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The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression towards severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4-6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an “early” inflammatory/immune signature preceding a “late” type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

Serum titers of SARS-CoV-2 neutralizing antibodies (nAb) correlate well with protection from symptomatic COVID-19, but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are life-long after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We therefore sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensivein vivomouse testing demonstrated potent induction of high titer nAb in measles-immune mice and confirmed the significant incremental contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin-display of the SARS-CoV-2 spike glycoprotein, and vaccine resurfacing. In animals primed and boosted with a MeV vaccine encoding the ancestral SARS-CoV-2 spike, high titer nAb responses against ancestral virus strains were only weakly cross-reactive with the omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the omicron BA.1 spike, antibody titers to both ancestral and omicron strains were robustly elevated and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that antigen engineering can enable the development of potent measles-based SARS-CoV-2 vaccine candidates.

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic—Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection bothin vitro, and in the Syrian golden hamster modelin vivo. In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants.ONE SENTENCE SUMMARYSARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

SUMMARYThe currently expanding monkeypox epidemic is caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. In contrast to monkeypox cases caused by clade I and subclade IIa MPXV, the prognosis of current cases is generally favorable, but person-to-person transmission is much more efficient. MPXV evolution is driven by selective pressure from hosts and loss of virus–host interacting genes. However, there is no satisfactory genetic explanation using single-nucleotide polymorphisms (SNPs) for the observed increased MPXV transmissibility. We hypothesized that key genomic changes may occur in the genome’s low-complexity regions (LCRs), which are highly challenging to sequence and have been dismissed as uninformative. Using a combination of highly sensitive techniques, we determined a first high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This effort revealed significant variation in short-tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of SNPs and that LCRs are not randomly distributed.In silicoanalyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs) 153, 204, and 208 could be affected in a manner consistent with the established “genomic accordion” evolutionary strategies of orthopoxviruses. Consequently, we posit that genomic studies focusing on phenotypic MPXV clade-/subclade-/lineage-/strain differences should change their focus to the study of LCR variability instead of SNP variability.

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat (Rhinolophus ferrumequinum) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats’ unique traits.

Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulates the immune response against many RNA viruses. We identified a small molecule compound, KIN1148, that directly binds RIG-I to drive IRF3activation to impart the expression of IRF3-target genes, including specific immunomodulatory cytokines and chemokines. KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I interferon (IFN) treatment. When administered in combination with a vaccine against influenza A virus (IAV), KIN1148 induces both neutralizing antibody and broadly cross-protective IAV-specific T cell responses compared to vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal H1N1 and H5N1 IAV challenge. Importantly, KIN1148 also augments human CD8+ T cell activation. Thus, we have identified a small molecule RIG-I agonist that serves as an effective adjuvant in inducing non-canonical RIG-I activation for induction of innate immune programs that enhance adaptive immune protection of antiviral vaccination.SummaryHemann, et. al. identify a small-molecule RIG-I agonist (KIN1148) that directly binds RIG-I for non-canonical activation and adjuvants pandemic and avian influenza virus vaccination. KIN1148 augments broadly neutralizing antibody and T cell responses in mice and enhances human DC maturation and CD8+ T cell activation.

Antiviral therapeutics to treat SARS-CoV-2 are much desired for the on-going pandemic. A well-precedented viral enzyme is the main protease (MPro), which is now targeted by an approved drug and by several investigational drugs. With the inevitable liabilities of these new drugs, and facing viral resistance, there remains a call for new chemical scaffolds against MPro. We virtually docked 1.2 billion non-covalent and a new library of 6.5 million electrophilic molecules against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC50 of 29 μM and 20 μM, respectively. Several series were optimized, resulting in inhibitors active in the low micromolar range. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. Together, these compounds reveal new chemotypes to aid in further discovery of MPro inhibitors for SARS-CoV-2 and other future coronaviruses.

