Mechanisms Of Interferon-inducible Transmembrane Proteins Inhibit Vaccinia Virus Infection

Notably, IFITM proteins are the only class of known host restriction factorsshown to have a bona fide role in blocking virus entry. To date, IFITM proteins havebeen reported to restrict the virus entry and infection of multiple pathogenic viruses,including enveloped viruses and non-enveloped virus, sunch as influenza A virus(IAV), HIV-1and reovirus and so on. These viruses restricted by IFITM proteinsbelong to RNA viruses, however, no antiviral activity of IFITM proteins againstmammalian DNA virus has been demonstrated to date. Therefore, to explore the roleof IFITM proteins in poxvirus infection, this study mainly focus on the interactionbetween these ISGs and vaccinia virus (VACV) to analyse the effect of IFITMs onVACV infection, replication and entry and the kinetics of IFITM proteins expressionregulated by VACV infection and to preliminarily to explore the role mechanism andpotential interaction moleculars of IFITM3by a variety of methods includingmolecular biology, the lentiviral packaging technology, eukaryotic expression,immunofluorescence, fluorescence microscope, flow cytometry, plaque assay,real-time quantitative PCR (qPCR), fluorescent molecule labelling virus tracertechnique, siRNA silencing, immunoprecipitation-mass spectrometry (IP-MS), laserscanning confocal microscope (LSCM) and bioinformatics methods.Firstly, based on our previous studies, the homology analysis of IFITM genesfrom HEK293T, HeLa and PBMCs cells was performed with the released sequenceson GenBank. We found that the amino acid sequence of IFITM1from HEK293T andPBMCs was consistent with that on GenBank, and there was one amino aciddifference of IFITM3from HeLa cells compared with that form other sources (HeLa:R71, others: C71), and there were three amino acid difference among the IFITM2genes from the different cell types. The human IFITM2and IFITM3proteins werevery highly homologous about90%and shared similar structure, but the structure ofIFITM1was different with the two formers. Then, the IFITM genes amplified fromhuman PBMCs were chose as target genes, and Flag tag was added in their N terminal by the designed primers. The expression of IFITM proteins was thenverified in eukaryotic cells, providing the basis for further antiviral research. Theantiviral activities of IFITM proteins against VACV were evaluated by transientexpression. We found that overexpression of IFITM proteins may exhibitedinhibitory activity to VACV infection.To more precisely evaluate the role of IFITMs in VACV infection, HEK293T,HeLa, A549, BHK-21and Vero cells stably expressing Flag-N-tagged IFITMproteins or vector were generated respectively. And the expression of IFITMs wasanalyzed in mRNA or protein levels by RT-qPCR or Western blot respectively.Furthermore, the membrane and cytosol proteins were isolated and extracted fromIFITM3+HEK293T and HeLa. Consistent with the previous finding, IFITM3proteins mainly localized to the cell membrane by western blot, which may berelated to its function. Based on these cell lines, the VACV infection assays wereperformed by fluorescence microscope, flow cytometry, plaque assay, etc. We foundthat the IFITM proteins inhibited VACV infection at low PFU/cell in HEK293T,A549, BHK-21or Vero cell lines, but the significantly restriction of IFITM was notobserved in HeLa-IFITM cell lines (except for IFITM1), and the antiviral activitiesof IFITM3in HEK293T, BHK-21and Vero cells were superior to that in A549cellsagainst VACV infection. These results suggested that the restriction efficiency ofIFITM proteins was cell-type-dependent. The viral gene expression at thetranscriptional and translational levels was analyzed by quantifying with RT-qPCRand Western blot at different time points post-infection. IFITMs delayed virustranscription and translation by suppressing VACV replication. In the growth curveexperiment, exogenous expression of IFITM3suppressed VACV replication andproliferation and impacted the production of progeny virions. Plaque analysisrevealed that IFITM3could inhibit not only the initial infection but also VACVcell-to-cell transmission and diffusion. In addition, IFITM proteins suppressedfowlpox virus (FPV) infection