Construction of hlFITM3 eukaryotic expression vectors
Through gene cloning and vector construction, the results were verified by western blot, PCR and sequencing, and two types of eukaryotic fusion expression vectors pcDNA3.1-HA-hlFITM3 and pEGFP-N1-hlFITM3 were successfully constructed (Fig1. A-C).
The PCR results showed (Fig1. D) that the vectors used to knock out the hlFITM3 gene were constructed successfully: pHMG-hRNA-hhRNAlFITM3.
Overexpression of hlFITMs has an inhibitory effect on influenza virus replication
After transfecting HEK293 cells with pEGFP-N1-hlFITM3 and pcDNA3.1-HA-hlFITM3 for 24h, they were infected with influenza virus at 1 MOI, and the culture supernatant was taken at different time points after infection. Fluorescence quantitative PCR was used to detect virus copy number. The results all showed that hIFITM3 had obvious anti-influenza virus effects. Virus amplification was significantly reduced at 24h and 48h after infection, when compared with controls. The difference became even more pronounced at 72h. (Fig1. E).
The constructed knockout plasmid was transfected into HEK293 cells, and a virus infection experiment was carried out. The results of fluorescence quantitative measurement showed that the pHMG-hRNA-hhRNAIFITM3 knockout plasmid was transfected, and the influenza virus infected the hlFITMl3 knockout cells. In contrast, the viral copy number was significantly increased (Fig1. F).
Subcellular localization of hlFITM3 and hABHD16A
In the yeast transformation test of hIFITM3 and hABHD16A, colonies grew on the SD-Leu-Trp solid medium lacking leucine (Leu) and tryptophan (Trp), but in the lack of leucine (Leu), Colonies were grown on pGBKT7-hIFITM3/pGADT7-hABHD16A on SD-Leu-Trp-His solid medium containing tryptophan (Trp) and histidine (His). It shows that hIFITM3 and hABHD16A can interact (Fig2. A) .
The successfully constructed fusion fluorescent expression vector was transfected into HEK293 cells, and the pEGFP-N1/pdhRed-monomer-N1 empty vector was transfected as a control group. Hoechest33342 nuclear staining was performed and observed with a laser confocal microscope. The results showed that hlFITM3 and hABHDl6A were aggregated and localized in the cell membrane structure, respectively. The superimposed pictures show hlFITM3 and hABHDl6A were co-located in the cell membrane structure (Fig2. B). After the plasmid was co-transfected for 24h, the plasmid-transfected cells were infected with 1MOI influenza virus, and culturing was continued for 48h. They were subsequently stained by Hoechest33342, and observed by laser confocal microscopy. The results showed that after influenza virus infection, there was a clear change in the location of hlFITM3 and hABHDl6A as compared to before infection, and the co-localized area tended to move towards the cell's nuclear area.
hABHDl6A has an inhibitory effect on influenza virus
The positive clones were verified by bacterial solution PCR, and the results showed that the pHMG-hRNA-hhRNAABHD16A was successfully constructed (Fig3. A). When hABHD16A was knocked out of HEK293 cells, the copy number of influenza virus increased significantly, compared with the control, and the difference observed was significant. This result indicates that hABHD16A has an inhibitory effect on influenza virus (Fig3. B). Once again, in HEK293 cells with an overexpression of hABHDl6A protein, there was no significant difference with the control group after 24 hours of infection with the virus. hABHDl6A has an effect on influenza virus replication at 48h after infection, which can also inhibit the replication of JEV virus at 72h atter infection (Fig3. C).
In HEK293 cells, when there was overexpression of hlFITM3 or hABHD16A alone compared to normal expressing cells, the results showed that hlFlTM3 significantly inhibited influenza virus replication at 24h, 48h, and 72h after infection. When hABHD16A was overexpressed, it also significantly inhibited influenza virus replication at 24h, 48h, and 72h after infection. After co-transformation of pcDNA3.1(+)-HA-hlFITM3/pcDNA3.1(+)-HA-hABHDl6A, the amount of virus that was detected at three time points of infection showed that the inhibitory effect from co-expression of the two proteins against influenza virus was significantly stronger than when they were expressed alone. These findings show that hlFITM3 and hABHDl6A can synergistically inhibit the replication of influenza virus (Fig3. D).
The inhibition of influenza virus replication by hlFITM3 is closely related to the S-palmitoylation of hABHDl6A
In order to determine whether the anti-influenza virus effect of hlFITM3 is related to its palmitoylation, PCR point mutation technology was used to amplify the palmitoylation site mutation (Fig4.A), and construct the mutant vector pcDNA3.1(+)-HA-hlFITM3-C50A-C5IA-C84A, and pcDNA3.1(+)-HA-hlFITM3-C84A eukaryotic expression vector. M-hlFITM3, hlFITM3\hABHDl6A overexpressing hlFITM3, hABHDl6A, C50A-C51A-C84A site mutations were infected with influenza virus, and the culture supernatant was taken after 24 h, and the viral copy number was detected by RT-PCR. Compared with the transfection control group, after transfection of hlFITM3, hABHDl6A, and co-transfection of hlFITM3/hABHDl6A, the replication of influenza virus was significantly inhibited. In the cells transfected with mutant M-hlFITM3, the virus copy number was significantly higher than that in the wild transfected hlFITM3 group, and with co-transfected mutant M-hlFITM3\hABHDl6A, the virus copy number was higher than that of co-transfected hlFITM3/hABHDl6A. These results indicate that the antiviral effect of hIFITM3 depends on its palmitoylation site, and the function of this site is related to hABHD16A interaction (Fig4. B).
Co-transfection of hlFITM3 and hABHDl6A can reduce the expression of inflammatory factors caused by influenza virus
Relative to uninfected cells, in flu virus-infected HEK293 cells, the hlFITM3 gene was significantly increased, and hABHDl6A gene expression was significantly reduced (Fig 5. A and B).
When IL6, TNF-α, IL-1β and IL-10 were transfected with hlFITM3 and hABHDl6A respectively, their expressions increased significantly, while with co-transfection, they decreased significantly, indicating that the interaction of hlFITM3 and hABHDl6A can reduce the expression of inflammatory factors. In the virus-infected group, the expression levels of IL6, TNF-α, IL-1β and IL-10 were increased compared with the empty vector control, but the co-expression of hlFITM3 and hABHDl6A decreased IL6, TNF-α, IL- 1β and IL-10 expression (Fig5. C-F). In HEK293 cells, compared with control cells transfected with empty vectors, the expression level of the MCP-1 gene was significantly reduced in single transfection and co-transfection; in the infected virus group, the expression level of MCP-1 gene was lower (Fig5. G). The expression level of the COX2 gene was significantly increased when transfected with hlFITM3 and hABHDl6A as well as when co-transfected, respectively, while infection with influenza virus could reduce the expression level of COX2 (Fig5. H).