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SummaryThe global emergence of SARS-CoV-2 urgently requires an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1–10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. We therefore conducted a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactome of both viruses, as well as their influence on transcriptome, proteome, ubiquitinome and phosphoproteome in a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon SARS-CoV-2 and SARS-CoV infections at different layers and identified unique and common molecular mechanisms of these closely related coronaviruses. The TGF-β pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org) highlights many hotspots that can be targeted by existing drugs and it can guide rational design of virus- and host-directed therapies, which we exemplify by identifying kinase and MMPs inhibitors with potent antiviral effects against SARS-CoV-2.

The severe‐acute‐respiratory‐syndrome‐coronavirus‐2 (SARS‐CoV‐2) is the causative agent of COVID‐19, but host cell factors contributing to COVID‐19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS‐CoV‐2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID‐19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS‐CoV‐2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease‐targeted inhibitors severely impair lung cell infection by the SARS‐CoV‐2 variants of concern alpha, beta, delta, and omicron and also reduce SARS‐CoV‐2 infection of primary human lung cells in a TMPRSS2 protease‐independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.

A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5′ and 3′ end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy., Graphical Abstract Graphical AbstractExclusive targeting of SARS-CoV-2 genomic RNA enables most efficient suppression of viral replication and virus-induced cell death.

T cell immunity is crucial for the control of SARS-CoV-2 infections and has been widely studied on a quantitative level. However, quality of responses, in particular of CD8+ T cells, has only been marginally investigated so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common Human Leucocyte Antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8+ T cells are detected up to twelve months from infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity towards virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8+ TCRs – classical features of protective immunity - are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality and to potentially restore functional CD8+ T cell immunity., Graphical Abstract, Wagner et al. detect CD8+ T cell responses to diverse immunodominant SARS-CoV-2 specific epitopes. ScRNA-sequencing of epitope responsive CD8+ T cells reveal a polyclonal T cell receptor repertoire. State of the art TCR re-expression confirm a highly specific and functional T cell response in convalescent mild COVID-19.

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'‐O‐ribose cap needed for viral immune escape. We find that the host cap 2'‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.

T cell immunity is crucial for the control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and has been widely characterized on a quantitative level. In contrast, the quality of such T cell responses has been poorly investigated, in particular in the case of CD8+T cells. Here, we explored the quality of SARS-CoV-2-specific CD8+T cell responses in individuals who recovered from mild symptomatic infections, through which protective immunity should develop, by functional characterization of their T cell receptor (TCR) repertoire. CD8+T cell responses specific for SARS-CoV-2-derived epitopes were low in frequency but could be detected robustly early as well as late - up to twelve months - after infection. A pool of immunodominant epitopes, which accurately identified previous SARS-CoV-2 infections, was used to isolate TCRs specific for epitopes restricted by common HLA class I molecules. TCR-engineered T cells showed heterogeneous functional avidity and cytotoxicity towards virus-infected target cells. High TCR functionality correlated with gene signatures of T cell function and activation that, remarkably, could be retrieved for each epitope:HLA combination and patient analyzed. Overall, our data demonstrate that highly functional HLA class I TCRs are recruited and maintained upon mild SARS-CoV-2 infection. Such validated epitopes and TCRs could become valuable tools for the development of diagnostic tests determining the quality of SARS-CoV-2-specific CD8+T cell immunity, and thereby investigating correlates of protection, as well as to restore functional immunity through therapeutic transfer of TCR-engineered T cells.

The SARS‐CoV‐2 pandemic imposed a large burden on health and society. Therapeutics targeting different components and processes of the viral infection replication cycle are being investigated, particularly to repurpose already approved drugs. Spike protein is an important target for both vaccines and therapeutics. Insights into the mechanisms of spike‐ACE2 binding and cell fusion could support the identification of compounds with inhibitory effects. Here, we demonstrate that the integrity of disulfide bonds within the receptor‐binding domain (RBD) plays an important role in the membrane fusion process although their disruption does not prevent binding of spike protein to ACE2. Several reducing agents and thiol‐reactive compounds are able to inhibit viral entry. N‐acetyl cysteine amide, L‐ascorbic acid, JTT‐705, and auranofin prevented syncytia formation, viral entry into cells, and infection in a mouse model, supporting disulfides of the RBD as a therapeutically relevant target.

