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.

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.

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.