Objective To explore the effect of the knockdown of transmembrane 9 superfamily protein member 2 (TM9SF2) on the replication of vesicular stomatitis virus (VSV), and investigate its role in the mechanism of antiviral innate immunity. Methods Small interfering RNA (siRNA) was used to knock down the TM9SF2 gene in human non-small cell lung cancer A549 cells. The CCK-8 method was used to assess cell proliferation. A VSV-green fluorescent protein (VSV-GFP) infected cell model was established. The plaque assay was used to measure the viral titer in the supernatant. RT-qPCR and Western blotting were employed to quantify the mRNA and protein levels of VSV genome replication in A549 cells following VSV infection, as well as the expression of interferon β (IFN-β) mRNA and interferon regulatory factor 3 (IRF3) protein phosphorylation following polyinosinic-polycytidylic acid (poly(I:C)) stimulation. Results Compared to the negative control, the knockdown of TM9SF2 exhibited a significant effect, with no observed impact on A549 cell proliferation. The VSV-GFP infected A549 cell model was successfully established. After viral stimulation, fluorescence intensity was reduced following TM9SF2 knockdown, and the mRNA and protein levels of VSV were significantly downregulated. The viral titer of VSV was decreased. After poly(I:C) stimulation, TM9SF2 knockdown significantly upregulated the mRNA level of IFN-β and the phosphorylation level of IRF3 protein. Conclusion The knockdown of TM9SF2 inhibits the replication of vesicular stomatitis virus, and positively regulates the type I interferon signaling pathway, thus enhancing the host's antiviral innate immune response.
Humans ; Virus Replication/genetics* ; Signal Transduction ; Membrane Proteins/metabolism* ; A549 Cells ; Vesiculovirus/physiology* ; Interferon-beta/metabolism* ; Interferon Regulatory Factor-3/genetics* ; Interferon Type I/metabolism* ; Vesicular Stomatitis/immunology* ; Gene Knockdown Techniques ; Vesicular stomatitis Indiana virus/physiology* ; RNA, Small Interfering/genetics*

In order to ensure the biosafety of the IFN-gamma antiviral activity assay, we used a replication-deficient VSV carrying GFP as an interferon sensitive indicator virus (VSVdeltaG*G). The antiviral activities of porcine IFN-gamma expressed in Escherichia coli and in baculovirus on MDBK cells were assessed. The results showed that the antiviral activity of porcine IFN-gamma expressed in baculovirus could reach 10(5) IU/mL, while the porcine IFN-gamma expressed in E. coli showed some antiviral activity (32 IU/mL) after refolding. The results of the VSVdeltaG*G-based antiviral assay were almost identical to that of the VSV*GFP-based assay, suggesting it is highly feasible to use VSVdeltaG*G as a substitute for VSV*GFP, making assays for IFN-gamma antiviral activity safer and more accurate.
Animals ; Antiviral Agents ; pharmacology ; Baculoviridae ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Interferon-gamma ; biosynthesis ; genetics ; metabolism ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Swine ; Vesiculovirus ; drug effects ; physiology