MHC class II proteins mediate cross-species entry of bat influenza viruses.

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.

Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir.

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC50 value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

Unraveling virus-induced cellular signaling cascades by label-free quantitative phosphoproteomics.

Due to the low stoichiometry and highly transient nature of protein phosphorylation it is challenging to capture the dynamics and complexity of phosphorylation events on a systems level. Here, we present an optimized protocol to measure virus-induced phosphorylation events with high sensitivity using label free quantification-based phosphoproteomics. Specifically, we describe filter assisted protein digestion (FASP), enrichment of phosphopeptides, mass spectrometry, and subsequent bioinformatic analysis. For complete details on the use and execution of this protocol, please refer to Hunziker et al. (2022).

Phosphoproteomic profiling of influenza virus entry reveals infection-triggered filopodia induction counteracted by dynamic cortactin phosphorylation.

Binding of influenza virus to its receptor triggers signaling cascades that reprogram the cell for infection. To elucidate global virus-induced changes to the cellular signaling landscape, we conducted a quantitative phosphoproteomic screen with human and avian influenza viruses. Proteins with functions in cell adhesion and cytoskeletal remodeling are overrepresented among the hits, and the majority of factors undergoing phosphorylation changes have a significant impact on infection efficiency. We show that influenza virus induces the formation of filopodia through Cdc42 signaling, which results in enhanced virus endocytosis. The host cell counteracts this mechanism with cortactin, a regulator of actin polymerization that becomes phosphorylated in response to virus binding and translocates to the cell cortex, where it limits filopodia formation and virus uptake. Overall, our study reveals the signaling cascades induced by influenza virus receptor engagement and uncovers virus-induced filopodia formation that is counteracted by the host cell.

The dengue virus 4 component of NIAID's tetravalent TV003 vaccine drives its innate immune signature.

Annually, roughly 2.5 billion people are at risk for dengue virus (DENV) infection, and the incidence of infection has increased 30-fold since its discovery in the 1900s. At present, there are no globally licensed antiviral treatments or vaccines that protect against all four of the DENV serotypes. The NIAID Live Attenuated Tetravalent Vaccine (LATV) dengue vaccine candidate is composed of variants of three DENV serotypes attenuated by a 30 nucleotide (Δ30) deletion in the 3' untranslated region and a fourth component that is a chimeric virus in which the prM and E genes of DENV-2 replace those of DENV-4 on the rDEN4Δ30 backbone. The vaccine candidate encodes the non-structural proteins of DENV-1, DENV-3, and DENV-4, which could be of critical importance in the presentation of DENV-specific epitopes in a manner that facilitates antigen presentation and confers higher protection. Our findings demonstrate that the attenuation mechanism (Δ30) resulted in decreased viral infectivity and replication for each vaccine virus in monocyte-derived dendritic cells but were able to generate a robust innate immune response. When tested as monovalent viruses, DEN-4Δ30 displayed the most immunogenic profile. In addition, we found that the tetravalent DENV formulation induced a significantly greater innate immune response than the trivalent formulation. We demonstrate that the presence of two components with a DENV-4Δ30 backbone is necessary for the induction of RANTES, CD40, IP-10, and Type I IFN by the tetravalent formulation. Finally, we found that the DEN-4Δ30 backbone in the DENV-2 component of the vaccine enhanced its antigenic properties, as evidenced by enhanced ability to induce IP-10 and IFNα2 in monocyte-derived dendritic cells. In sum, our study shows that the Δ30 and Δ30/Δ31 mutations attenuate the DENV vaccine strains in terms of replication and infectivity while still allowing the induction of a robust innate immune response.

IFITM3 incorporation sensitizes influenza A virus to antibody-mediated neutralization.

The disease severity of influenza is highly variable in humans, and one genetic determinant behind these differences is the IFITM3 gene. As an effector of the interferon response, IFITM3 potently blocks cytosolic entry of influenza A virus (IAV). Here, we reveal a novel level of inhibition by IFITM3 in vivo: We show that incorporation of IFITM3 into IAV particles competes with incorporation of viral hemagglutinin (HA). Decreased virion HA levels did not reduce infectivity, suggesting that high HA density on IAV virions may be an antagonistic strategy used by the virus to prevent direct inhibition. However, we found that IFITM3-mediated reduction in HA content sensitizes IAV to antibody-mediated neutralization. Mathematical modeling predicted that this effect decreases and delays peak IAV titers, and we show that, indeed, IFITM3-mediated sensitization of IAV to antibody-mediated neutralization impacts infection outcome in an in vivo mouse model. Overall, our data describe a previously unappreciated interplay between the innate effector IFITM3 and the adaptive immune response.

Multifactorial seroprofiling dissects the contribution of pre-existing human coronaviruses responses to SARS-CoV-2 immunity.

Determination of SARS-CoV-2 antibody responses in the context of pre-existing immunity to circulating human coronavirus (HCoV) is critical for understanding protective immunity. Here we perform a multifactorial analysis of SARS-CoV-2 and HCoV antibody responses in pre-pandemic (N = 825) and SARS-CoV-2-infected donors (N = 389) using a custom-designed multiplex ABCORA assay. ABCORA seroprofiling, when combined with computational modeling, enables accurate definition of SARS-CoV-2 seroconversion and prediction of neutralization activity, and reveals intriguing interrelations with HCoV immunity. Specifically, higher HCoV antibody levels in SARS-CoV-2-negative donors suggest that pre-existing HCoV immunity may provide protection against SARS-CoV-2 acquisition. In those infected, higher HCoV activity is associated with elevated SARS-CoV-2 responses, indicating cross-stimulation. Most importantly, HCoV immunity may impact disease severity, as patients with high HCoV reactivity are less likely to require hospitalization. Collectively, our results suggest that HCoV immunity may promote rapid development of SARS-CoV-2-specific immunity, thereby underscoring the importance of exploring cross-protective responses for comprehensive coronavirus prevention.

TIGIT limits immune pathology during viral infections.

Co-inhibitory pathways have a fundamental function in regulating T cell responses and control the balance between promoting efficient effector functions and restricting immune pathology. The TIGIT pathway has been implicated in promoting T cell dysfunction in chronic viral infection. Importantly, TIGIT signaling is functionally linked to IL-10 expression, which has an effect on both virus control and maintenance of tissue homeostasis. However, whether TIGIT has a function in viral persistence or limiting tissue pathology is unclear. Here we report that TIGIT modulation effectively alters the phenotype and cytokine profile of T cells during influenza and chronic LCMV infection, but does not affect virus control in vivo. Instead, TIGIT has an important effect in limiting immune pathology in peripheral organs by inducing IL-10. Our data therefore identify a function of TIGIT in limiting immune pathology that is independent of viral clearance.

Phosphoproteomic-based kinase profiling early in influenza virus infection identifies GRK2 as antiviral drug target.

Although annual influenza epidemics affect around 10% of the global population, current treatment options are limited and development of new antivirals is needed. Here, using quantitative phosphoproteomics, we reveal the unique phosphoproteome dynamics that occur in the host cell within minutes of influenza A virus (IAV) infection. We uncover cellular kinases required for the observed signaling pattern and find that inhibition of selected candidates, such as the G protein-coupled receptor kinase 2 (GRK2), leads to decreased IAV replication. As GRK2 has emerged as drug target in heart disease, we focus on its role in IAV infection and show that it is required for viral uncoating. Replication of seasonal and pandemic IAVs is severely decreased by specific GRK2 inhibitors in primary human airway cultures and in mice. Our study reveals the IAV-induced changes to the cellular phosphoproteome and identifies GRK2 as crucial node of the kinase network that enables IAV replication.