Biology subjects

Limpens, R. W. A. L.

Publications and source records attributed to Limpens, R. W. A. L..

8 recordsLinked to original sources

bioRxiv2026

Conserved structural features of RNA export pores spanning the double membrane of arterivirus and coronavirus replication organelles

Corona- and arteriviruses are distantly related positive-stranded RNA virus families within the order Nidovirales. Both transform intracellular membranes into double-membrane vesicles (DMVs) that serve as replication organelles. Newly made viral RNA presumably exits DMVs through double-membrane-spanning molecular pores formed by coronavirus nsp3-nsp4 and arterivirus nsp2-nsp3. Although sub-nm resolution structural information is available for the coronavirus DMV pore, only low-resolution data exist for the pore of the prototypic arterivirus equine arteritis virus (EAV). Here, we modeled arterivirus nsp2 and nsp3 to define their domain architecture, membrane topology, and potential organization into a DMV pore containing 12 copies of each protein. Despite the much smaller dimensions of the arterivirus pore, our analysis suggests striking conservation of features also found in coronaviruses, including conserved positively-charged residues lining analogous pore channel constrictions implicated in RNA export. We investigated their importance in arteriviruses using EAV reverse genetics and nsp2-nsp3 expression systems. While none of these mutations impaired DMV formation, charge-neutralizing substitutions were lethal, whereas conservative substitutions were partially tolerated. We also identified an additional conserved positively-charged residue in the nsp2 C-terminal domain, likely facing the outer DMV membrane, that is essential for DMV formation. Together, these findings reveal nidovirus-wide conservation of important DMV pore features despite the large evolutionary distance between arteri- and coronaviruses. ImportanceFor eukaryotic positive-strand RNA viruses, efficient replication depends on the spatial organization of viral RNA synthesis within replication organelles in the infected cells cytoplasm. These virus-induced, membrane-bound compartments are thought to provide an optimized environment for viral RNA synthesis. In corona- and arteriviruses, distantly related families within the nidovirus order, unusual double-membrane vesicles (DMVs) have been identified as the primary site of viral RNA synthesis. A hallmark of these structures is a double-membrane-spanning pore complex that connects their interior to the cytosol, thereby presumably enabling export of newly synthesized viral RNA. Here, we analyze the arterivirus DMV pore complex and identify several conserved structural features, including positively-charged residues in the pore-forming proteins nsp2 and nsp3, which are essential for viral replication, likely due to their role in RNA export. Together, our findings underscore the functional conservation and fundamental importance of DMV pore complexes across evolutionary distant nidovirus families.

microbiology↗

bioRxiv2026

Double-membrane-spanning RNA export pores are a conserved feature in nidovirus replication organelles

Upon infection, arteriviruses, coronaviruses, and other nidoviruses transform endoplasmic reticulum membranes into viral replication organelles. These include large numbers of double-membrane vesicles (DMVs) whose interior is considered the primary site of viral RNA synthesis. Early studies characterized nidovirus DMVs as sealed compartments, leaving it unclear how newly synthesized viral RNA could be exported to the cytosol. The discovery of DMV-spanning pore complexes in coronavirus-infected cells provided a plausible solution for this topological challenge. However, their structural organization, functional features, and evolutionary conservation across the nidovirus order, have remained unclear. Here, we investigated the macromolecular architecture of DMVs induced by two prototypic arteriviruses using cellular cryo-electron tomography. Despite the substantial evolutionary distance separating arteriviruses and coronaviruses, we observed DMV-spanning pore complexes with striking structural similarities to those previously described in coronaviruses. These pores appear to facilitate both export and encapsidation of viral RNA. In the absence of viral RNA synthesis, ectopic expression of the arterivirus transmembrane nonstructural proteins nsp2 and nsp3 sufficed to induce the formation of pore-containing DMVs. Together, our findings reveal the conservation of key structural features of DMV pores across two distantly related nidovirus families and support a central role for these pores in nidovirus replication.

microbiology↗

bioRxiv2025

Nanoscale imaging of native symbiotic animal tissue using amultimodal large volume imaging pipeline for cryo-electrontomography

