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.

SARS-CoV-2 variants reveal features critical for replication in primary human cells.

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.

Breaking the Convention: Sialoglycan Variants, Coreceptors, and Alternative Receptors for Influenza A Virus Entry.

The influenza A virus (IAV) envelope protein hemagglutinin binds α2,6- or α2,3-linked sialic acid as a host cell receptor. Bat IAV subtypes H17N10 and H18N11 form an exception to this rule and do not bind sialic acid but enter cells via major histocompatibility complex (MHC) class II. Here, we review current knowledge on IAV receptors with a focus on sialoglycan variants, protein coreceptors, and alternative receptors that impact IAV attachment and internalization beyond the well-described sialic acid binding.

The impact of S2 mutations on Omicron SARS-CoV-2 cell surface expression and fusogenicity.

SARS-CoV-2 Omicron subvariants are still emerging and spreading worldwide. These variants contain a high number of polymorphisms in the spike (S) glycoprotein that could potentially impact their pathogenicity and transmission. We have previously shown that the S:655Y and P681H mutations enhance S protein cleavage and syncytia formation. Interestingly, these polymorphisms are present in Omicron S protein. Here, we characterized the cleavage efficiency and fusogenicity of the S protein of different Omicron sublineages. Our results showed that Omicron BA.1 subvariant is efficiently cleaved but it is poorly fusogenic compared to previous SARS-CoV-2 strains. To understand the basis of this phenotype, we generated chimeric S protein using combinations of the S1 and S2 domains from WA1, Delta and Omicron BA.1 variants. We found that the S2 domain of Omicron BA.1 hindered efficient cell-cell fusion. Interestingly, this domain only contains six unique polymorphisms never detected before in ancestral SARS-CoV-2 variants. WA1614G S proteins containing the six individuals S2 Omicron mutations were assessed for their fusogenicity and S surface expression after transfection in cells. Results showed that the S:N856K and N969K substitutions decreased syncytia formation and impacted S protein cell surface levels. However, we observed that "first-generation" Omicron sublineages that emerged subsequently, had convergently evolved to an enhanced fusogenic activity and S expression on the surface of infected cells while "second-generation" Omicron variants have highly diverged and showed lineage-specific fusogenic properties. Importantly, our findings could have potential implications in the improvement and redesign of COVID-19 vaccines.

MHC class II proteins mediate sialic acid independent entry of human and avian H2N2 influenza A viruses.

Influenza A viruses (IAV) pose substantial burden on human and animal health. Avian, swine and human IAV bind sialic acid on host glycans as receptor, whereas some bat IAV require MHC class II complexes for cell entry. It is unknown how this difference evolved and whether dual receptor specificity is possible. Here we show that human H2N2 IAV and related avian H2N2 possess dual receptor specificity in cell lines and primary human airway cultures. Using sialylation-deficient cells, we reveal that entry via MHC class II is independent of sialic acid. We find that MHC class II from humans, pigs, ducks, swans and chickens but not bats can mediate H2 IAV entry and that this is conserved in Eurasian avian H2. Our results demonstrate that IAV can possess dual receptor specificity for sialic acid and MHC class II, and suggest a role for MHC class II-dependent entry in zoonotic IAV infections.

H19 influenza A virus exhibits species-specific MHC class II receptor usage.

Avian influenza A virus (IAV) surveillance in Northern California, USA, revealed unique IAV hemagglutinin (HA) genome sequences in cloacal swabs from lesser scaups. We found two closely related HA sequences in the same duck species in 2010 and 2013. Phylogenetic analyses suggest that both sequences belong to the recently discovered H19 subtype, which thus far has remained uncharacterized. We demonstrate that H19 does not bind the canonical IAV receptor sialic acid (Sia). Instead, H19 binds to the major histocompatibility complex class II (MHC class II), which facilitates viral entry. Unlike the broad MHC class II specificity of H17 and H18 from bat IAV, H19 exhibits a species-specific MHC class II usage that suggests a limited host range and zoonotic potential. Using cell lines overexpressing MHC class II, we rescued recombinant H19 IAV. We solved the H19 crystal structure and identified residues within the putative Sia receptor binding site (RBS) that impede Sia-dependent entry.

Restriction factor screening identifies RABGAP1L-mediated disruption of endocytosis as a host antiviral defense.

Host interferons (IFNs) powerfully restrict viruses through the action of several hundred IFN-stimulated gene (ISG) products, many of which remain uncharacterized. Here, using RNAi screening, we identify several ISG restriction factors with previously undescribed contributions to IFN-mediated defense. Notably, RABGAP1L, a Tre2/Bub2/Cdc16 (TBC)-domain-containing protein involved in regulation of small membrane-bound GTPases, robustly potentiates IFN action against influenza A viruses (IAVs). Functional studies reveal that the catalytically active TBC domain of RABGAP1L promotes antiviral activity, and the RABGAP1L proximal interactome uncovered its association with proteins involved in endosomal sorting, maturation, and trafficking. In this regard, RABGAP1L overexpression is sufficient to disrupt endosomal function during IAV infection and restricts an early post-attachment, but pre-fusion, stage of IAV cell entry. Other RNA viruses that enter cells primarily via endocytosis are also impaired by RABGAP1L, while entry promiscuous SARS-CoV-2 is resistant. Our data highlight virus endocytosis as a key target for host defenses.