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs. ispartof: bioRxiv ispartof: location:United States status: Published online

NDV-HXP-S is a recombinant Newcastle disease virus based-vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which expresses an optimized (HexaPro) spike protein on its surface. The vaccine can be produced in embryonated chicken eggs using the same process as that employed for the production of influenza virus vaccines. Here we performed a secondary analysis of the antibody responses after vaccination with inactivated NDV-HXP-S in a Phase I clinical study in Thailand.The SARS-CoV-2 neutralizing and spike binding activity of NDV-HXP-S post-vaccination serum samples was compared to that of matched samples from mRNA BNT162b2 (Pfizer) vaccinees. Neutralizing activity of sera from NDV-HXP-S vaccinees was comparable to that of individuals vaccinated with BNT162b2. Interstingly, the spike binding activity of the NDV-HXP-S vaccinee samples was lower than that of sera obtained from individuals vaccinated with the mRNA vaccine. This let us to calculate ratios between binding and neutralizing antibody titers. Samples from NDV-HXP-S vaccinees had binding to neutralizing activity ratios similar to those of convalescent sera suggesting a very high proportion of neutralizing antibodies and low non-neutralizing antibody titers. Further analysis showed that, in contrast to mRNA vaccination, which induces strong antibody titers to the receptor binding domain (RBD), the N-terminal domain, and the S2 domain, NDV-HXP-S vaccination induces a very RBD focused response with little reactivity to S2. This explains the high proportion of neutralizing antibodies since most neutralizing epitopes are located in the RBD. In conclusion, vaccination with inactivated NDV-HXP-S induces a high proportion of neutralizing antibodies and absolute neutralizing antibody titers comparable to those after mRNA vaccination.

The continual emergence of SARS-CoV-2 variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant, has rendered ineffective a number of previously EUA approved SARS-CoV-2 neutralizing antibody therapies. Furthermore, even those approved antibodies with neutralizing activity against Omicron are reportedly ineffective against the subset of Omicron variants that contain a R346K substitution, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. Following a campaign of antibody discovery based on the vaccination of Harbour H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of Spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against the Omicron and Omicron + R346K variants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for use in human clinical trials.

Ly6Chi inflammatory monocytes show high IFN responses, and contribute to both protective and pathogenic functions following influenza virus infection. In order to understand the significance of IFN responses in this subset, we examined monocytes during infection with a lethal H5N1 virus that induces high levels of IFN and a low-pathogenicity H1N1 virus that induces low levels of IFN. We show that H5N1 infection results in early recruitment of high numbers of Ly6Chi monocytes and induction of chemokines and Ifnb1. Using unbiased transcriptomic and proteomic approaches, we also find that monocytes are significantly enriched during H5N1 infection and are associated with chemokine and IFN signatures in mice, and with severity of symptoms after influenza virus infection in humans. Recruited Ly6Chi monocytes subsequently become infected in the lung, produce IFN-β, and mature into FasL+ monocyte-derived cells (FasL+MCs) expressing dendritic cell markers. Both Ccr2-/- and Faslgld mice are protected from lethal infection, indicating that monocytes contribute to pathogenesis. Global loss of type I and type III IFN signaling in Stat2-/- mice results in loss of monocyte recruitment, likely reflecting a requirement for IFN-dependent chemokine induction. Here we show that IFN is not directly required for monocyte recruitment on an IFN-sufficient background, but is required for maturation to FasL+MCs. Loss of IFN signaling skews to a Ly6Clo phenotype associated with tissue repair, suggesting that IFN signaling in monocytes is a critical determinant of influenza virus pathogenesis.

