Downregulation of MHC class I expression by influenza A and B viruses

Manipulation of the MHC-I presentation pathway, and thus limiting MHC-I cell surface expression, is used by many viruses to evade immune recognition. In particular, downregulation of MHC-I molecules at the cell surface can reduce the ability of CD8+ T cells to recognize viral peptides presented by MHC-I molecules and thereby delay viral clearance by CD8+ T cells. To date, MHC-I downregulation by influenza viruses has not been reported. Given that influenza virus infections are a global health concern and that CD8+ T cells play an important role in promoting influenza virus clearance and recovery from influenza disease, we investigated whether influenza A and B viruses (IAV, IBV) downregulated MHC-I as a novel mechanism to evade cellular immunity. Here, we showed that infection of several cell types, including epithelial A549 cells, with a panel of IAV and IBV viruses downregulated the surface MHC-I expression on IAV/IBV-infected cells during the late stages of influenza virus infection in vitro. This observation was consistent across a panel of class I-reduced (C1R) cell lines expressing 14 different HLA-A or -B alleles and a panel of 721.221 cell lines expressing 11 HLA-C alleles. Interestingly, IBV infection caused more pronounced reduction in surface MHC-I expression compared to IAV. Importantly, the two viruses utilized two distinct mechanisms for MHC-I downregulation. Our data demonstrated that while IAV caused a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the preferential loss of MHC-I molecules from the cell surface, consequent of delayed MHC-I trafficking to the cell surface, resulting from retaining MHC-I intracellularly during IBV infection. Overall, our study suggests that influenza viruses across both IAV and IBV subtypes have the potential to downregulate MHC-I surface expression levels. Our findings provide new insights into the host-pathogen interaction of influenza A and B viruses and inform the design of novel vaccine strategies against influenza viruses

Downregulation of MHC Class I Expression by Influenza A and B Viruses

Manipulation of the MHC-I presentation pathway, and thus limiting MHC-I cell surface expression, is used by many viruses to evade immune recognition. In particular, downregulation of MHC-I molecules at the cell surface can reduce the ability of CD8+ T cells to recognize viral peptides presented by MHC-I molecules and thereby delay viral clearance by CD8+ T cells. To date, MHC-I downregulation by influenza viruses has not been reported. Given that influenza virus infections are a global health concern and that CD8+ T cells play an important role in promoting influenza virus clearance and recovery from influenza disease, we investigated whether influenza A and B viruses (IAV, IBV) downregulated MHC-I as a novel mechanism to evade cellular immunity. Here, we showed that infection of several cell types, including epithelial A549 cells, with a panel of IAV and IBV viruses downregulated the surface MHC-I expression on IAV/IBV-infected cells during the late stages of influenza virus infection in vitro. This observation was consistent across a panel of class I-reduced (C1R) cell lines expressing 14 different HLA-A or -B alleles and a panel of 721.221 cell lines expressing 11 HLA-C alleles. Interestingly, IBV infection caused more pronounced reduction in surface MHC-I expression compared to IAV. Importantly, the two viruses utilized two distinct mechanisms for MHC-I downregulation. Our data demonstrated that while IAV caused a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the preferential loss of MHC-I molecules from the cell surface, consequent of delayed MHC-I trafficking to the cell surface, resulting from retaining MHC-I intracellularly during IBV infection. Overall, our study suggests that influenza viruses across both IAV and IBV subtypes have the potential to downregulate MHC-I surface expression levels. Our findings provide new insights into the host-pathogen interaction of influenza A and B viruses and inform the design of novel vaccine strategies against influenza viruses

Interaction of mouse norovirus (MNV) with the cellular immune response of host cells

