Luis Martínez-Sobrido

Recovery of proofreading-impaired SARS-CoV-2 reveals a mutator phenotype and an ExoN activity threshold for viability

ABSTRACT Coronaviruses (CoVs) replicate unusually large RNA genomes that necessitate proofreading by the 3′-to-5′ exoribonuclease (ExoN) formed by nonstructural proteins 14 (nsp14) and 10 (nsp10). Previous studies suggested that inactivation of the ExoN catalytic site in severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is lethal, leaving unresolved whether the virus can tolerate impaired proofreading activity. Here, we investigated the functional requirement for ExoN in SARS-CoV-2 replication by combining a continuous fluorescence-based biochemical assay with an optimized single-bacmid reverse genetics system. Mutational analysis of residues involved in RNA binding or catalysis revealed graded effects on ExoN activity in vitro. Alanine substitution of Lys9, a residue positioned near the RNA-binding interface, did not reduce ExoN activity, whereas charge reversal at this position (K9E) impaired activity more strongly than alanine substitutions of the catalytic motif I residues D90 and E92 (D90A/E92A). Correspondingly, recombinant SARS-CoV-2 carrying K9A was readily recovered, whereas the D90A/E92A mutant was recovered only after an extended delay, and K9E could not be rescued despite repeated attempts. The D90A/E92A mutant exhibited reduced replication while maintaining the engineered ExoN substitutions during serial passage. Deep sequencing of viral populations revealed a marked increase in genome-wide sequence variation in the D90A/E92A mutant, demonstrating a stable mutator phenotype. Together, these findings indicate that SARS-CoV-2 can tolerate substantial impairment of ExoN activity but depends on a minimal activity threshold for viability. This system provides a platform for defining how SARS-CoV-2 proofreading controls genome stability, viral fitness, and sensitivity to antiviral strategies that exploit reduced replication fidelity. IMPORTANCE Coronaviruses have unusually large RNA genomes because they encode a proofreading enzyme that removes copying errors during replication. It has been unclear whether SARS-CoV-2 can survive when this proofreading function is strongly weakened because earlier studies suggested that loss of the enzyme’s catalytic activity is lethal. We show that SARS-CoV-2 can tolerate substantial impairment of proofreading but only when residual exonuclease activity remains above a minimal threshold. A virus with impaired proofreading replicates less efficiently and accumulates mutations across its genome, whereas a more severe defect prevents virus recovery. These findings clarify how coronavirus proofreading balances genome stability with viral fitness and provide a useful system for studying how reduced replication fidelity affects viral evolution, antiviral sensitivity, and attenuation. Defining this activity threshold may also help guide antiviral strategies that target coronavirus proofreading. Coronaviruses have unusually large RNA genomes because they encode a proofreading enzyme that removes copying errors during replication. It has been unclear whether SARS-CoV-2 can survive when this proofreading function is strongly weakened because earlier studies suggested that loss of the enzyme’s catalytic activity is lethal. We show that SARS-CoV-2 can tolerate substantial impairment of proofreading but only when residual exonuclease activity remains above a minimal threshold. A virus with impaired proofreading replicates less efficiently and accumulates mutations across its genome, whereas a more severe defect prevents virus recovery. These findings clarify how coronavirus proofreading balances genome stability with viral fitness and provide a useful system for studying how reduced replication fidelity affects viral evolution, antiviral sensitivity, and attenuation. Defining this activity threshold may also help guide antiviral strategies that target coronavirus proofreading.

SARS-CoV-2 endoribonucleaseU and ORF6 work synergistically to antagonize innate immune signaling 2327626

Coronaviruses (CoVs) produce dsRNA during genome replication and mRNA synthesis. Upon sensing dsRNA, host cells activate numerous antiviral pathways. CoVs encode multiple proteins that antagonize antiviral responses. The conserved CoV nsp15 contains an endoribonuclease (EndoU) that cleaves viral ssRNA to limit dsRNA accumulation. Similarly, the viral protein, NS6, encoded by open reading frame (ORF)6 is expressed by severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 and has been shown to block STAT translocation, but its absence has minimal impact on IFN-stimulated genes (ISGs). A viral genome has limited coding capacity, and ORFs encoding proteins without a significant function are typically mutated or deleted over time. Therefore, we infer that ORF6 plays an essential role in immune evasion, perhaps dependent upon other viral proteins. A recombinant SARS-CoV-2 encoding an inactive EndoU (nsp15mut) and an interrupted ORF6 (ORF6stop) was constructed. Viral kinetics and innate immune induction were assessed in both Calu-3 cells and primary nasal air-liquid interface (ALI) cultures using RT-qPCR and Western blot analysis. Replication of SARS-CoV-2 nsp15mut/ORF6stop in nasal ALI cultures was attenuated at both 33 °C and 37 °C compared to WT SARS-CoV-2. A significant increase in type I and III IFN transcripts was also observed in nasal ALI cultures infected with nsp15mut/ORF6stop compared to WT and each single mutant. Robust induction of various ISGs were also observed via Western blot analysis, with a faster induction observed at 37 °C. The synergistic relationship between nsp15 and ORF6 is essential for efficient SARS-CoV-2 replication. We hypothesize that since nsp15 functions by reducing dsRNA upstream of ORF6, nsp15 acts as a more potent antagonist leaving little activity for ORF6 to account for. However, when both EndoU activity and NS6 are absent, a robust induction of IFN and ISGs are induced to levels higher than that of the nsp15mut alone. This work was supported by National Institutes of Health grants R01 AI140442 (SRW), R01AI169537 (SRW&NAC), RO1A1AI161175 (LM-S); Department of Veterans Affairs Merit Review 1-I01-BX005432-01 (NAC&SRW); the Penn Center for Research on Emerging Viruses (SRW Viral Immunology (VIR)

Mutations in SARS-CoV-2 Nsp1 are critical determinants of viral pathogenicity in mice

Pre-existing and Cross-Reactive Immunity to Avian Influenza H5N1 in Humans: Implications for Pandemic Risk and Vaccine Strategies.

Due to the continuous evolution of Influenza A viruses (IAVs), novel strains with efficient human-to-human transmission may emerge and cause future pandemics. Among these, highly pathogenic avian influenza (HPAI) H5N1 remains a major concern because of its impact on wildlife, livestock, and human health. The widespread circulation of H5N1 clade 2.3.4.4b, detected in hundreds of bird species and numerous mammals worldwide, highlights important changes in viral ecology and transmission, increasing its zoonotic and pandemic potential. This review summarizes current evidence on cross-reactive and cross-protective immunity to H5N1 in humans, focusing primarily on humoral immune responses. We examine the presence of pre-existing H5N1-reactive antibodies in individuals without known exposure and discuss how previous seasonal influenza infection or vaccination may contribute to their development. Particular attention is given to antibodies targeting conserved regions of hemagglutinin (HA), especially the stalk domain, as well as neuraminidase (NA), which may provide heterosubtypic protection. We also evaluate the ability of seasonal influenza vaccines and infections to induce cross-reactive responses against H5N1 and their potential role in partial protection or immune priming. Finally, we review current and emerging H5N1 vaccination strategies, including adjuvanted and mRNA-based platforms, and identify priorities for surveillance, population immunity assessment, and the development of broadly protective influenza vaccines.

Scarless SARS-CoV-2 Genome Engineering and Variant Analysis

A genome engineering technology is used to change a single amino acid in the viruses’ main protease enzyme to match that of circulating Omicron isolates to demonstrate antiviral efficacy of approved drugs and uncover mutants with reduced drug sensitivity.