SUMMARYCyclic GMP-AMP synthase (cGAS) is a primary sensor of aberrant DNA that governs an innate immune signaling cascade, leading to the induction of the type-I interferon response. We have previously identified polyglutamine binding protein 1, PQBP1, as an adaptor molecule required for cGAS-mediated innate immune response of lentiviruses, including the human immunodeficiency virus 1 (HIV-1), but dispensable for the recognition of DNA viruses. HIV-1- encoded DNA is synthesized as a single copy from its RNA genome, and is subsequently integrated into the host chromatin. HIV-1 then produces progeny through amplification and packaging of its RNA genome, thus, in contrast to DNA viruses, HIV-1 DNA is both transient and of low abundance. However, the molecular basis for the detection and verification of this low abundance HIV-1 DNA pathogen-associated molecular pattern (PAMP) is not understood. Here, we elucidate a two-factor authentication strategy that is employed by the innate immune surveillance machinery to selectively respond to the low concentration of PAMP, while discerning these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates intact viral capsid, which serves as a primary verification step for the viral nucleic acid cargo. As the reverse transcription and capsid disassembly initiate, cGAS protein is then recruited to the capsid in a PQBP1-dependent manner, enabling cGAS molecules to be co-positioned at the site of PAMP generation. Thus, these data indicate that PQBP1 recognition of the HIV-1 capsid sanctions a robust cGAS-dependent response to a limited abundance and short-lived DNA PAMP. Critically, this illuminates a molecular strategy wherein the modular recruitment of co-factors to germline encoded pattern recognition receptors (PRRs) serves to enhance repertoire of pathogens that can be sensed by the innate immune surveillance machinery.

Influenza A viruses (IAV) remain emerging threats to human public health. Live-attenuated influenza vaccines (LAIV) are one of the most effective prophylactic options to prevent disease caused by influenza infections. However, licensed LAIV remain restricted for use in 2- to 49-year old healthy and non-pregnant people. Therefore, development of LAIV with increased safety, immunogenicity, and protective efficacy is highly desired. The United States (U.S.) licensed LAIV is based on the master donor virus (MDV) A/Ann Arbor/6/60 H2N2 backbone, which was generated by adaptation of the virus to growth at low temperatures. Introducing the genetic signature of the U.S. MDV into the backbone of other IAV strains resulted in varying levels of attenuation. While the U.S. MDV mutations conferred an attenuated phenotype to other IAV strains, the same amino acid changes did not significantly attenuate the pandemic A/California/04/09 H1N1 (pH1N1) strain. To attenuate pH1N1, we replaced the conserved leucine at position 319 with glutamine (L319Q) in PB1 and analyzed the in vitro and in vivo properties of pH1N1 viruses containing either PB1 L319Q alone or in combination with the U.S. MDV mutations using two animal models of influenza infection and transmission, ferrets and guinea pigs. Our results demonstrated that L319Q substitution in the pH1N1 PB1 alone or in combination with the mutations of the U.S. MDV resulted in reduced pathogenicity (ferrets) and transmission (guinea pigs), and an enhanced temperature sensitive phenotype. These results demonstrate the feasibility of generating an attenuated MDV based on the backbone of a contemporary pH1N1 IAV strain.IMPORTANCEVaccination represents the most effective strategy to reduce the impact of seasonal IAV infections. Although LAIV are superior in inducing protection and sterilizing immunity, they are not recommended for many individuals who are at high risk for severe disease. Thus, development of safer and more effective LAIV are needed. A concern with the current MDV used to generate the U.S. licensed LAIV is that it is based on a virus isolated in 1960. Moreover, mutations that confer the temperature sensitive, cold-adapted, and attenuated phenotype of the U.S. MDV resulted in low level of attenuation in the contemporary pandemic A/California/04/09 H1N1 (pH1N1). Here, we show that introduction of PB1 L319Q substitution, alone or in combination with the U.S. MDV mutations, resulted in pH1N1 attenuation. These findings support the development of a novel LAIV MDV based on a contemporary pH1N1 strain as a medical countermeasure against currently circulating H1N1 IAV.

