Construction and bioinformatics analysis of a PK-15 cell line with stable knockdown of porcine G3BP1 gene

Abstract:

[Background] Ras-GTPase-activating protein SH3 domain binding protein 1 (G3BP1), an important component and iconic protein of stress granules (SGs), plays a role in host resistance to pathogen invasion. [Objective] To construct a PK-15 cell line with stable knockdown of G3BP1 gene, and to explore the effects of knocking down G3BP1 gene on the replication of vesicular stomatitis virus (VSV), seneca virus (SVV), and vaccinia virus (VACV) and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), myxovirus resistance protein 1 (Mx1), interferon-stimulated gene 54 (ISG54), and interferon-β (IFN-β). [Methods] The lentiviral vector was used to construct a PK-15 cell line with stable knockdown of porcine G3BP1 gene. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and quantitative real-time polymerase chain reaction (qPCR) were carried out to analyze the effects of G3BP1 knockdown on the replication of VSV, SVV, and VACV and the expression of TNF-α, IL-6, Mx1, ISG54, and IFN-β after infection with VSV, SVV and VACV. The bioinformatics tools were used to characterize the G3BP1 gene. [Results] A PK-15 cell line with stable knockdown of G3BP1 was successfully constructed. RT-qPCR and qPCR results showed that knockdown of G3BP1 gene promoted the replication of VSV and SVV (P<0.000 1), while it had no significant effect on the replication of VACV (P>0.05). RT-qPCR results showed that VSV and SVV infections up-regulated the mRNA levels of TNF-α, IL-6, Mx1, ISG54, and IFN-β (P<0.05, P<0.000 1), and knocking down G3BP1 gene down-regulated the mRNA levels of these cytokines (P<0.01, P<0.000 1). RT-qPCR results showed that VACV infection inhibited the production of these cytokines (P<0.01, P<0.000 1), and knocking down G3BP1 gene had no significant effect on the expression of the cytokines inhibited by VACV (P>0.05). The deduced porcine G3BP1 protein was an unstable, hydrophilic, and non-secretory protein with 30 phosphorylation sites, with no transmembrane region. The secondary structure of the protein was mainly composed of random coils (52.47%). The protein was mainly located in the cytoplasm (34.38%) and nucleus (31.25%). [Conclusion] In this study, a PK-15 cell line with stable knockdown of porcine G3BP1 gene was obtained. Knocking down this gene promoted the replication of VSV and SVV but had no significant effect on the VACV replication. Furthermore, it significantly inhibited the production of TNF-α, IL-6, Mx1, ISG54, and IFN-β. The protein had 30 phosphorylation sites and was an unstable, hydrophilic, and non-secretory protein with no transmembrane region. The results help to further reveal the role of G3BP1 in viral infection.

Key words:PK-15 cells;G3BP1 gene;cytokine;bioinformatics