Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories (preprint)

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.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis. (preprint)

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 both in vitro, and in the Syrian golden hamster model in 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.

SARS-CoV-2 Variants of Concern Have Acquired Mutations Associated With an Increased Spike Cleavage (preprint)

Several SARS-CoV-2 lineages have emerged leading to the divergence of more transmissible variants termed Variants of Concern (VOCs). The natural selection of mutations in the spike protein can impact viral cell entry, transmission, and pathogenesis. Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to demonstrate that the substitution S:655Y, included in the highly prevalent Gamma variant, enhances viral replication and spike protein cleavage. Moreover, viral competition experiments demonstrate that the S:655Y transmits more efficiently than the ancestor 655H in the hamster model. Finally, we analyze a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibit an increased spike cleavage and fusogenic capacity. This 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 show a convergent evolution that promotes the spike protein processing.Funding Information: This research was partly funded by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a NIAID funded Center of Excellence for Influenza Research and Response (CEIRR, contract #75N93021C00014) (AGS), NCI SeroNet grant U54CA260560 (AGS), NIAID grants U19AI135972 and U19AI142733 (AGS), DARPA grant HR0011-19-2-0020 (AGS), JPB Foundation (AGS), Open Philanthropy Project (research grant 2020-215611 (5384) (AGS), anonymous donors to AGS, NBAF Transition Funds from the State of Kansas (JAR), NIAID Centers of Excellence for Influenza Research and Surveillance under contract number HHSN 272201400006C (JAR), AMP Core of the Center for Emerging and Zoonotic Infectious Diseases of the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number P20GM130448 (JAR) and Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases under grant number HSHQDC 16-A-B0006 (JAR). AGR is funded by Marion Alban MSCIC Scholars Award and the 2020 Robin Chemers Neustein Postdoctoral fellowship. ML is funded by a fellowship of the Belgian American Education FoundationDeclaration of Interests: The A.G.-S. laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, N-fold LLC, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer and Merck, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories and Pfizer, outside of the reported work. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections, owned by the Icahn School of Medicine at Mount Sinai, New York. The Icahn School of Medicine at Mount Sinai has filed a patent application relating to SARS-CoV-2 serological assays, which lists Viviana Simon as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. All other authors have nothing to declare. Ethics Approval Statement: Human SARS-CoV-2: Nasopharyngeal swab specimens were collected as part of the routine SARS-CoV-2 surveillance conducted by the Mount Sinai Pathogen Surveillance program (IRB approved, HS#13-00981).All hamster animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Icahn School of Medicine at Mount Sinai (ISMMS).The Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai (ISMMS) reviewed and approved the mink model of COVID-19.

SARS-CoV-2 variants of concern have acquired mutations associated with an increased spike cleavage (preprint)

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.

Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant (preprint)

Emergence of SARS-CoV-2 variants, including the globally successful B.1.1.7 lineage, suggests viral adaptations to host selective pressures resulting in more efficient transmission. Although much effort has focused on Spike adaptation for viral entry and adaptive immune escape, B.1.1.7 mutations outside Spike likely contribute to enhance transmission. Here we used unbiased abundance proteomics, phosphoproteomics, mRNA sequencing and viral replication assays to show that B.1.1.7 isolates more effectively suppress host innate immune responses in airway epithelial cells. We found that B.1.1.7 isolates have dramatically increased subgenomic RNA and protein levels of Orf9b and Orf6, both known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation, and Orf9b binding and activity was regulated via phosphorylation. We conclude that B.1.1.7 has evolved beyond the Spike coding region to more effectively antagonise host innate immune responses through upregulation of specific subgenomic RNA synthesis and increased protein expression of key innate immune antagonists. We propose that more effective innate immune antagonism increases the likelihood of successful B.1.1.7 transmission, and may increase in vivo replication and duration of infection.