Single cell RNA sequencing (scRNAseq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNAseq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. The performance of different scRNAseq methods to study SARS-CoV-2 RNAs has not been thoroughly evaluated. Here, we compare different scRNAseq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs), which are produced only during active viral replication and not present in viral particles. We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') and Chromium Next GEM Single Cell V(D)J (10X 5') sequencing, we find that 10X 5' with an extended R1 sequencing strategy maximizes the unambiguous detection of sgmRNAs by increasing the number of reads spanning leader-sgmRNA junction sites. Differential gene expression testing and KEGG enrichment analysis of infected cells compared with bystander or mock cells showed an enrichment for COVID19-associated genes, supporting the ability of our method to accurately identify infected cells. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.Single cell RNA sequencing (scRNAseq) has emerged as a valuable tool to study host-viral interactions particularly in the context of COronaVIrus Disease-2019 (COVID-19). scRNAseq has been developed and optimized for analyzing eukaryotic mRNAs, and the ability of scRNAseq to measure RNAs produced by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has not been fully characterized. Here we compare the performance of different scRNAseq methods to detect and quantify SARS-CoV-2 RNAs and develop an analysis workflow to specifically quantify unambiguous reads derived from SARS-CoV-2 genomic RNA and subgenomic mRNAs. Our work demonstrates the strengths and limitations of scRNAseq to measure SARS-CoV-2 RNA and identifies experimental and analytical approaches that allow for SARS-CoV-2 RNA detection and quantification. These developments will allow for studies of coronavirus RNA biogenesis at single-cell resolution to improve our understanding of viral pathogenesis.

SARS-CoV-2, a novelBetacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, andin silicoandin vitrostudies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult white-tailed deer are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.

For efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2’ by different cellular proteases such as furin and TMPRSS2. Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis. Here, we investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, we showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates. To better understand the impact of this polymorphism, we analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds. Results demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals. Finally, we investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins. Results showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein. Our study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.

Rapid development of coronavirus disease 2019 (COVID-19) vaccines and expedited authorization for use and approval has been proven beneficial to mitigate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread and given hope in this desperate situation. It is believed that sufficient supplies and equitable allocations of vaccines are necessary to limit the global impact of the COVID-19 pandemic and the emergence of additional variants of concern. We have developed a COVID-19 vaccine based on Newcastle disease virus (NDV) that can be manufactured at high yields in embryonated eggs. Here we provide evidence that the NDV vector expressing an optimized spike antigen (NDV-HXP-S), upgraded from our previous construct, is a versatile vaccine that can be used live or inactivated to induce strong antibody responses and to also cross-neutralize variants of concern. The immunity conferred by NDV-HXP-S effectively counteracts SARS-CoV-2 infection in mice and hamsters. It is noteworthy that vaccine lots produced by existing egg-based influenza virus vaccine manufacturers in Vietnam, Thailand and Brazil exhibited excellent immunogenicity and efficacy in hamsters, demonstrating that NDV-HXP-S vaccines can be quickly produced at large-scale to meet global demands.

SummaryBroadly neutralizing antibodies against influenza virus hemagglutinin (HA) have the potential to provide universal protection against influenza virus infections. Here, we report a distinct class of broadly neutralizing antibodies targeting an epitope toward the bottom of the HA stalk domain where HA is “anchored” to the viral membrane. Antibodies targeting this membrane-proximal anchor epitope utilized a highly restricted repertoire, which encode for two conserved motifs responsible for HA binding. Anchor targeting B cells were common in the human memory B cell repertoire across subjects, indicating pre-existing immunity against this epitope. Antibodies against the anchor epitope at both the serological and monoclonal antibody levels were potently induced in humans by a chimeric HA vaccine, a potential universal influenza virus vaccine. Altogether, this study reveals an underappreciated class of broadly neutralizing antibodies against H1-expressing viruses that can be robustly recalled by a candidate universal influenza virus vaccine.

There is an urgent need to identify which COVID-19 patients will develop life-threatening illness so that scarce medical resources can be optimally allocated and rapid treatment can be administered early in the disease course, when clinical management is most effective. To aid in the prognostic classification of disease severity, we performed untargeted metabolomics profiling of 341 patients with plasma samples collected at six longitudinal time points. Using the temporal metabolic profiles and machine learning, we then built a predictive model of disease severity. We determined that the levels of 25 metabolites measured at the time of hospital admission successfully predict future disease severity. Through analysis of longitudinal samples, we confirmed that these prognostic markers are directly related to disease progression and that their levels are restored to baseline upon disease recovery. Finally, we validated that these metabolites are also altered in a hamster model of COVID-19. Our results indicate that metabolic changes associated with COVID-19 severity can be effectively used to stratify patients and inform resource allocation during the pandemic.

