Biology subjects

Sahler, J.

Publications and source records attributed to Sahler, J..

6 recordsLinked to original sources

bioRxiv2025

Deletion of a KSF Motif Attenuates NSP1 Host Cell Translation Shutoff and Impairs SARS-CoV-2 Virulence

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), triggered a global pandemic with profound social and economic consequences. The viral spike (S) protein has been identified as a key determinant of SARS-CoV-2 pathogenicity. In this study, we demonstrate that the Omicron BA.4 and BA.5 variants, which have closely related S proteins, exhibit different virulence in K18-hACE2 transgenic mice. A comparison of genomic sequences revealed key differences between variants BA.4 and BA.5, including a three amino acid deletion ({Delta}KSF) in the linker region of the non-structural protein 1 (NSP1) in BA.4. Using reverse genetic systems, we engineered a recombinant (r)SARS-CoV-2 BA.5 expressing BA.4 NSP1, which was significantly attenuated in vivo, similar to the natural BA.4 isolate, compared to rBA.5 wild-type (WT). This finding indicates that NSP1 is responsible, at least in part, for the differences in virulence between BA.4 and BA.5. Mechanistically, BA.4 NSP1 showed a reduced ability to inhibit host gene translation compared to BA.5 NSP1. Notably, a rSARS-CoV-2 WA1 original strain containing the same {Delta}KSF in NSP1 was also attenuated in vivo compared to rWA1 WT. Together, these findings highlight the contributions of the NSP1 linker region to inhibiting host gene expression and SARS-CoV-2 pathogenicity, as well as the feasibility of targeting NSP1 for the rational design of live-attenuated vaccines and/or antivirals. IMPORTANCEUnderstanding why some SARS-CoV-2 variants cause more severe disease than others is crucial for proper management or for the rational design of prophylactic and therapeutic treatments. While most studies focus on the spike (S) protein, we found that another viral protein, NSP1, also plays a key role in disease severity among SARS-CoV-2 variants. A small deletion in NSP1, present in the Omicron BA.4 variant, weakens the virus ability to shut down the hosts immune response, making BA.4 less severe than BA.5. When we introduced the same deletion into the original SARS- CoV-2 WA1 strain, the virus also became less harmful. This discovery suggests that NSP1 is an important virulence factor and supports the feasibility of targeting NSP1 for the development of new prophylactic and therapeutic treatments against SARS-CoV-2. By uncovering NSP1s role in pathogenesis, our study provides insights that could help in designing better strategies to combat future variants of SARS-CoV-2.

microbiology↗

bioRxiv2025

Diverse microbial exposure exacerbates the development of allergic airway inflammation in adult mice

BackgroundExposure to a diversity of microbes has been implicated in playing a major role in susceptibility to the development of allergic lung type diseases. The hygiene hypothesis suggests that those exposed to a broad diversity of microbes are more likely to be protected against developing allergic type diseases. However, changes in exposure to microbial diversity can occur in both younger individuals, as well as in adults, and the effects are not always understood. ObjectiveWe investigated the effect of exposure to broad microbial diversity on the airway T cell response in house dust mite (HDM) induced allergic airway disease (AAD, a model of allergic asthma). MethodsWe increased exposure to broad microbial diversity by co-housing specific pathogen free (SPF) adult or newborn mice with pet store mice (PSE or BiPSE, respectively). Mice were then exposed to HDM to induce AAD. ResultsWe found that the effect of increased microbial exposure on the development of allergic airway inflammation differs by age. Increasing exposure to diverse microbes as adults exacerbates the development of allergic airway inflammation, whereas this was not observed when exposure occurred at birth. ConclusionWe suggest that experimental evaluation of the hygiene hypothesis in inflammation, particularly those using mouse models, may need to consider age of the host and time of microbial exposure. Capsule SummaryMouse models of increased exposure to diverse microbial environment shown to differentially affect the development of allergic airway inflammation, depending on the age of microbial exposure.

