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

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.

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

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

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.

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. One-Sentence SummaryPlitidepsin, an inhibitor of SARS-Cov-2 in vitro, is safe and positively influences the outcome of patients hospitalized with COVID-19.

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

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

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.

Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

The Coronaviridae are a family of viruses that causes disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors that are common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted parallel genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E) and glycosaminoglycans (for OC43). Additionally, we discovered phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle as well as the potential development of host-directed therapies.

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.