Role of WNV NS4A and NS4B Proteins in Reducing Type I IFN Response via Interactions with Host IKKe and TBK1 Proteins
Date
2022
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West Nile virus (WNV) is a zoonotic flavivirus, with the potential to cause severe virally induced meningitis and encephalitis in humans. Currently, there are no specific antivirals or vaccine options for treatment or prevention of West Nile virus infection. Research on the closely related Dengue virus (DENV) has shown that both nonstructural proteins 4A and 4B (NS4A and NS4B) play key roles in the inhibition of type I interferon (IFN) by infected cells, aiding in the virus’ ability to evade the innate immune system. Despite this, the mechanism by which NS4A and NS4B inhibits the production of IFN-β is inadequately understood, especially for WNV. Recent findings for DENV suggest that the virus may target TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa B kinase subunit epsilon (IKKe), both potent activators of the type I IFN pathway. The expression of IKKe or TBK1 is required for full expression of IFN-β in cell models, and are a potential target for many viruses. To elucidate if WNV NS4A and/or NS4B proteins interacted with TBK1 and/or IKKe, we examined potential interactions between viral and host cell proteins and determined the degree of IFN inhibition in TBK1 and IKKe overexpressing cells transfected with WNV protein. Human endothelial kidney 293T (HEK293T) cells were transfected with WNV NS4A or NS4B, and IKKe or TBK1 plasmids (n=10-15). Pearson correlation coefficient (PCC) values were used to determine degree of colocalization of WNV proteins to host cell proteins shown in immunofluorescence assay (IFA) images. Calculations of PCC were done using FIJI (version 2.1.0/1.53c), an application within ImageJ, utilizing the coloc2 plugin. To corroborate the findings from the IFA imaging, Western blots of sucrose gradients from transfected cell lysate were performed to visualize recruitment of IKKe and TBK1 away from its intended location in the cytoplasm towards fractions containing WNV protein. To determine whether NS4A and NS4B were able to inhibit type I IFN production in cells overexpressing IKKe and/or TBK1, a dual-reporter luciferase assay was conducted to determine the degree to which these proteins could inhibit IKKe and TBK1 activation of IFN-β production.
IFA imaging data indicated that cells expressing WNV proteins saw a significant degree of colocalization with IKKe and TBK1 (PCC ≥0.5) with NS4B seeing the highest degree of colocalization. Additional wWestern blot imaging taken from sucrose gradients conducted on transfected cells lysates showed a clear recruitment of IKKe and TBK1 proteins from the cytoplasm in control cells, towards the rough endoplasmic reticulum (RER) in cells expressing WNV protein. Luciferase assay data showed that in the presence of NS4A or NS4B, in comparison to other WNV NS proteins, IFN-β production was remarkably reduced (p<0.0001).
Our study has identified interactions between NS4A and NS4B with both IKKe and TBK1 proteins and potential sequestration of these cytoplasmic host cell proteins towards the RER where the WNV proteins are located. Additionally, we have demonstrated that NS4A and NS4B significantly inhibit IFN-β production in cells expressing high levels of TBK1 and/or IKKe, likely via the targeting of said proteins. These findings provide further evidence that NS4A and NS4B may be optimal candidates for further antiviral development to combat WNV infection.
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Virology, Immunology, Cellular biology, IKKe, NS4A, NS4B, TBK1, West Nile Virus
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71 pages
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