Analysis of Recombinant VP22-SOX2 Fusion Protein Subcellular Location in HepG2 Cell Culture

Cell penetrating peptides (CPPs) are cell permeable proteins that help facilitate impermeable molecules into the cells. Herpes simplex virus protein VP22 is one of the CPP with mechanism of facilitating proteins into the cells by non-classical Golgi-independent. SRY-related HMG-box (SOX) family of transcription factors are well associated with the regulation of the development of embryonic cells. Recombinant fusion protein VP22 is hoped to translocate the protein into the cell. The research conducted is an experimental study using VP22-SOX2 as intervention and human hepatoma HepG2 cells as subject to determine the efficacy of VP22-SOX2. There are 2 main groups: HepG2 cells with VP22-SOX2 incubated for 6 hours and 1 hours, where both are accompanied with the control group in the absence of VP22-SOX2. Both undergo immunostaining using indirect immunostaining method with antibody specific against SOX2 and observation is done using confocal microscope. Counting the total number of cells and number of cells with the fluorescence is performed using ImageJ program. Based on statistic, there is no significant difference between the experiment and the control group incubated for 6 hours. In addition, there is no significant difference between experimental group incubated in 6 hours and experimental group incubated in 1 hour. VP22-SOX2 do not achieve statistically significant protein translocation and incubation period has no effect in the rates of protein translocation. Possible explanation is the requirement of media supplements such as serum replacement needed to stabilize the protein in the prevention of precipitation.

Herpes Simplex Virus 1 Tegument Protein VP22 Abrogates cGAS/STING-Mediated Antiviral Innate Immunity

ABSTRACTCytosolic DNA arising from intracellular pathogens is sensed by cyclic GMP-AMP synthase (cGAS) and triggers a powerful innate immune response. However, herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, has developed multiple mechanisms to attenuate host antiviral machinery and facilitate viral infection and replication. In the present study, we found that HSV-1 tegument protein VP22 acts as an inhibitor of cGAS/stimulator of interferon genes (cGAS/STING)-mediated production of interferon (IFN) and its downstream antiviral genes. Our results showed that ectopic expression of VP22 decreased cGAS/STING-mediated IFN-β promoter activation and IFN-β production. Infection with wild-type (WT) HSV-1, but not VP22-deficient virus (ΔVP22), inhibited immunostimulatory DNA (ISD)-induced activation of the IFN signaling pathway. Further study showed that VP22 interacted with cGAS and inhibited the enzymatic activity of cGAS. In addition, stable knockdown of cGAS facilitated the replication of ΔVP22 virus but not the WT. In summary, our findings indicate that HSV-1 VP22 acts as an antagonist of IFN signaling to persistently evade host innate antiviral responses.IMPORTANCEcGAS is very important for host defense against viral infection, and many viruses have evolved ways to target cGAS and successfully evade the attack by the immune system of their susceptible host. This study demonstrated that HSV-1 tegument protein VP22 counteracts the cGAS/STING-mediated DNA-sensing antiviral innate immunity signaling pathway by inhibiting the enzymatic activity of cGAS. The findings in this study will expand our understanding of the interaction between HSV-1 replication and the host DNA-sensing signaling pathway.