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.

Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization.

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.

Antiviral Activity of Type I, II, and III Interferons Counterbalances ACE2 Inducibility and Restricts SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is a recently emerged respiratory coronavirus that has infected >23 million people worldwide with >800,000 deaths. Few COVID-19 therapeutics are available, and the basis for severe infections is poorly understood. Here, we investigated properties of type I (ß), II (γ), and III (λ1) interferons (IFNs), potent immune cytokines that are normally produced during infection and that upregulate IFN-stimulated gene (ISG) effectors to limit virus replication. IFNs are already in clinical trials to treat COVID-19. However, recent studies highlight the potential for IFNs to enhance expression of host angiotensin-converting enzyme 2 (ACE2), suggesting that IFN therapy or natural coinfections could exacerbate COVID-19 by upregulating this critical virus entry receptor. Using a cell line model, we found that beta interferon (IFN-ß) strongly upregulated expression of canonical antiviral ISGs, as well as ACE2 at the mRNA and cell surface protein levels. Strikingly, IFN-λ1 upregulated antiviral ISGs, but ACE2 mRNA was only marginally elevated and did not lead to detectably increased ACE2 protein at the cell surface. IFN-γ induced the weakest ISG response but clearly enhanced surface expression of ACE2. Importantly, all IFN types inhibited SARS-CoV-2 replication in a dose-dependent manner, and IFN-ß and IFN-λ1 exhibited potent antiviral activity in primary human bronchial epithelial cells. Our data imply that type-specific mechanisms or kinetics shape IFN-enhanced ACE2 transcript and cell surface levels but that the antiviral action of IFNs against SARS-CoV-2 counterbalances any proviral effects of ACE2 induction. These insights should aid in evaluating the benefits of specific IFNs, particularly IFN-λ, as repurposed therapeutics.IMPORTANCE Repurposing existing, clinically approved, antiviral drugs as COVID-19 therapeutics is a rapid way to help combat the SARS-CoV-2 pandemic. Interferons (IFNs) usually form part of the body's natural innate immune defenses against viruses, and they have been used with partial success to treat previous new viral threats, such as HIV, hepatitis C virus, and Ebola virus. Nevertheless, IFNs can have undesirable side effects, and recent reports indicate that IFNs upregulate the expression of host ACE2 (a critical entry receptor for SARS-CoV-2), raising the possibility that IFN treatments could exacerbate COVID-19. Here, we studied the antiviral- and ACE2-inducing properties of different IFN types in both a human lung cell line model and primary human bronchial epithelial cells. We observed differences between IFNs with respect to their induction of antiviral genes and abilities to enhance the cell surface expression of ACE2. Nevertheless, all the IFNs limited SARS-CoV-2 replication, suggesting that their antiviral actions can counterbalance increased ACE2.

Propagation and Titration of Influenza Viruses.

Influenza viruses are constantly circulating among humans, in which they cause seasonal epidemics of severe respiratory disease. Additionally, these zoonotic viruses infect different mammals and birds, from which new antigenic variants are occasionally transmitted to humans leading to devastating global pandemics. Surveillance programs, in which viruses from the main reservoir (waterfowl), intermediate hosts (like pigs and other farm animals), and other affected species are isolated and characterized, are crucial for the global influenza prevention strategy. This chapter gives an overview of the most commonly used methods for the propagation and titration of influenza viruses, which are key steps in surveillance procedures, as well as in vaccine development and basic research. Depending on the host and the viral strain, primary isolates are obtained from biological samples of different origin and subsequently amplified in embryonated chicken eggs or cell cultures. These propagation procedures are the focus of the first part of this chapter. Once the initial isolates have been amplified, virus titration methods based on particular characteristics of influenza viruses, such as their ability to agglutinate red blood cells (RBCs) or to induce cytopathic effects (CPE) in cell monolayers, are used to estimate the amount of viral particles. Such approaches, like the hemagglutination assay (HA assay), 50% tissue culture infectious dose (TCID50), or plaque assay, are included in the second part of this chapter. Although they are simple and cost-effective, some of these techniques have been partially replaced by faster and more sensitive methods based on the quantification of viral genomes, such as the quantitative real-time reverse transcription PCR (RT-qPCR), which is presented at the end of this section. The different protocols are explained in detail in order to facilitate the preparation and quantification of infectious virus stocks.

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.