One year in the coronavirus disease 2019 (COVID-19) pandemic, the first vaccines are being rolled out under emergency use authorizations. It is of great concern that newly emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can escape antibody-mediated protection induced by previous infection or vaccination through mutations in the spike protein. The glutamate (E) to Lysine (K) substitution at position 484 (E484K) in the receptor binding domain (RBD) of the spike protein is present in the rapidly spreading variants of concern belonging to the B.1.351 and P.1 lineages. We performed in vitro microneutralization assays with both the USA-WA1/2020 virus and a recombinant (r)SARS-CoV-2 virus that is identical to USA-WA1/2020 except for the E484K mutation introduced in the spike RBD. We selected 34 sera from study participants based on their SARS-CoV-2 spike ELISA antibody titer (negative [N=4] versus weak [N=8], moderate [N=11] or strong positive [N=11]). In addition, we included sera from five individuals who received two doses of the Pfizer SARS-CoV-2 vaccine BNT162b2. Serum neutralization efficiency was lower against the E484K rSARS-CoV-2 (vaccination samples: 3.4 fold; convalescent low IgG: 2.4 fold, moderate IgG: 4.2 fold and high IgG: 2.6 fold) compared to USA-WA1/2020. For some of the convalescent donor sera with low or moderate IgG against the SARS-CoV-2 spike, the drop in neutralization efficiency resulted in neutralization ID50 values similar to negative control samples, with low or even absence of neutralization of the E484K rSARS-CoV-2. However, human sera with high neutralization titers against the USA-WA1/2020 strain were still able to neutralize the E484K rSARS-CoV-2. Therefore, it is important to aim for the highest titers possible induced by vaccination to enhance protection against newly emerging SARS-CoV-2 variants. Two vaccine doses may be needed for induction of high antibody titers against SARS-CoV-2. Postponing the second vaccination is suggested by some public health authorities in order to provide more individuals with a primer vaccination. Our data suggests that this may leave vaccinees less protected against newly emerging variants.

The current COVID-19 (coronavirus disease 19) pandemic, caused by SARS-CoV-2, disproportionally affects the elderly and people with comorbidities like obesity and associated type 2 diabetes mellitus. Small animal models are crucial for the successful development and validation of antiviral vaccines, therapies and to study the role that comorbidities have on the outcome of viral infections. The initially available SARS-CoV-2 isolates require adaptation in order to use the mouse angiotensin converting enzyme 2 (mACE-2) entry receptor and to productively infect the cells of the murine respiratory tract. We have “mouse-adapted” SARS-CoV-2 by serial passaging a clinical virus isolate in the lungs of mice. We then used low doses of this virus in mouse models for advanced age, diabetes and obesity. Similar to SARS-CoV-2 infection in humans, the outcome of infection with mouse-adapted SARS-CoV-2 resulted in enhanced morbidity in aged and diabetic obese mice. Mutations associated with mouse adaptation occurred in the S, M, N and ORF8 genes. Interestingly, one mutation in the receptor binding domain of the S protein results in the change of an asparagine to tyrosine residue at position 501 (N501Y). This mutation is also present in the newly emerging SARS-CoV-2 variant viruses reported in the U.K. (20B/501Y.V1, B1.1.7 lineage) that is epidemiologically associated with high human to human transmission. We show that human convalescent and post vaccination sera can neutralize the newly emerging N501Y virus variant with similar efficiency as that of the reference USA-WA1/2020 virus, suggesting that current SARS-CoV-2 vaccines will protect against the 20B/501Y.V1 strain.

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naïve cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Histologic lesions that characterized primary SARS-CoV-2 infected cats at 4 DPC were absent in the re-challenged cats. Naïve sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naïve sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against reinfection.

