| Influenza A virus belongs to the family of Orthomyxoviridae and contains eight segments of single stranded negative sense viral RNA(vRNA) as its genome. Genome which encodes PA, PB1, PB2 and NP proteins is highly conserved. And the vRNP, composed by PA, PB1, PB2, NP and vRNA, is responsible for transcription and replication of influenza viruse. Virus-host interactions constitute a molecular basis of infection, replication, pathogenicity and immunity of virus. Therefore, understanding theseinteractions not only helps us understand the life cycle of the influenza virus, but also provides new direction for developing antivirals.In early studies, the laboratory had screened eIF4A3(Eukaryotic translation initiation factor 4A isoform 3)by tandem affinity purification experiment. And it had been confirmed that eIF4A3 could interact with the influenza virus polymerase subunit PB1, and further study found that it contributed to the replication of influenza virus and promoted the transcription and replication of influenza viruse. To explore the mechanism, we found that eIF4A3 overexpression could promote polymerase activity of influenza viruse, and the promotion for polymerase activity was due to stimulating influenza virus RNP assembly. Another we found knockdown of eIF4A3 could inhibite vRNP nuclear export. This not only expand the existing knowledge of interaction network between influenza virus and host factors, but also provide ideas and directions for future prevention and control of influenza virus. The main results were as follows: 1 eIF4A3 had a positive regulation role in influenza virus replication in A549 cellsWe identified e IF4A3 couuld interact with the polymerase complex by tandem affinity purification in early protein screening. To check the role of eIF4A3 in influenza viral replication, we observed a significant decrease in NP mRNA level in A549 cells treated with eIF4A3 siRNA in comparison with control cells. And the result was consistent with above after TCID50 experiment. 2 eIF4A3 targeted influenza viral transcription and replication machinery during influenza A virus infectionTo examine the role of eIF4A3 in viral RNA and protein synthesis in infected cells, A549 cells were treated with eIF4A3 siRNA or eIF4A3 expression followed by infection of HM virus at a MOI of 5, total RNAs were subjected to quantitative RT-PCR to measure the mRNA, vRNA, and cRNA levels, and protein was subjected to western blot analyses. These datas indicated that eIF4A3 can target influenza viral transcription and replication machinery. 3 eIF4A3 could interact with PB1 subunit of the viral polymeraseBy coimmunoprecipitation assays we demonstrated that eIF4A3 could interact with PB1 subunit of influenza virus RNA polymerase and e IF4A3 had a colocalisation with PB1 in cells with ZEISS confocal microscopy. And endogenous e IF4A3 could interact with PB1 in HM-infected cells. 4 eIF4A3 upregulated the viral RNA polymerase activityTo detect the effect of eIF4A3 on influenza virus polymerase activity in a minigenome assay, we found overexpression of e IF4A3 could upregulate polymerase activity of the influenza A virus-like minigenome, and knockdown of e IF4A3 leaded to an inhibition of luciferase activity. 5 eIF4A3 could stimulate influenza virus RNP assembly in the minigenome assayTo explore the reason for eIF4A3 promoting influenza virus polymerase activity, we first detected the effect of eIF4A3 on the expression level of RNA polymerase subunits and NP proteins in the minigenome assay, we confirmed that the levels of the polymerase subunits or NP proteins had no obvious change. Further study we found eIF4A3 could significantly promote the influenza virus RNP assembly 6 eIF4A3 knockdown leaded to nuclear retention of RNP in infected cellsA549 cells treated with sieIF4A3 or control NC were infected with influenza virus HM at a MOI of 5. At 5 hpi, A549 cells were fixed, permeabilized, stained. It was clear that the intracellular localisation of the NP was mostly nucleus in cells treated with sieIF4A3 in comparison with control cells by indirect immunofluorescence. That is to say, eIF4A3 knockdown leaded to nuclear retention of NP in infected cells. 7 The helicase function of eIF4A3 played an important role in its stimulating viral replicationTo study the role of helicase activity of eIF4A3 in its influence on influenza virus replication, we mutated the DEAD area of eIF4A3, and examined its role in influenza virus replication by quantitative RT-PCR. The results showed that mutation of the DEAD area caused the properties of enhancing replication of influenza virus losed. |