SARS-CoV-2 enters host cells after binding through its spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor. Soluble ACE2 ectodomains bind and neutralize the virus, yet their short in vivo half-live limits their therapeutic use. This limitation can be overcome by fusing the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain, but this bears the risk of unwanted Fc-receptor activation and antibody-dependent disease enhancement. Here, we describe optimized ACE2-IgG4-Fc fusion constructs that avoid Fc-receptor activation, preserve the desired ACE-2 enzymatic activity and show promising pharmaceutical properties. The engineered ACE2-IgG4-Fc fusion proteins neutralize the original SARS-CoV, pandemic SARS-CoV-2 as well as the rapidly spreading SARS-CoV-2 variants-of-concern, B.1.17 and B.1.351. Importantly, these variants-of-concern are inhibited at picomolar concentrations proving that ACE-2-IgG4 maintains – in contrast to therapeutic antibodies - its full antiviral potential. Thus, ACE2-IgG4-Fc fusion proteins are promising candidate anti-antivirals to combat the current and future pandemics.

SARS-CoV-2 enters host cells after binding through its spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor. Soluble ACE2 ectodomains bind and neutralize the virus, yet their short in vivo half-live limits their therapeutic use. This limitation can be overcome by fusing the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain, but this bears the risk of Fc-receptor activation and antibody-dependent cellular cytotoxicity. Here, we describe optimized ACE2-IgG4-Fc fusion constructs that avoid Fc-receptor activation, preserve the desired ACE-2 enzymatic activity and show promising pharmaceutical properties. The engineered ACE2-IgG4-Fc fusion proteins neutralize the original SARS-CoV, pandemic SARS-CoV-2 as well as the rapidly spreading SARS-CoV-2 alpha, beta and delta variants-of-concern. Importantly, these variants-of-concern are inhibited at picomolar concentrations proving that ACE-2-IgG4 maintains – in contrast to therapeutic antibodies - its full antiviral potential. Thus, ACE2-IgG4-Fc fusion proteins are promising candidate anti-antivirals to combat the current and future pandemics., Graphical abstract Image 1

Zika virus (ZIKV) infection can cause developmental and neurological defects and represents a threat for human health. Type I/III interferon responses control ZIKV infection and pathological processes, yet the virus has evolved various mechanisms to defeat these host responses. Here, we established a pipeline to delineate at high-resolution the genetic evolution of ZIKV in a controlled host cell environment. We uncovered that serially passaged ZIKV acquired increased infectivity, defined as the probability for one virus to initiate infection, and simultaneously developed a resistance to TLR3-induced restriction. We built a mathematical model that suggests that the increased infectivity is due to a reduced time-lag between infection and viral replication. We found that this adaptation is cell-type specific, suggesting that different cell environments may drive viral evolution along different routes. Deep-sequencing of ZIKV quasi-species pinpointed mutations whose increased frequencies temporally coincide with the acquisition of the adapted phenotype. We functionally validated a point-mutation in ZIKV envelope (E) protein recapitulating the adapted phenotype. Its positioning on the E structure suggests a putative function in protein refolding/stability. Altogether, our results uncovered ZIKV adaptations to the cell environment leading to an accelerated replication onset coupled with resistance to TLR3-induced antiviral response. Our work provides insights into viral escape mechanisms and interactions with host cell and can serve as a framework to study other viruses.

A promising approach to tackle the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. The proof of concept that SARS-CoV-2 can be inhibited with siRNAs, however, is missing. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, terminating replication before start of transcription and preventing cytopathic effects. Coronaviruses replicate via negative sense intermediate transcripts using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5’ and 3’ end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting sequences shared by different viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs targeting open reading frame 1 (ORF1) which is solely part of gRNA. We propose two independent mechanisms for this: An increased accessibility of translational-active ORF1 before the start of transcription, as well as highly abundant sgRNAs out-competing siRNAs that target common sequences of transcripts. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that an early therapy start, together with targeting ORF1, might be key for high antiviral efficacy.

As the COVID-19 pandemic continues to spread, research on SARS-CoV-2 is also rapidly evolving and the number of associated manuscripts on preprint servers is soaring. To facilitate proteomic research, Zecha et al. present protein expression profiles of four model cell lines and investigate the response of Vero E6 cells to viral infection. Further, they evaluate the feasibility of proteomic analysis for SARS-CoV-2 diagnostics and critically discuss the merits of proteomic approaches in the context of the COVID-19 research., Graphical Abstract Highlights • In-depth proteomes of 4 SARS-CoV-2 cell line models (Vero E6, Calu-3, Caco-2, A549). • Proteomic evidence for thousands of Chlorocebus sabaeus proteins. • Proteomic response of Vero E6 cells to SARS-CoV-2 infection. • Synthetic peptides, spectral libraries, and targeted assays for SARS-CoV-2 proteins., As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC–MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.