Resistance mechanisms of SARS-CoV-2 3CLpro to the non-covalent inhibitor WU-04.

Drug resistance poses a significant challenge in the development of effective therapies against SARS-CoV-2. Here, we identified two double mutations, M49K/M165V and M49K/S301P, in the 3C-like protease (3CLpro) that confer resistance to a novel non-covalent inhibitor, WU-04, which is currently in phase III clinical trials (NCT06197217). Crystallographic analysis indicates that the M49K mutation destabilizes the WU-04-binding pocket, impacting the binding of WU-04 more significantly than the binding of 3CLpro substrates. The M165V mutation directly interferes with WU-04 binding. The S301P mutation, which is far from the WU-04-binding pocket, indirectly affects WU-04 binding by restricting the rotation of 3CLpro's C-terminal tail and impeding 3CLpro dimerization. We further explored 3CLpro mutations that confer resistance to two clinically used inhibitors: ensitrelvir and nirmatrelvir, and revealed a trade-off between the catalytic activity, thermostability, and drug resistance of 3CLpro. We found that mutations at the same residue (M49) can have distinct effects on the 3CLpro inhibitors, highlighting the importance of developing multiple antiviral agents with different skeletons for fighting SARS-CoV-2. These findings enhance our understanding of SARS-CoV-2 resistance mechanisms and inform the development of effective therapeutics.

Targeting G9a translational mechanism of SARS-CoV-2 pathogenesis for multifaceted therapeutics of COVID-19 and its sequalae.

By largely unknown mechanism(s), SARS-CoV-2 hijacks the host translation apparatus to promote COVID-19 pathogenesis. We report that the histone methyltransferase G9a noncanonically regulates viral hijacking of the translation machinery to bring about COVID-19 symptoms of hyperinflammation, lymphopenia, and blood coagulation. Chemoproteomic analysis of COVID-19 patient peripheral mononuclear blood cells (PBMC) identified enhanced interactions between SARS-CoV-2-upregulated G9a and distinct translation regulators, particularly the N 6 -methyladenosine (m 6 A) RNA methylase METTL3. These interactions with translation regulators implicated G9a in translational regulation of COVID-19. Inhibition of G9a activity suppressed SARS-CoV-2 replication in human alveolar epithelial cells. Accordingly, multi-omics analysis of the same alveolar cells identified SARS-CoV-2-induced changes at the transcriptional, m 6 A-epitranscriptional, translational, and post-translational (phosphorylation or secretion) levels that were reversed by inhibitor treatment. As suggested by the aforesaid chemoproteomic analysis, these multi-omics-correlated changes revealed a G9a-regulated translational mechanism of COVID-19 pathogenesis in which G9a directs translation of viral and host proteins associated with SARS-CoV-2 replication and with dysregulation of host response. Comparison of proteomic analyses of G9a inhibitor-treated, SARS-CoV-2 infected cells, or ex vivo culture of patient PBMCs, with COVID-19 patient data revealed that G9a inhibition reversed the patient proteomic landscape that correlated with COVID-19 pathology/symptoms. These data also indicated that the G9a-regulated, inhibitor-reversed, translational mechanism outperformed G9a-transcriptional suppression to ultimately determine COVID-19 pathogenesis and to define the inhibitor action, from which biomarkers of serve symptom vulnerability were mechanistically derived. This cell line-to-patient conservation of G9a-translated, COVID-19 proteome suggests that G9a inhibitors can be used to treat patients with COVID-19, particularly patients with long-lasting COVID-19 sequelae.

A Murine Model of Post-acute Neurological Sequelae Following SARS-CoV-2 Variant Infection.

Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. Surviving mice showed no detectable viral RNA in the brain and minimal neuroinflammation post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and reduced levels of genes associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent T helper 1 prone cellular immune responses and high neutralizing antibodies against Delta and Omicron variants in the periphery for months post-acute infection. Overall, infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long COVID patients and may be useful for future assessment of the efficacy of vaccines and therapeutics against SARS-CoV-2 variants.

A murine model of post-acute neurological sequelae following SARS-CoV-2 variant infection.

Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. In the brain, no detectable viral RNA and minimal residential immune cell activation was observed in the surviving mice post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and gene expression levels associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent systemic T helper 1 prone cellular immune responses and strong sera neutralizing antibodies against Delta and Omicron variants months post-acute infection. Overall, our findings suggest that infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long-COVID patients and provides new insights into the role of systemic and brain residential immune factors in PASC pathogenesis.

The alarmin IL-33 exacerbates pulmonary inflammation and immune dysfunction in SARS-CoV-2 infection.

Dysregulated host immune responses contribute to disease severity and worsened prognosis in COVID-19 infection and the underlying mechanisms are not fully understood. In this study, we observed that IL-33, a damage-associated molecular pattern molecule, is significantly increased in COVID-19 patients and in SARS-CoV-2-infected mice. Using IL-33-/- mice, we demonstrated that IL-33 deficiency resulted in significant decreases in bodyweight loss, tissue viral burdens, and lung pathology. These improved outcomes in IL-33-/- mice also correlated with a reduction in innate immune cell infiltrates, i.e., neutrophils, macrophages, natural killer cells, and activated T cells in inflamed lungs. Lung RNA-seq results revealed that IL-33 signaling enhances activation of inflammatory pathways, including interferon signaling, pathogen phagocytosis, macrophage activation, and cytokine/chemokine signals. Overall, these findings demonstrate that the alarmin IL-33 plays a pathogenic role in SARS-CoV-2 infection and provides new insights that will inform the development of effective therapeutic strategies for COVID-19.

Integrated multi-omics analyses identify anti-viral host factors and pathways controlling SARS-CoV-2 infection.

Host anti-viral factors are essential for controlling SARS-CoV-2 infection but remain largely unknown due to the biases of previous large-scale studies toward pro-viral host factors. To fill in this knowledge gap, we perform a genome-wide CRISPR dropout screen and integrate analyses of the multi-omics data of the CRISPR screen, genome-wide association studies, single-cell RNA-Seq, and host-virus proteins or protein/RNA interactome. This study uncovers many host factors that are currently underappreciated, including the components of V-ATPases, ESCRT, and N-glycosylation pathways that modulate viral entry and/or replication. The cohesin complex is also identified as an anti-viral pathway, suggesting an important role of three-dimensional chromatin organization in mediating host-viral interaction. Furthermore, we discover another anti-viral regulator KLF5, a transcriptional factor involved in sphingolipid metabolism, which is up-regulated, and harbors genetic variations linked to COVID-19 patients with severe symptoms. Anti-viral effects of three identified candidates (DAZAP2/VTA1/KLF5) are confirmed individually. Molecular characterization of DAZAP2/VTA1/KLF5-knockout cells highlights the involvement of genes related to the coagulation system in determining the severity of COVID-19. Together, our results provide further resources for understanding the host anti-viral network during SARS-CoV-2 infection and may help develop new countermeasure strategies.

TRIM7 ubiquitinates SARS-CoV-2 membrane protein to limit apoptosis and viral replication.

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 -/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M-K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.