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Biology subjects

Sparrer, K. M. J.

Publications and source records attributed to Sparrer, K. M. J..

10 recordsLinked to original sources
bioRxiv2026

Laudanosine restricts Ebola virus entry by targeting TPC2-dependent endolysosomal trafficking

Late endosome-dependent viruses, including filo- and arenaviruses, rely on host endolysosomal trafficking for productive infection. Here, we used a dual-colour Vesicular stomatitis virus (VSV) based pseudoparticle screen of CytoSorb-derived fractions to identify inhibitors of the Zaire Ebolavirus glycoprotein (GP)-mediated entry. Iterative chromatographic purification and mass spectrometry identified Laudanosine, a degradation product of the clinically used neuromuscular blocker Atracurium, as the antiviral compound. Laudanosine specifically inhibited entry mediated by Ebola, Marburg, Lymphocytic choriomeningitis and Lassa virus glycoproteins without affecting VSV-G-dependent entry. Importantly, Laudanosine inhibited authentic Ebola virus infection without detectable cytotoxicity in cell culture and embryonic zebrafish. Molecular dynamics simulations suggest stable association of Laudanosine with the allosteric inhibitory pocket of the lysosomal two-pore channel (TPC2). Consistently, Laudanosine impairs autophagic flux and disrupts endolysosomal trafficking. Together, our findings identify Laudanosine as a previously unrecognised inhibitor of TPC2-dependent entry of highly lethal viral pathogens.

microbiology↗
bioRxiv2025

Structural Basis for OAS2 Regulation and its Antiviral Function

Oligoadenylate synthetase (OAS) proteins are immune sensors for double-stranded RNA and critical for restricting viruses. OAS2 comprises two OAS domains, only one of which can synthesize 2-5-oligoadenylates for RNase L activation. Existing structures of OAS1 provide a model for enzyme activation, but do not explain how multiple OAS domains discriminate RNA length. Here, we discover that OAS2 exists in an autoinhibited state as a zinc-mediated dimer and present a mechanism for RNA length discrimination: the catalytically deficient domain acts as a molecular ruler that prevents autoreactivity to short RNAs. We demonstrate that dimerization and myristoylation localize OAS2 to Golgi membranes and that this is required for OAS2 activation and restriction of viruses that exploit the endomembrane system for replication, e.g. coronaviruses. Finally, our results highlight the non-redundant role of OAS proteins and emphasize the clinical relevance of OAS2 by identifying a patient with a loss-of-function mutation leading to autoimmune disease.

biochemistry↗
bioRxiv2023

Determinants of species-specific utilization of ACE2 by human and animal coronaviruses

Utilization of human ACE2 allowed several bat coronaviruses (CoVs), including the causative agent of COVID-19, to infect humans either directly or via intermediate hosts. Here, we analyzed the ability of Spike proteins from 24 human or animal CoVs to use ACE2 receptors across nine reservoir, potential intermediate and human hosts. We show that overall SARS-CoV-2 Omicron variants evolved more efficient ACE2 usage but mutation of R493Q in BA.5 Spike disrupts utilization of ACE2 from Greater horseshoe bats. Spikes from most CoVs showed species-specific differences in ACE2 usage, partly due to variations in ACE2 residues 31, 41 or 354. Mutation of T403R allowed the RaTG13 bat CoV Spike to use all ACE2 orthologs analysed for viral entry. Sera from COVID-19 vaccinated individuals neutralized the Spike proteins of a range of bat Sarbecoviruses. Our results define determinants of ACE2 receptor usage of diverse CoVs and suggest that COVID-19 vaccination may protect against future zoonoses of SARS-CoV-related bat viruses. HighlightsO_LIMutation of R493Q in BA.5 Spike disrupts utilization of ACE2 from Greater horseshoe bats C_LIO_LIVariations in ACE2 residues 31, 41 or 354 affect utilization by coronavirus Spike proteins C_LIO_LIResidue R403 in the Spike protein of bat coronavirus allow broad and effective ACE2 usage C_LIO_LISera from COVID-19 vaccinated individuals neutralize Spike proteins of bat Sarbecoviruses C_LI

molecular biology↗
bioRxiv2022

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques

Type-I interferons (IFN-I) are critical mediators of innate control of viral infections, but also drive recruitment of inflammatory cells to sites of infection, a key feature of severe COVID-19. Here, and for the first time, IFN-I signaling was modulated in rhesus macaques (RMs) prior to and during acute SARS-CoV-2 infection using a mutated IFN2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. In SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs. Notably, IFNmod treatment resulted in a potent reduction in (i) SARS-CoV-2 viral load in Bronchoalveolar lavage (BAL), upper airways, lung, and hilar lymph nodes; (ii) inflammatory cytokines, chemokines, and CD163+MRC1-inflammatory macrophages in BAL; and (iii) expression of Siglec-1, which enhances SARS-CoV-2 infection and predicts disease severity, on circulating monocytes. In the lung, IFNmod also reduced pathogenesis and attenuated pathways of inflammasome activation and stress response during acute SARS-CoV-2 infection. This study, using an intervention targeting both IFN- and IFN-{beta} pathways, shows that excessive inflammation driven by type 1 IFN critically contributes to SARS-CoV-2 pathogenesis in RMs, and demonstrates the potential of IFNmod to limit viral replication, SARS-CoV-2 induced inflammation, and COVID-19 severity.

