Biology subjects

de Jong, A. W. M.

Publications and source records attributed to de Jong, A. W. M..

7 recordsLinked to original sources

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↗

bioRxiv2026

VAPB and its binding partner AKAP11 promote lipid droplet degradation

The endoplasmic reticulum (ER) is the master regulator of various cellular processes. To achieve its diverse functions, the ER interacts with other organelles at membrane contact sites, regions where organelles are brought into proximity. Most ER membrane contact sites are facilitated by the ER-resident VAP proteins. To address the role of VAP proteins in regulating cellular lipid homeostasis, we performed a targeted lipidomic screen after silencing individual VAPs. The loss of VAPB increases cellular levels of neutral lipids stored in lipid droplets (LDs). The increase in neutral lipids is reflected in the size, number and motility of LDs, and is due to the impaired degradation of these organelles. VAPB requires its contact site forming ability to regulate LDs, prompting the identification of protein kinase A (PKA) anchor AKAP11 as a VAPB interaction partner in regulating LD degradation and dynamics. Collectively, our findings identify a role for the ER-resident VAPB-AKAP11 interaction and PKA activity in regulating LD homeostasis. SummaryThe ER-resident membrane contact site protein VAPB and its interaction partner AKAP11 regulate lipid droplet size and motility by mediating neutral lipid degradation. VAPB requires its ability to form membrane contact sites and interact with AKAP11, protein kinase A anchor protein, for mediating lipid droplet homeostasis. This paper uncovers a role for VAPB-AKAP11-PKA axis in regulating the homeostasis of lipid droplets.

cell biology↗

bioRxiv2025

A human sensory neuron model for varicella-zoster virus latency and reactivation in vitro.

Varicella-zoster virus (VZV) is a ubiquitous human neurotropic alphaherpesvirus that establishes lifelong latency in sensory ganglionic neurons. Subsequent viral reactivation causes herpes zoster, a morbid disease often complicated by neuropathic pain. Mechanisms underlying VZV latency and reactivation are not understood, mostly due to the lack of permissive animal models and challenges of current in vitro latency modelling. Here, we evaluated HD10.6 cells, a simplified and easily expandable human sensory neuron line to model VZV latency and reactivation. Mature HD10.6 (mHD10.6) differentiated neurons supported productive VZV infection, viral DNA replication, production of infectious progeny, and viral spread in cultures. VZV infection was associated with limited cytopathic effects and ultrastructural changes. Infecting mHD10.6 neurons with cell-free VZV in the presence of antivirals resulted in a quiescent-persistent state, characterized by persistent VZV genomes with restricted VZV gene expression and absence of infectious virus. Importantly, VZV could be reactivated by treatment with capsaicin, as evidenced by increased lytic viral transcription and virus spread. In conclusion, this study establishes human HD10.6 neurons as a novel and scalable in vitro model for studying VZV latency and reactivation to identify virus and host factors governing latency that may serve as therapeutic targets to restrict VZV reactivation. ImportanceMost individuals carry latent varicella-zoster virus (VZV) in their dorsal root ganglia (DRG), which can reactivate to cause shingles and chronic pain. The mechanisms by which VZV establishes latency and triggers of reactivation are incompletely understood. Current platforms for the study of VZV latency do not easily support functional experiments (ganglia) or are difficult to expand and complicated by mixed neuronal populations (stem cell-derived neurons). Here, we demonstrate that matured HD10.6 (mHD10.6) cells derived from immortalized human DRG-derived neurons provide a clonal, scalable, and easy-to-expand platform for studying lytic, latent, and reactivated VZV infection in sensory neurons. We propose that the HD10.6 platform could provide the basis to conduct studies on the viral latent state that have hitherto not been possible.

microbiology↗

bioRxiv2024

Perturbation of de novo lipogenesis hinders MERS-CoV assembly and release, but not the biogenesis of viral replication organelles

Coronaviruses hijack host cell metabolic pathways and resources to support their replication. They induce extensive host endomembrane remodeling to generate viral replication organelles and exploit host membranes for assembly and budding of their enveloped progeny virions. Because of the overall significance of host membranes, we sought to gain insight into the role of host factors involved in lipid metabolism in cells infected with Middle East respiratory syndrome coronavirus (MERS-CoV). We employed a single-cycle infection approach in combination with pharmacological inhibitors, biochemical assays, lipidomics, light and electron microscopy. Pharmacological inhibition of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN), key host factors in de novo fatty acid biosynthesis, led to pronounced inhibition of MERS-CoV particle release. Inhibition of ACC led to a profound metabolic switch in Huh7 cells, altering their lipidomic profile and inducing lipolysis. However, despite the extensive changes induced by the ACC inhibitor, the biogenesis of viral replication organelles remained unaffected. Instead, ACC inhibition appeared to affect the trafficking and post-translational modifications of the MERS-CoV envelope proteins. Electron microscopy revealed an accumulation of nucleocapsids in early budding stages, indicating that MERS-CoV assembly is adversely impacted by ACC inhibition. Notably, inhibition of palmitoylation resulted in similar effects, while supplementation of exogenous palmitic acid reversed the compounds inhibitory effects, possibly reflecting a crucial need for palmitoylation of the MERS-CoV Spike and Envelope proteins for their role in virus particle assembly. ImportanceMiddle East respiratory syndrome coronavirus (MERS-CoV) is the etiological agent of a zoonotic respiratory disease of limited transmissibility between humans. However, MERS-CoV is still considered a high-priority pathogen and is closely monitored by WHO due to its high lethality rate of around 35% of laboratory-confirmed infections. Like other positive-strand RNA viruses, MERS-CoV relies on the host cells endomembranes to support various stages of its replication cycle. However, in spite of this general reliance of MERS-CoV replication on host cell lipid metabolism, mechanistic insights are still very limited. In our study, we show that pharmacological inhibition of acetyl-CoA carboxylase (ACC), a key enzyme in the host cells fatty acid biosynthesis pathway, significantly disrupts MERS-CoV particle assembly without exerting a negative effect on the biogenesis of viral replication organelles. Furthermore, our study highlights the potential of ACC as a target for the development of host-directed antiviral therapeutics against coronaviruses.

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

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

Inhibition of arterivirus RNA synthesis by cyclophilin inhibitors is counteracted by mutations in replicase transmembrane subunits

Previously, the cyclophilin inhibitors cyclosporin A (CsA) and Alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales. Here we aimed to identify arterivirus proteins involved in the mode-of-action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to a [~]2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAVs CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA-resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely-related drug ALV CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.\n\nImportanceCurrently, no approved treatments are available to combat infections with nidoviruses, a group of plus-stranded RNA viruses including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporin A (CsA) and Alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and may thus represent a class of pan-nidovirus inhibitors. Here, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two trans-membrane subunits of the viral replication complex, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of nidoviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and other RNA viruses inhibited by these compounds.

microbiology↗