The COVID-19 pandemic was a stark reminder that a barren global antiviral pipeline has grave humanitarian consequences. Pandemics could be prevented in principle by accessible, easily deployable broad-spectrum oral antivirals. Here we report the results of theCOVID Moonshot, a fully open-science, crowd sourced, structure-enabled drug discovery campaign targeting the SARS-CoV-2 main protease. We discovered a novel chemical series that is differentiated from current Mpro inhibitors in that it maintains a new non-covalent, non-peptidic scaffold with nanomolar potency. Our approach leveraged crowdsourcing, high-throughput structural biology, machine learning, and exascale molecular simulations and high-throughput chemistry. In the process, we generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. In a first for a structure-based drug discovery campaign, all compound designs (>18,000 designs), crystallographic data (>840 ligand-bound X-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2,400 compounds) for this campaign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.

While the current pandemic remains a thread to human health, the polyclonal nature of the antibody response against SARS-CoV-2 is not fully understood. Other than SARS-CoV-2, humans are susceptible to six different coronaviruses, and previous exposure to antigenically related and divergent seasonal coronaviruses is frequent. We longitudinally profiled the early humoral immune response against SARS-CoV-2 on hospitalized COVID-19 patients, and quantify levels of pre-existing immunity to OC43, HKU1 and 223E seasonal coronaviruses. A strong back-boosting effect to conserved, but not variable regions of OC43 and HKU1 betacoronaviruses spike protein was observed. All patients developed antibodies against SARS-CoV-2 spike and nucleoprotein, with peak induction at day 7 post hospitalization. However a negative correlation was found between antibody memory boost to human coronaviruses and induction of IgG and IgM against SARS-CoV-2 spike. Our findings provide evidence of immunological imprinting that determine the antibody profile to COVID-19 patients in an original antigenic sin fashion.

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the virus that causes coronavirus disease 2019 (COVID‐19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ‐PEG‐CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol‐poly(ethylene glycol) macromolecular amphiphile. It is water‐soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ‐PEG‐CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS‐CoV‐2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model., Amphiphilic TRL7/8 agonist adjuvanted recombinant Spike protein vaccine protects against SARS‐CoV‐2 infection in mouse models.

A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors that inhibited viral entry, nucleic acid binding proteins that suppressed viral RNA synthesis, and a highly enriched cluster of ER and Golgi-resident ISGs that inhibited viral translation and egress. These included the type II integral membrane protein BST2/tetherin, which was found to impede viral release, and is targeted for immune evasion by SARS-CoV-2 Orf7a protein. Overall, these data define the molecular basis of early innate immune control of viral infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19. Funding: This work was supported by the following grants to the Sanford Burnham Prebys Medical Discovery Institute and the Icahn School of medicine at Mount Sinai: DoD: W81XWH-20-10270; DHIPC: U19 AI118610; Fluomics/NOSI: U19 AI135972. This work was also supported by generous philanthropic donations from Dinah Ruch and Susan & James Blair, from the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)) and anonymous donors. Additional support has been provided by DARPA grant HR0011-19-2-0020 and by CRIP (Center for research on Influenza Pathogenesis), a NIAID-funded Center of Excellence for Influenza Research and Surveillance (CEIRS, contract # HHSN272201400008C). This work was additionally supported by the following grants to Northwestern University Feinberg School of Medicine: a CTSA supplement to NCATS: UL1 TR002389; a CTSA supplement to NUCATS with the generous support of the Dixon family: UL1 TR001422; and a Cancer Center supplement: P30 CA060553, and the following grant to JG at UC San Diego: NIH grant R37AI081668. This work was also supported by a generous grant from the James B. Pendleton Charitable Trust. Conflict of Interest: The authors declare no competing interests. Ethical Approval: All experiments involving live SARS-CoV-2 followed the approved standard operating procedures of the Biosafety Level 3 facility at the Sanford Burnham Prebys Medical Discovery Institute.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively instable, a feature that might be enhanced by the presence of a polybasic cleavage site in the SARS-CoV-2 spike. Exchange of K986 and V987 to prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle Eastern respiratory syndrome coronavirus spikes. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin converting enzyme 2 via adenovirus transduction. Variants tested include spike protein with a deleted polybasic cleavage site, the proline mutations, a combination thereof, as well as the wild type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the PP mutations completely protected from challenge in this mouse model.ImportanceA vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validates the choice of antigens that contain the PP mutation and suggests that deletion of the polybasic cleavage site could lead to a further optimized design.