immunology↗
bioRxiv2022

Determinants of Spike Infectivity, Processing and Neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2

The SARS-CoV-2 Omicron variant rapidly outcompeted other variants and currently dominates the COVID-19 pandemic. Its enhanced transmission, immune evasion and pathogenicity is thought to be driven by numerous mutations in the Omicron Spike protein. Here, we examined the impact of amino acid changes that are characteristic for the BA.1 and/or BA.2 Omicron lineages on Spike function, processing and susceptibility to neutralization. Individual mutations of S371F/L, S375F and T376A in the ACE2 receptor-binding domain as well as Q954H and N969K in the hinge region 1 impaired infectivity, while changes of G339D, D614G, N764K and L981F moderately enhanced it. Most mutations in the N-terminal region and the receptor binding domain reduced sensitivity of the Spike protein to neutralization by sera from individuals vaccinated with the BNT162b2 vaccine or therapeutic antibodies. Our results represent a systematic functional analysis of Omicron Spike adaptations that allowed this SARS-CoV-2 variant to overtake the current pandemic. HIGHLIGHTSO_LIS371F/L, S373P and S375F impair Spike function and revert in some BA. 1 isolates C_LIO_LIChanges of Q954H and N969K in HR1 reduce while L981F enhances S-mediated infection C_LIO_LIOmicron-specific mutations in the NTD and RBD of Spike reduce neutralization C_LIO_LIN440K, G446S, E484A and Q493K confer resistance to bamlanivimab or imdevimab C_LI

microbiology↗
bioRxiv2021

Spike mutation T403R allows bat coronavirus RaTG13 to use human ACE2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, most likely emerged from bats1. A prerequisite for this devastating zoonosis was the ability of the SARS-CoV-2 Spike (S) glycoprotein to use human angiotensin-converting enzyme 2 (ACE2) for viral entry. Although the S protein of the closest related bat virus, RaTG13, shows high similarity to the SARS-CoV-2 S protein it does not efficiently interact with the human ACE2 receptor2. Here, we show that a single T403R mutation allows the RaTG13 S to utilize the human ACE2 receptor for infection of human cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S significantly reduced ACE2-mediated virus infection. The S protein of SARS-CoV-1 that also uses human ACE2 also contains a positive residue (K) at this position, while the S proteins of CoVs utilizing other receptors vary at this location. Our results indicate that the presence of a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2. This finding could help to predict the zoonotic potential of animal coronaviruses.

microbiology↗
bioRxiv2020

Systematic analysis of innate immune antagonism reveals vulnerabilities of SARS-CoV-2

The innate immune system constitutes a powerful barrier against viral infections. However, it may fail because successful emerging pathogens, like SARS-CoV-2, evolved strategies to counteract it. Here, we systematically assessed the impact of 29 SARS-CoV-2 proteins on viral sensing, type I, II and III interferon (IFN) signaling, autophagy and inflammasome formation. Mechanistic analyses show that autophagy and type I IFN responses are effectively counteracted at different levels. For example, Nsp14 induces loss of the IFN receptor, whereas ORF3a disturbs autophagy at the Golgi/endosome interface. Comparative analyses revealed that antagonism of type I IFN and autophagy is largely conserved, except that SARS-CoV-1 Nsp15 is more potent in counteracting type I IFN than its SARS-CoV-2 ortholog. Altogether, however, SARS-CoV-2 counteracts type I IFN responses and autophagy much more efficiently than type II and III IFN signaling. Consequently, the virus is relatively resistant against exogenous IFN-/{beta} and autophagy modulation but remains highly vulnerable towards IFN-{gamma} and -{lambda} treatment. In combination, IFN-{gamma} and -{lambda} act synergistically, and drastically reduce SARS-CoV-2 replication at exceedingly low doses. Our results identify ineffective type I and II antagonism as weakness of SARS-CoV-2 that may allow to devise safe and effective anti-viral therapies based on targeted innate immune activation.

microbiology↗
bioRxiv2020

IFITM proteins promote SARS-CoV-2 infection in human lung cells

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.

microbiology↗
bioRxiv2020

Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2

SARS-CoV-2 is the causative agent of the current COVID-19 pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1) which suppresses host gene expression by ribosome association via an unknown mechanism. Here, we show that Nsp1 from SARS-CoV-2 binds to 40S and 80S ribosomes, resulting in shutdown of capped mRNA translation both in vitro and in cells. Structural analysis by cryo-electron microscopy (cryo-EM) of in vitro reconstituted Nsp1-40S and of native human Nsp1-ribosome complexes revealed that the Nsp1 C-terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks RIG-I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.

molecular biology↗