immunology↗

bioRxiv2024

Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6

The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation enhanced host lipid droplet consumption by NSP6 without impacting its known ER-zippering function or double-membrane vesicle morphology. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6s key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications. Author SummaryAs SARS-CoV-2 continues to spread and adapt in humans, viral variants with enhanced spread and immune evasion have emerged throughout the COVID-19 pandemic. While most of the mutations occur in the Spike protein and have been extensively studied, non-Spike mutations have been accumulating and are not as well understood. Here, we constructed Spike-defective genomes of recent Omicron variants and systematically compared their RNA replication capabilities independently from Spike. We also performed single-round infection assays with various Omicron variant Spike proteins to quantify viral entry capabilities. Interestingly, viral entry and RNA replication were negatively correlated, suggesting that as variants evolved reduced entry functions under growing immune pressure on Spike, RNA replication increased as a compensatory mechanism. We focused on a viral protein NSP6 that acquired mutations that significantly enhanced RNA replication. We validated that a frequently accessed L260F mutation in NSP6 enhanced viral infection in cells and increased pathogenesis in mice. While the mutation did not alter NSP6s role in maintaining a membranous network of viral replication factories, the mutation enhanced viral hijacking of the host lipid droplet machinery. Collectively, we highlight the important role of non-Spike mutations in the evolutionary trajectories of SARS-CoV-2 variants with possible monitoring and therapeutic implications.

microbiology↗

bioRxiv2024

Cell-free expression of Nipah virus transmembrane proteins for proteoliposome vaccine design

Membrane proteins expressed on the surface of enveloped viruses are potent antigens in a vaccine, yet are difficult to produce and present due to their instability without a lipid scaffold. Current vaccination strategies that incorporate viral membrane proteins, such as live attenuated viruses, inactivated viruses, or extracellular vesicles, have limitations including lengthy production time, poor immunogenicity, extensive processing steps, and/or poor stability. Cell-free protein synthesis of viral membrane proteins offers a rapid, one-step method to assemble vaccine nanoparticles via cotranslational folding of membrane proteins into nanoscale liposomes. Here, we develop a vaccine candidate for the deadly Nipah virus (NiV), a highly lethal virus listed by the World Health Organization as a priority pathogen, by cell-free expressing two full-length Nipah virus membrane proteins. We demonstrate that both NiV fusion protein (NiV F) and NiV glycoprotein (NiV G) can be expressed and cotranslationally integrated into liposomes and that they fold into their native conformation. We find the removal of a signal peptide sequence and alteration of liposome lipid composition improves viral membrane protein incorporation. Furthermore, a lipid adjuvant, monophosphoryl lipid A (MPLA), can be readily added to liposomes without disrupting protein-vesicle loading or protein folding conformations. Finally, we demonstrate that our generated liposomal formulations lead to enhanced humoral responses in mice compared to empty and single-protein controls. This work establishes a platform to quickly assemble and present membrane antigens as multivalent vaccines that will enable a rapid response to the broad range of emerging pathogenic threats.

synthetic biology↗

bioRxiv2021

SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice

Lethal COVID-19 is associated with respiratory failure that is thought to be caused by acute respiratory distress syndrome (ARDS) secondary to pulmonary infection. To date, the cellular pathogenesis has been inferred from studies describing the expression of ACE2, a transmembrane protein required for SARS-CoV-2 infection, and detection of viral RNA or protein in infected humans, model animals, and cultured cells. To functionally test the cellular mechanisms of COVID-19, we generated hACE2fl animals in which human ACE2 (hACE2) is expressed from the mouse Ace2 locus in a manner that permits cell-specific, Cre-mediated loss of function. hACE2fl animals developed lethal weight loss and hypoxemia within 7 days of exposure to SARS-CoV-2 that was associated with pulmonary infiltrates, intravascular thrombosis and patchy viral infection of lung epithelial cells. Deletion of hACE2 in lung epithelial cells prevented viral infection of the lung, but not weight loss, hypoxemia or death. Inhalation of SARS-CoV-2 by hACE2fl animals resulted in early infection of sustentacular cells with subsequent infection of neurons in the neighboring olfactory bulb and cerebral cortex-- events that did not require lung epithelial cell infection. Pharmacologic ablation of the olfactory epithelium or Foxg1Cre mediated deletion of hACE2 in olfactory epithelial cells and neurons prevented lethality and neuronal infection following SARS-CoV-2 infection. Conversely, transgenic expression of hACE2 specifically in olfactory epithelial cells and neurons in Foxg1Cre; LSL-hACE2 mice was sufficient to confer neuronal infection associated with respiratory failure and death. These studies establish mouse loss and gain of function genetic models with which to genetically dissect viral-host interactions and demonstrate that lethal disease due to respiratory failure may arise from extrapulmonary infection of the olfactory epithelium and brain. Future therapeutic efforts focused on preventing olfactory epithelial infection may be an effective means of protecting against severe COVID-19.

microbiology↗

bioRxiv2021

A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) 1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs 3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein 5-7. Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >106 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 8. Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.

microbiology↗