Biology subjects

Estes, L. K.

Publications and source records attributed to Estes, L. K..

6 recordsLinked to original sources

bioRxiv2026

Antagonism of stress granules key for SARS-CoV-2 infection and pathogenesis.

Viruses must subvert host responses to facilitate successful infection. While most antiviral responses are associated with interferons, stress granules (SG) are another barrier to viral infection by inducing translation arrest. To combat SG activity, viruses have evolved mechanisms to disrupt formation and disassemble these complexes. Our prior studies identified residues in SARS-CoV-2 NSP3 (Y138/F145) and nucleocapsid (F17) that independently antagonize SG activity. Disrupting these key residues in NSP3 or nucleocapsid attenuated viral replication, but only modestly impacted in vivo pathogenesis suggesting overlap in SG antagonism partially compensate for the individual losses. In this study, we evaluated a SARS-CoV-2 mutant (YF/F17A) that combines the NSP3 and N mutations. We find that loss of both SG antagonizing functions attenuates SARS-CoV-2 replication in vitro. While no changes are seen in type I IFN sensitivity, attenuation corresponds to increased induction of SGs. Importantly, the SARS-CoV-2 YF/F17A mutant has significant attenuation in vivo with reduced viral replication, less weight loss, and limited immune pathology. Notably, infection with the YF/F17A mutant stimulated less interferon and inflammation. Despite these muted host responses, the YF/F17A mutant stimulated robust protection against subsequent challenge with WT SARS-CoV-2. Overall, the study highlights the importance of SG control for SARS-CoV-2 infection and offers a novel, interferon independent target for vaccination and therapeutic treatment going forward. ImportanceThis study demonstrates that SARS-CoV-2 uses multiple mechanisms to block host stress granules during infection. Knocking out both N and NSP3 mediated antagonism of stress granules attenuates viral replication and disease caused by SARS-CoV-2. Importantly, while most therapeutics target key viral processes or induce interferon pathways, this study shows stress granule activation as a novel approach to attenuate and treat coronavirus infection.

microbiology↗

bioRxiv2025

Key residues in SARS-CoV-2 NSP3 hyper variable region are necessary to modulate early stress granule activity.

Antagonism of the host responses that limits viral replication is critical to the success of infection. Recently, we identified that the hypervariable region (HVR) of SARS-CoV-2 NSP3 binds to FXR1 and disrupts stress granule formation during the early stages of infection. Despite variation across the rest of the HVR, a 20-amino acid region, highly conserved in the Sarbecovirus family, is required for NSP3-FXR1 binding, but the critical residues remained unresolved. In this study, we explore the individual residues in NSP3 driving FXR1 binding and determine their impact on viral replication, pathogenesis, and stress granule formation. Our results indicate that the tyrosine at position 138 (Y138) and a phenylalanine at position 145 (F145) are required for FXR1 binding and affinity. Using reverse genetics, we showed mutating NSP3 Y138A/F145A (YF mutant) reduced viral replication in vitro and in vivo. Importantly, we demonstrate that attenuation is not due to differential type I interferon responses, but rather loss of stress granule control by the NSP3 mutant as compared to WT. Together, our findings demonstrate the importance of Y138 and F145 within the NSP3-HVR in regulating stress granule formation at the early times post infection. IMPORTANCEStress granules play a key role in host-antiviral defenses and viruses have developed strategies to antagonize their activity. For SARS-CoV-2, the virus has two proteins that antagonize stress granules with NSP3 acting early and nucleocapsid acting at late times. Here, we show that key NSP3 residues Y138 and F145, conserved across the Sarbecovirus family, are necessary to bind FXR1 and disrupt its activity in stress granule formation. Mutating these residues results in attenuation of SARS-CoV-2 replication and induces stress granule formation at early times post infection. These results show the importance of these NSP3 residues in disrupting stress granule formation early and highlight multiple approaches SARS-CoV-2 uses to antagonize stress granule activation.

microbiology↗

bioRxiv2025

The furin cleavage site is required for pathogenesis, but not transmission of SARS-CoV-2

The SARS-CoV-2 spike, key to viral entry, has two features that differentiate it from other sarbecoviruses: the presence of a furin cleavage site (FCS; PRRAR sequence) and an extended S1/S2 loop characterized by an upstream QTQTN amino acid motif. Our prior works show that shortening the S1/S2 loop by deleting either the FCS ({Delta}PRRA) or deleting an upstream sequence ({Delta}QTQTN), ablates spike processing, alters host protease usage, and attenuates infection in vitro and in vivo. With the importance of the loop length established, here we evaluated the impact of disrupting the FCS, but preserving the S1/S2 loop length. Using reverse genetics, we generated a SARS-CoV-2 mutant that disrupts the FCS (PQQAR) but maintains its extended S1/S2 loop. The SARS-CoV-2 PQQAR mutant has reduced replication, decreased spike processing, and attenuated disease in vivo compared to wild-type SARS-CoV-2. These data, similar to the FCS deletion mutant, indicate that loss of the furin cleavage site attenuates SARS-CoV-2 pathogenesis. Importantly, we subsequently found that the PQQAR mutant is transmitted in the direct contact hamster model despite lacking an intact FCS. However, competition transmission showed that the mutant was attenuated compared to WT SARS-CoV-2. Together, the data argue that the FCS is required for SARS-CoV-2 pathogenesis but is not strictly required for viral transmission.

microbiology↗

bioRxiv2024

SARS-CoV-2 EndoU-ribonuclease regulates RNA recombination and impacts viral fitness.

Coronaviruses (CoVs) maintain large RNA genomes that frequently undergoes mutations and recombination, contributing to their evolution and emergence. In this study, we find that SARS-CoV-2 has greater RNA recombination frequency than other human CoVs. In addition, coronavirus RNA recombination primarily occurs at uridine (U)-enriched RNA sequences. Therefore, we next evaluated the role of SARS-CoV-2 NSP15, a viral endonuclease that targets uridines (EndoU), in RNA recombination and virus infection. Using a catalytically inactivated EndoU mutant (NSP15H234A), we observed attenuated viral replication in vitro and in vivo. However, the loss of EndoU activity also dysregulated inflammation resulting in similar disease in vivo despite reduced viral loads. Next-generation sequencing (NGS) demonstrated that loss of EndoU activity disrupts SARS-CoV-2 RNA recombination by reducing viral sub-genomic message but increasing recombination events that contribute to defective viral genomes (DVGs). Overall, the study demonstrates that NSP15 plays a critical role in regulating RNA recombination and SARS-CoV-2 pathogenesis.

microbiology↗

bioRxiv2024

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

microbiology↗

bioRxiv2023

Loss-of-function mutation in Omicron variants reduces spike protein expression and attenuates SARS-CoV-2 infection

SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel amino acid mutations in the spike protein alone. While most studies focus on receptor binding domain changes, mutations in the C-terminus of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. In this study, we examined three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we found that the mutant increased spike processing, consistent with prior reports for H655Y and P681H individually. Next, we generated a single N679K mutant, finding reduced viral replication in vitro and less disease in vivo. Mechanistically, the N679K mutant had reduced spike protein in purified virions compared to wild-type; spike protein decreases were further exacerbated in infected cell lysates. Importantly, exogenous spike expression also revealed that N679K reduced overall spike protein yield independent of infection. Although a loss-of-function mutation, transmission competition demonstrated that N679K had a replication advantage in the upper airway over wild-type SARS-CoV-2 in hamsters, potentially impacting transmissibility. Together, the data show that N679K reduces overall spike protein levels during Omicron infection, which has important implications for infection, immunity, and transmission.

microbiology↗