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

Due to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus. Here, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type or a pre-fusion membrane anchored format. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, we report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.Research in contextEvidence before this studyThe spike (S) protein of the SARS-CoV-2 is the major antigen that notably induces neutralizing antibodies to block viral entry. Many COVID-19 vaccines are under development, among them viral vectors expressing the S protein of SARS-CoV-2 exhibit many benefits. Viral vector vaccines have the potential of being used as both live or inactivated vaccines and they can induce Th1 and Th2-based immune responses following different immunization regimens. Additionally, viral vector vaccines can be handled under BSL-2 conditions and they grow to high titers in cell cultures or other species restricted-hosts. For a SARS-CoV-2 vaccine, several viral vectors are being tested, such as adenovirus, measles virus and Modified vaccinia Ankara.Added value of this studyThe NDV vector vaccine against SARS-CoV-2 described in this study has advantages similar to those of other viral vector vaccines. But the NDV vector can be amplified in embryonated chicken eggs, which allows for high yields and low costs per dose. Also, the NDV vector is not a human pathogen, therefore the delivery of the foreign antigen would not be compromised by any pre-existing immunity in humans. Finally, NDV has a very good safety record in humans, as it has been used in many oncolytic virus trials. This study provides an important option for a cost-effective SARS-CoV-2 vaccine.Implications of all the available evidenceThis study informs of the value of a viral vector vaccine against SARS-CoV-2. Specifically, for this NDV based SARS-CoV-2 vaccine, the existing egg-based influenza virus vaccine manufactures in the U.S. and worldwide would have the capacity to rapidly produce hundreds of millions of doses to mitigate the consequences of the ongoing COVID-19 pandemic.

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2)(1-3). Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in both the lung and brain leading to lethality. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the lung, and no clinical signs of infection with a challenge dose of 104 plaque forming units. The K18-hACE2 model provides a stringent model for testing the ability of vaccines and antivirals to protect against disease, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.

Blighted Gotham Deaths caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in New York City (NYC) during the spring of 2020 have vastly exceeded those reported in China and many other countries. What were the early events that led to such a severe outbreak? Gonzalez-Reiche et al. sampled some of the early patients seeking assistance in February and March of 2020 at the Mount Sinai Health System. Phylogenetic analysis of virus sequences in these people, who were drawn from across NYC, showed that the virus had been independently introduced many times from Europe and elsewhere in the United States. Subsequent clusters of community transmission occurred. The focus of infection in NYC is a marker of the role this city plays as a two-way hub for human movement. Science this issue p. 297

SummaryDeubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host innate immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, DUBs that regulate this pathway have not been identified. Using a ubiquitin C-terminal electrophile, we profiled DUBs that function during influenza A virus (IAV) infection, and isolated OTUB1 as a key regulator of RIG-I dependent antiviral responses. OTUB1 was interferon-inducible, and interacted with RIG-I, viral PB2 and NS1. Upon infection, OTUB1 relocalised from the nucleus to mitochondrial membranes, and activated the RIG-I signaling complex via hydrolysis of K48 polyubiquitin chains and by forming a repressive complex with UBCH5c. Using a reconstituted system composed of in vitro translated [35S]IRF3, purified RIG-I, mitochondrial membranes and cytosol expressing OTUB1 variants, we recapitulated the mechanism of OTUB1-dependent RIG-I activation. A wide range of IAV NS1 proteins triggered proteasomal degradation of OTUB1, thereby antagonizing the RIG-I signaling cascade and antiviral responses.HighlightsOTUB1 is induced during influenza A virus infections in an IFN-I dependent mannerOTUB1 regulates the RIG-I complex by hydrolysing K48-linked polyubiquitin chains and by sequestering UBCH5c to prevent K48 polyubiquitylationOptimal K63 versus K48 polyubiquitin chain concentrations determine RIG-I activationInfluenza NS1 targets OTUB1 for proteasomal degradation