© 2017 Dr. Svenja FritzlarHuman noroviruses (HuNoV) cause the majority of non-bacterial gastroenteritis cases worldwide and generate an economic burden of 60 billion USD every year. Noroviruses are highly infectious and predominantly cause issues in closed environments such as cruise ships, hospitals and nursing homes. Due to the lack of a tissue culture or small animal model, HuNoV research has been impaired and so far no drug treatment or vaccine is available. Despite recent advances in the field and the successful replication of HuNoV in B cells and human intestinal organoids, models of HuNoV replication in vitro still remain to be established. Fortunately, murine norovirus (MNV) was discovered in 2003 and has since been used as a model system to investigate NoV infections. In this study we show that MNV infection reduces the surface expression of MHC class I proteins. The reduction in MHC class I levels on the cell surface is based on reduced intracellular levels of the protein. We reveal that MHC class I transcription is not reduced during MNV infection, implying that either MHC class I translation is affected or MHC class I proteins are degraded during MNV infections. We were able to partially rescue the surface expression of MHC class I proteins on MNV infected cells with MG132, a proteasome inhibitor. These findings indicate that MNV interferes with the MHC class I pathway in either directly degrading the protein or targeting it for the degradation pathway within the cell. Furthermore, we identified MNV NS3 as the viral protein which is essential and sufficient for the MHC class I surface reduction when separately expressed in cells. Additionally, we investigated the effect of MNV on cytokine secretion. The secretion of the cytokines IFNβ and TNFα is significantly reduced in MNV infected cells, which is not due to a down regulation of cytokine mRNA transcription. Analysis of the intracellular expression of cytokines and host cell translation in general, revealed a continuous decrease in global host cell translation in MNV infected cells. The translational shutdown seems to be induced by the dsRNA-sensitive regulator PKR. PKR becomes phosphorylated and phosphorylates the translation initiation factor eIF2α, impeding host cell translation. Whilst the translation of host proteins is stalled, viral proteins are still able to be translated due to a cap-independent mechanism. Furthermore, we interrogated the interaction of MNV with the microtubules and the microtubule-associated protein GEF-H1. We discovered an interaction of GEF-H1 with the viral protein MNV NS3, which leads to changes in the expression levels and location of GEF-H1 within the cell and prevents the formation of GEF-H1 induced microtubule fibres. This indicates a potential interference of MNV NS3 with GEF-H1, which has been proposed to play a major role in the immune detection of viral replication. Despite various approaches to identify a similar role of GEF-H1 during MNV infection, we have so far not been able to support the proposed function of the protein. Considering the multiple roles of GEFs like GEF-H1, it is possible that MNV and specifically NS3 acts on a different GEF-H1-regulated pathway during MNV infection

The Microtubule-Associated Innate Immune Sensor GEF-H1 Does Not Influence Mouse Norovirus Replication in Murine Macrophages

Norovirus is an acute infection of the gastrointestinal tract causing rapid induction of vomiting and diarrhoea. The infection is sensed and controlled by the innate immune system, particularly by the RNA helicase MDA-5 and type I and III interferons (IFNs). We have observed that intracellular replication of murine norovirus (MNV) occurs in membranous clusters proximal to the microtubule organising centre, a localisation dependent on intact microtubules. Recently, it was shown that the host protein guanine nucleotide exchange factor-H1 (GEF-H1) is a microtubule-associated innate immune sensor that activates interferon Regulatory Factor 3 to induce the production of type I IFNs. Thus, we interrogated the potential role of GEF-H1 in controlling MNV infections. We observed that GEF-H1 was recruited to the MNV replication complex; however RNAi-mediated suppression of GEF-H1 did not outwardly affect replication. We furthered our studies to investigate the impact of GEF-H1 on MNV innate detection and observed that GEF-H1 did not contribute to type I IFN induction during MNV infection or influenza virus infection but did result in a small reduction of interferon–β (IFNβ) during West Nile virus infection. Intriguingly, we discovered an interaction of GEF-H1 with the viral MNV non-structural protein 3 (NS3), an interaction that altered the location of GEF-H1 within the cell and prevented the formation of GEF-H1-induced microtubule fibres. Thus, our results indicate that GEF-H1 does not contribute significantly to the innate immune sensing of MNV, although its function may be modulated via interaction with the viral NS3 protein

Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity

Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity