C. Ye

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

Enhanced Pathogenicity and Contact Transmissibility of Human-origin Avian Influenza H5N1 Clade 2.3.4.4b Genotype B3.13 Compared to D1.1 in Ferrets

Since its emergence in 2020, multiple genotypes of the H5N1 clade 2.3.4.4b have been identified, with B3.13 and D1.1 emerging in the USA as two major and concerning genotypes. However, their relative pathogenicity and transmissibility in mammals have not been fully elucidated. We compared the pathogenicity and transmissibility of the first two human H5N1 clade 2.3.4.4b cases caused by B3.13 in Texas (A/Texas/37/2024; HPhTX B3.13) and D1.1 in Louisiana (A/Louisiana/12/2024; HPhLA D1.1) in a ferret model of infection and transmission. HPhTX B3.13 infection resulted in more severe clinical disease and enhanced viral shedding, with evidence of increased transmission relative to HPhLA D1.1. Histopathological analysis revealed more extensive lung pathology in animals infected with HPhTX B3.13, consistent with increased viral loads and inflammatory responses. Importantly, both genotypes showed no significant differences in reactivity to ferret sera raised against candidate vaccine virus (CVV) strains, receptor binding properties, or neuraminidase (NA) activity and thermostability. Whole-genome sequencing revealed no adaptive mutations in HPhTX B3.13 following infection or transmission. In contrast, HPhLA D1.1 showed rapid acquisition of the mammalian-adaptive mutation E627K in infected ferrets and both E627K and Q194K in the only fatal contact animal. Both mutations were associated with enhanced polymerase activity and computational analyses suggested that they enhance interactions with the mammalian host factors ANP32A and B. Our findings indicate that B3.13 is already well adapted for mammalian infection and transmission whereas D1.1 retains evolutionary potential through the rapid acquisition of adaptive mutations, highlighting important genotype-specific differences relevant to zoonotic risk assessment and pandemic preparedness. Significance Influenza H5N1 viruses continue to diversify genetically while expanding into mammalian hosts, increasing opportunities for viral adaptation and zoonotic transmission, including humans. However, whether the predominant clade 2.3.4.4b genotype differs in its capacity to infect, transmit, and evolve in mammals remains poorly understood. Using the ferret model of influenza infection and transmission, we demonstrated that the currently circulating B3.13 and D1.1 genotypes exhibit distinct pathogenic and transmission characteristics despite retaining similar receptor-binding characteristics, NA functions, and antigenic profiles. While B3.13 readily infects and transmits in ferrets and does not develop further adaptive mutations associated with increased replication and transmission, D1.1 rapidly acquires mammalian-adaptive mutations after a single infection and/or transmission event, highlighting its evolutionary potential. These findings show that genotype-specific biological properties can influence zoonotic risk independently of antigenic similarity and emphasize the importance of integrating phenotypic characterization with genomic surveillance to improve pandemic preparedness and guide public health risk assessment.

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.