The transcription of interferon-stimulated genes (ISGs) is classically triggered via activation of the JAK-STAT pathway, and together, ISGs raise a multifaceted antiviral barrier. An increasing body of evidence reports the existence of additional, non-canonical pathways and transcription factors that coordinate ISG expression. Detailed knowledge of how heterogenous mechanisms regulate ISG expression is crucial for the rational design of drugs targeting the type I interferon response. Here, we characterize the first ETS transcription factor family member as a regulator of non-canonical ISG expression: E74-like ETS transcription factor 1 (ELF1). Using high-content microscopy to quantify viral infection over time, we found that ELF1, itself an ISG, inhibits eight diverse RNA and DNA viruses uniquely at multi-cycle replication. ELF1 did not regulate expression of type I or II interferons, and ELF1’s antiviral effect was not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNAseq revealed that the ELF1 transcriptional program is distinct from, and delayed with respect to, the immediate interferon response. Finally, knockdown experiments demonstrated that ELF1 is a critical component of the antiviral interferon response in vitro and in vivo. Our findings reveal a previously overlooked mechanism of non-canonical ISG regulation that both amplifies and prolongs the initial interferon response by expressing broadly antiviral restriction factors.AUTHOR SUMMARYOver 60 years after their discovery, we still struggle to understand exactly how interferons inhibit viruses. Our gap in knowledge stems, on one hand, from the sheer number of interferon-stimulated effector genes, of which only few have been characterized in mechanistic detail. On the other hand, our knowledge of interferon-regulated gene transcription is constantly evolving. We know that different regulatory mechanisms greatly influence the quality, magnitude, and timing of interferon-stimulated gene expression, all of which may contribute to the antiviral mechanism of interferons. Deciphering these regulatory mechanisms is indispensable for understanding this critical first line of host defense, and for harnessing the power of interferons in novel antiviral therapies. Here, we report a novel mechanism of interferon-induced gene regulation by an interferon-stimulated gene, which, paradoxically, inhibits viruses in the absence of additional interferon signaling: E74-like ETS transcription factor 1 (ELF1) raises an unusually delayed antiviral program that potently restricts propagation of all viruses tested in our study. Reduced levels of ELF1 significantly diminished interferon-mediated host defenses against influenza A virus in vitro and in vivo, suggesting a critical but previously overlooked role in the type I interferon response. The transcriptional program raised by ELF1 is vast and comprises over 400 potentially antiviral genes, which are almost entirely distinct from those known to be induced by interferon. Taken together, our data provide evidence for a critical secondary wave of antiviral protection that adds both “quality” and “time” to the type I interferon response.

Influenza A viruses (IAVs) are contagious pathogens and one of the leading causes of respiratory tract infections in both humans and animals worldwide. Upon infection, the innate immune system provides the first line of defense to neutralize or limit the replication of invading pathogens, creating a fast and broad response that brings the cells into an alerted state through the secretion of cytokines and the induction of the interferon (IFN) pathway. At the same time, IAVs have developed a plethora of immune evasion mechanisms in order to avoid or circumvent the host antiviral response, promoting viral replication. Herein, we will review and summarize already known and recently described innate immune mechanisms that host cells use to fight IAV viral infections as well as the main strategies developed by IAVs to overcome such powerful defenses during this fascinating virus-host interplay.

RIG-I-like receptors (RLRs) are cytoplasmic sensors of viral RNA that trigger the signaling cascade that leads to type I interferon (IFN) production. Transcriptional induction of RLRs by IFN is believed to play the role of positive feedback to further amplify viral sensing. We found that RLRs and several other IFN-stimulated genes (ISGs) are induced early in viral infection independent of IFN. Expression of these early ISGs requires IRF3/IRF7 and is highly correlated amongst them. Simultaneous detection of mRNA ofIFNB1, viral replicase, and ISGs revealed distinct populations ofIFNB1expressing and non-expressing cells which are highly correlated with the levels of early ISGs but are uncorrelated with IFN-dependent ISGs and viral gene expression. Individual expression of RLRs madeIFNB1expression more robust and earlier, suggesting a causal relation between levels of RLR and induction of IFN.