza on human.The main research contents are as follows: 1.e EF1A1 inhibited the propagation of HM virusA549 cells transfected plasmid overexpressing e EF1A1 or siRNA targeting e EF1A1 were inoculated with HM virus,supernatant and cells were collected at different time points.The supernatant was inoculated on MDCK cells for TCID50 test.The cells were used to detect the mRNA level of viral protien NP.The titer of virus reduced significantly when e EF1A1 overexpressed,the result was opposite when eEF1A1 silenced.2.The effect of e EF1A1 on viral polyme rase complexInfluenza viral polymerase plays a key role in the replication of virus,thus the activity of Rd Rp was tested by using an in vitro transcription assay under the condition of overexpressing or silencing e EF1A1.The results showed that e EF1A1 inhibited the activity of viral Rd Rp.As e EF1A1 affected the activity of polymerase,it seemed that e EF1A1 may have a connection with polymerase.Then the interactions between e EF1A1 and PB1,PB2 were detected through Co-IP assay.At the meantime,the colocalizations between eEF1A1 and PB1,PB2 were observed in IF assay.The minimal transcriptional and replicative machinery of influenza virus is vRNP,and the polymerase needs to form vRNP for its function,so e EF1A1 may affect the assembly of vRNP by interacting with the subunits.We constructed vRNP complex in vitro,then assembly of vRNP was detected when e EF1A1 overexpressed or silenced.The NP protien was chosen as the bait protien,coprecipitated PB2 was detected by Co-IP assay,the assembly situation of vRNP was judged according to the amount of coprecipitated PB2.The amount of coprecipitated PB2 was reduced when e EF1A1 overexpressed,and increased when e EF1A1 silenced.The results indicated that e EF1A1 inhibited the assembly of vRNP.The assembly of vRNP was complex,and it was not resolved completely.It is known that PA and PB1 transported to the nuclear by forming a dimer,PB2 transported to the nuclear alone,then they formed a trimer,but the binding process of the trimer and NP,vRNA was not clear.In this study,we explored the effect of e EF1A1 on the nuclear import of PA-PB1 dimer.We found that nuclear import of PA-PB1 dimer was decreased when e EF1A1 overexpressed,it indicated that e EF1A1 could inhibit the nuclear import of PA-PB1 dimer.At present,the results showed that e EF1A1 inhibited the assembly of vRNP by decreasing the nuclear import of PA-PB1 dimer,then the activity of viral polymerase was reduced,consequently,the viral propagation was affected.3.Ala206 of e EF1A1 was a key amino acid residue on affecting HM proliferationMeanwhile,we found that chicken eEF1A1 didn't affect the proliferation of HM virus,and ch-eEF1A1 only have three different amino acid residues compared with h-e EF1A1: 206 aa,405aa and 446 aa.Then we constructed relevant mutants and explored their effect on viral propagation.The results showed that h-eEF1A1 A206 S mutant decreased the inhibition on viral propagation of h-eEF1A1,and ch-e EF1A1 S206 A mutant could reduce the titer of HM virus.It indicated that Ala206 of eEF1A1 played a key role in affecting HM proliferation.4.The infection of influenza virus affected the expression of e EF1A1The expression level of e EF1A1 was upregulated firstly and reduced subsequently in A549 cells when HM infected.And when PA or PB1 overexpressed,the level of endogenous eEF1A1 decreased.It showed that the expression of e EF1A1 was upregulated by the host to resist the invasion of virus,then it was downregulated by a unkonwn mechanism of virus to inhibit its function on antivirus.It indicated that the impact between virus and host was interactive when t he virus infected the host.