The field of cryo-EM offers the possibility to gain high-resolution structural information of biomolecules in their native state. Advances in sample thinning of cryo-EM samples allows the study of proteins inside intact cells using tomography, opening the door for visual proteomics. However, thicker samples such as tissues or entire organisms are still largely unsuitable for cryo-electron tomography (cryo-ET). Therefore, significant efforts are directed toward developing and improving preparation methods to enable cryo-ET of such complex samples. We focused on the binary association between the Hawaiian bobtail squid Euprymna scolopes and the luminous bacteria Vibrio fischeri. The Squid-Vibrio system has long been studied to understand host-symbiont interactions. Our goal is to study the bacterial-host interface using cryo-ET, at a resolution previously unattainable by conventional EM methods. Here, we present a multi-modal preparation and correlative imaging workflow--including cryo- fluorescence microscopy, microCT, freeze-substitution electron tomography (FS-ET), and serial blockface SEM--to localize and prepare specific regions of the dissected symbiotic light organs for cryo-ET. This approach enabled us to directly visualize symbiotic V. fischeri within the internal host crypts at macromolecular resolution, revealing spatial organization, physical contact, and putative exchange interfaces between host and microbe. Our findings provide structural insights into a foundational model of host-microbe symbiosis and demonstrate the feasibility of cryo-ET for investigating intact tissues at the nanoscale.

microbiology↗

bioRxiv2025

ICP1 bacteriophage treatment antagonizes colonization of the zebrafish larval intestine by Vibrio cholerae

Outbreaks of cholera pose a major threat to human health. Currently, antibiotics are the most effective treatment against the causative agent, the bacterium Vibrio cholerae. However, the use of antibiotics eventually leads to the emergence of resistant strains, which necessitates the need for alternative approaches. The use of bacteriophages to target the infection by antibiotic resistant bacteria is one promising alternative. While clearance of Vibrio cholerae with the use of phages has been performed on several animal models, none of these models are natural hosts of V. cholerae. Therefore, we set out to investigate the interaction between V. cholerae and bacteriophage ICP1 both in vitro and in vivo in a natural host, the zebrafish model, Danio rerio. To study the interplay between host, bacteria and phages we used a combination of light and ultrastructural imaging techniques, including confocal fluorescence microscopy, serial block face scanning electron microscopy (EM) imaging and cryogenic EM, which allowed us to investigate both the colonization process by V. cholerae and clearance by the ICP1 bacteriophage. In addition, we determined the effects of the microbiome on this treatment by using germ-free, conventionalized and monoassociated zebrafish larvae as a host. Independent of the presence and composition of microbiomes used here, V. cholerae efficiently colonized the larval intestine. Finally, we demonstrate significant in vivo clearance of V. cholerae N16961-dsRED by ICP1, underscoring the role of phage-bacteria dynamics in shaping pathogen colonization within the zebrafish larval host. SIGNIFICANCECholera remains a life-threatening disease that causes recurring outbreaks and significant mortality, particularly in developing and conflict-affected regions. As antimicrobial resistance continues to rise, there is an urgent need to better understand the ecological and microbial dynamics that govern Vibrio cholerae colonization and persistence. This research investigates how V. cholerae interacts with bacteriophages, the host environment, and the resident microbiota within a natural vertebrate host, offering new insights into the factors that influence pathogen clearance and shaping of the gut ecosystem during infection. The powerful combination of serial block-face scanning and cryogenic electron microscopy, fluorescence microscopy, and traditional colony/plaque counting methods revealed previously unobserved aspects of the interplay between host, pathogen, phages, and the microbiome, highlighting phage-driven clearance of V. cholerae during colonization.

microbiology↗

bioRxiv2020

A molecular pore spans the double membrane of the coronavirus replication organelle

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored micro-environment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and mRNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. Here, using cellular electron cryo-microscopy, we unveiled a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a critical role in coronavirus replication and thus constitutes a novel drug target

microbiology↗

bioRxiv2020

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of nonstructural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (Remdesivir, Alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.

microbiology↗

bioRxiv2020

A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis

Zoonotic coronavirus (CoV) infections, like those responsible for the current SARS-CoV-2 epidemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). While double-membrane vesicles (DMVs) appear to be a pan-coronavirus RO element, studies to date describe an assortment of additional coronavirus-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labelling of newly-synthesized viral RNA followed by quantitative EM autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs MERS-CoV and SARS-CoV, and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in coronavirus infection.

microbiology↗

bioRxiv2019

Mind the gap: micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae

Cryo-focussed ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-lamella workflow is a low-throughput technique and can easily be obstructed by technical issues like the bending of the lamellae during the final cryo-FIB-milling steps. The severity of lamella bending seems to correlate with shrinkage of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin lamella, leading to its bending and breakage. To protect the lamellae from these forces, we milled \"micro-expansion joints\" alongside the lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of such micro-expansion joints drastically decreases lamella bending. Furthermore, we show that this adaptation does not create instabilities that could constrain subsequent parts of the cryo-lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them an easy solution against cryo-lamella bending in any biological sample milled on EM grids.

microbiology↗