Objectives: The molecular mechanisms underlying the pathogenesis of human coronavirus OC43 (HCoV-OC43) infection are poorly understood. In this study, we investigated the ability of HCoV-OC43 to antagonize the transcriptional activation of antiviral response elements. Methods: HCoV-OC43 structural (membrane M and nucleocapsid N) and accessory proteins (ns2a and ns5a) were expressed individually in human embryonic kidney 293 (HEK-293) cells. The transcriptional activation of antiviral response elements was assessed by measuring the levels of firefly luciferase expressed under the control of interferon (IFN)-stimulated response element (ISRE), IFN-β promoter, or nuclear factor kappa B response element (NF-κB-RE). The antiviral gene expression profile in HEK-293 cells was determined by PCR array. Results: The transcriptional activity of ISRE, IFN-β promoter, and NF-κB-RE was significantly reduced in the presence of HCoV-OC43 ns2a, ns5a, M, or N protein, following the challenge of cells with Sendai virus, IFN-α or tumor necrosis factor-α. The expression of antiviral genes involved in the type I IFN and NF-κB signaling pathways was also downregulated in the presence of HCoV-OC43 structural or accessory proteins. Conclusion: Both structural and accessory HCoV-OC43 proteins are able to inhibit antiviral response elements in HEK-293 cells, and to block the activation of different antiviral signaling pathways.

Human coronavirus OC43 (HCoV-OC43) is a respiratory virus that usually causes a common cold. However, it has the potential to cause severe infection in young children and immunocompromised adults. Both SARS-CoV and MERS-CoV were shown to express proteins with the potential to evade early innate immune responses. However, the ability of HCoV-OC43 to antagonise the intracellular antiviral defences has not yet been investigated. The potential role of the HCoV-OC43 structural (M and N) and accessory proteins (ns2a and ns5a) in the alteration of antiviral gene expression was investigated in this study. HCoV-OC43M, N, ns2a and ns5a proteins were expressed in human embryonic kidney 293 (HEK-293) cells before challenge with Sendai virus. The Human Antiviral Response PCR array was used to profile the antiviral gene expression in HEK-293 cells. Over 30 genes were downregulated in the presence of one of the HCoV-OC43 proteins, e.g. genes representing mitogen-activated protein kinases, toll-like receptors, interferons, interleukins, and signaling transduction proteins. Our findings suggest that similarly to SARS-CoV and MERS-CoV, HCoV-OC43 has the ability to downregulate the transcription of genes critical for the activation of different antiviral signaling pathways. Further studies are needed to confirm the role of HCoV-OC43 structural and accessory proteins in antagonising antiviral gene expression.

Background Human coronavirus OC43 (HCoV-OC43) causes common cold, and is associated with severe respiratory symptoms in infants, elderly and immunocompromised patients. HCoV-OC43 is a member of Betacoronavirus genus that includes also the Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS) coronaviruses. Both SARS-CoV and MERS-CoV were shown to express proteins with the potential to evade early innate immune responses. However, the ability of HCoV-OC43 to antagonise the intracellular antiviral defences has not yet been investigated. The objective of this study was to investigate the role of HCoV-OC43 structural (membrane and nucleocapsid) and accessory (ns5a and ns2a) proteins in the modulation of antiviral gene expression profile in human embryonic kidney 293 (HEK-293) cells using PCR array analysis. Methods HCoV-OC43 membrane (M), nucleocapsid (N), ns5a and ns2a mRNA were amplified and cloned into the pAcGFP1-N expression vector (Clontech), followed by transfection in HEK-293 cells. Expression of M, N, ns5a and ns2a proteins were confirmed by indirect immunofluorescence test. Three days post-transfection, the cells were challenged by Sendai virus. The Human Antiviral Response PCR array system (Qiagen) was used to profile the antiviral gene expression in HEK-293 cells, using the fold regulation comparison and the manual normalisation methods. Results Around 50–60 genes were downregulated by HCoV-OC43 proteins, the most prominent genes being those critical for the activation of transcription factors involved in the antiviral response like interferon regulatory factors (IRFs) and activator protein 1 (AP-1). Among the most important downregulated genes were those coding for Interferons (IFNs) mitogen-activated protein kinases (MAPKs), pro-apoptotic and pyroptotic proteins (Caspases, cathepsins, tumour necrosis factor), pro-inflammatory cytokines (Interleukins), pattern recognition receptors (PRRs; toll-like receptors and NOD-like receptors) and their signaling transduction proteins (TICAM1, MAVS). Conclusion This study shows for the first time that similarly to SARS-CoV and MERS-CoV, HCoV-OC43 has the ability to downregulate the transcription of genes critical for the activation of different antiviral signaling pathways. Disclosures All authors: No reported disclosures.

BackgroundIn Kuwait, human coronavirus OC43 (HCoV-OC43) causes 25–30% of common cold, and 8.8% of respiratory infections in hospitalised patients. It is also associated with severe respiratory symptoms in infants, elderly, and immunocompromised patients. Our previous results showed that the expression of antiviral genes in human embryonic kidney (HEK) 293 cells is downregulated in the presence of HCoV-OC43 proteins. To understand the role of HCoV-OC43 proteins in antagonizing antiviral responses of the host, we investigated the effect of HCoV-OC43 structural and accessory proteins on the transcriptional activation of interferon-stimulated response element (ISRE), interferon-β (IFN-β) promoter, and nuclear factor kappa B response element (NF-kappaB-RE).MethodsHCoV-OC43 ns2a, ns5a, membrane (M), and nucleocapsid (N) mRNA were amplified and cloned into the pAcGFP1-N expression vector, followed by transfection in HEK-293 cells. Two days post-transfection, the cells were co-transfected with a reporter vector containing firefly luciferase under the control of ISRE, IFN-β promoter, or NF-kappaB-RE. Renilla luciferase vector was used as an internal control for transfection efficiency. Following 24 hours of incubation, the cells were treated with either IFN or tumour necrosis factor (TNF) for 6 hours. Thereafter, promoter activity was assayed using the dual-luciferase reporter assay system. Influenza NS1 protein was used as positive control for antagonism.ResultsThe transcriptional activity of ISRE, IFN-β promoter, and NF-kappaB-RE was downregulated in the presence of ns2a, ns5a, M, or N protein as there was a sharp fall in firefly luciferase levels. Overall, HCoV-OC43 proteins reduced firefly luciferase levels for ISRE and IFN-β promoter by at least ten fold, whereas for NF-kappaB-RE the firefly luciferase levels were reduced by at least fivefold.ConclusionHCoV-OC43 has the ability to block the activation of different antiviral signaling pathways.Disclosures All authors: No reported disclosures.