of BHK-21cells.To further expose the role of endogenous IFITM proteins in cellular antiviralresponse or IFN-mediated antiviral response, siRNAs targeting to IFITM genes weredesigned, and siRNA silencing analysis was performed in HeLa and A549cellsexpressing considerable amounts of endogenous IFITM proteins. Results showedthat, however, an enhancement of VACV infection was not measured in HeLa and A549cells when endogenous IFITMs was silenced, but the depletion of IFITMpartially decreased the antiviral actions of IFN-with cell-type-dependent.Additionally, siRNA interference experiments in293T-IFITM3indicated thatincreased virus infection was observed in IFITM3overexpressing cells.Simultaneously, cell viability was evaluated after treating with siRNA or virusinfection by MTS assay. We found the silencing of endogenous IFITM proteinsenhanced cell toxicity produced by VACV infection and reduced cell viability, buthad no significant influence on the cell proliferation and viability without virusinfection.Based on the above data, in the case of IFITM3, we explore its role andmechanism of inhibition in virus infection. IFITM3suppressed viral early or lategene transcription initiation, suggested that a block of IFITM3occurs prior to thetranscriptional initiation of early genes. And entry of the core into the cytoplasmresults in the mRNA production of early genes, IFITM3thus was considered toinhibit VACV infection by preventing cytosolic entry of the virus core. Then, weused the laser scanning confocal microscopy and fluorescent molecule labellingvirus tracer technique to analyze the effect of IFITM3on virus-cell binding,observing that IFITM3interfered with VACV attachment or virus-cell binding.Simultaneously, confocal microscope was used to observe the viral entry process.According to the above data, IFITM3plays an antiviral role in the stage of virusentry. In addition, we investigated the cellular location and distribution of IFITM3protein by fluorescence microscopy. Results showed that, consistent with theprevious finding, IFITM3proteins mainly localized to the cell membrane,contributing to the role of IFITM3to virus attachment. Additionally, in endosomalacidification inhibition or endosomal acidification assays, IFITM3mainly blockedthe cytosolic entry of the low pH-dependent viruses and a low pH could antagonizethe antiviral activity of IFITM3.We next verified the impact of VACV infection on cell endogenous IFITMproteins to better understand the effect of IFITM proteins in virus infection andobserved that the expression of endogenous IFITM proteins was down-regulated atthe late phase of VACV infection in translation levels but not transcription levels.Moreover, the transcriptional analysis of some key molecules in IFN pathway suchas STING, IRF3and IRF7suggested that these molecules had no significant changes after VACV infection, but the mRNA transcription of IFN and IFNβ was increasedat the late phase of infection (48hpi). To further understand the role mechanism ofIFITM3, we identified the potential intercatin proteins with IFITM3under VACVinfection through a “Flag-mediated immunoprecipitation-mass spectrometry analysis”approach. Results showed65host proteins and6VACV proteins, which were notidentified in the control cells infected by VACV. These proteins are being validatedone by one. With more precision experiments and analysis in the future, the potentialinteraction moleculars cound be found to clarify the molecular mechanism ofIFITMs.In conclusion, this study provides further understanding of the antiviralspectrum of IFN-induced IFITM proteins and the complex interactions betweenvirus infection and cells. Although IFITM proteins have been demonstrated toprotect cells from diverse enveloped RNA virus infections by inhibiting virus-cellfusion, our study gives evidence for their antiviral activity against a DNA virus byinterfering with VACV-cell binding and restricting virus core entry. Simultaneously,we also analyzed the role of endogenous IFITM proteins in IFN-mediated innateimmunity against virus infections. These results encourage exploring the potentialapplication of IFITM proteins as viral entry inhibitors and further analysis of VACVentry pathways and the mechanism of IFITM3-mediated restriction.