Plitidepsin has a positive therapeutic index in adult patients with COVID-19 requiring hospitalization. (preprint)

Plitidepsin is a marine-derived cyclic-peptide that inhibits SARS-CoV-2 replication at low nanomolar concentrations by the targeting of host protein eEF1A (eukaryotic translation-elongation-factor-1A). We evaluated a model of intervention with plitidepsin in hospitalized COVID-19 adult patients where three doses were assessed (1.5, 2 and 2.5 mg/day for 3 days, as a 90-minute intravenous infusion) in 45 patients (15 per dose-cohort). Treatment was well tolerated, with only two Grade 3 treatment-related adverse events observed (hypersensitivity and diarrhea). The discharge rates by Days 8 and 15 were 56.8% and 81.8%, respectively, with data sustaining dose-effect. A mean 4.2 log10 viral load reduction was attained by Day 15. Improvement in inflammation markers was also noted in a seemingly dose-dependent manner. These results suggest that plitidepsin impacts the outcome of patients with COVID-19.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes. (preprint)

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes (preprint)

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

Host-directed therapies against early-lineage SARS-CoV-2 retain efficacy against B.1.1.7 variant (preprint)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths worldwide and massive societal and economic burden. Recently, a new variant of SARS-CoV-2, known as B.1.1.7, was first detected in the United Kingdom and is spreading in several other countries, heightening public health concern and raising questions as to the resulting effectiveness of vaccines and therapeutic interventions. We and others previously identified host-directed therapies with antiviral efficacy against SARS-CoV-2 infection. Less prone to the development of therapy resistance, host-directed drugs represent promising therapeutic options to combat emerging viral variants as host genes possess a lower propensity to mutate compared to viral genes. Here, in the first study of the full-length B.1.1.7 variant virus, we find two host-directed drugs, plitidepsin (aplidin; inhibits translation elongation factor eEF1A) and ralimetinib (inhibits p38 MAP kinase cascade), as well as remdesivir, to possess similar antiviral activity against both the early-lineage SARS-CoV-2 and the B.1.1.7 variant, evaluated in both human gastrointestinal and lung epithelial cell lines. We find that plitidepsin is over an order of magnitude more potent than remdesivir against both viruses. These results highlight the importance of continued development of host-directed therapeutics to combat current and future coronavirus variant outbreaks.

SARS-CoV-2 sensing by RIG-I and MDA5 links epithelial infection to macrophage inflammation (preprint)

SARS-CoV-2 infection causes broad-spectrum immunopathological disease, exacerbated by inflammatory co-morbidities. A better understanding of mechanisms underpinning virus-associated inflammation is required to develop effective therapeutics. Here we discover that SARS-CoV-2 replicates rapidly in lung epithelial cells despite triggering a robust innate immune response through activation of cytoplasmic RNA-ensors RIG-I and MDA5. The inflammatory mediators produced during epithelial cell infection can stimulate primary human macrophages to enhance cytokine production and drive cellular activation. Critically, this can be limited by abrogating RNA sensing, or by inhibiting downstream signalling pathways. SARS-CoV-2 further exacerbates the local inflammatory environment when macrophages or epithelial cells are primed with exogenous inflammatory stimuli. We propose that RNA sensing of SARS-CoV-2 in lung epithelium is a key driver of inflammation, the extent of which is influenced by the inflammatory state of the local environment, and that specific inhibition of innate immune pathways may beneficially mitigate inflammation-associated COVID-19. HighlightsO_LISARS-CoV-2 activates RNA sensors and consequent inflammatory responses in lung epithelial cells C_LIO_LIEpithelial RNA sensing responses drive pro-inflammatory macrophage activation C_LIO_LIExogenous inflammatory stimuli exacerbate responses to SARS-CoV-2 in both eplithelial cells and macrophages C_LIO_LIImmunomodulators inhibit RNA sensing responses and consequent macrophage inflammation C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/424169v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@b07adborg.highwire.dtl.DTLVardef@51ddf7org.highwire.dtl.DTLVardef@c38f9aorg.highwire.dtl.DTLVardef@108db57_HPS_FORMAT_FIGEXP M_FIG C_FIG