Biology subjects

Vlok, M.

Publications and source records attributed to Vlok, M..

4 recordsLinked to original sources

bioRxiv2025

Post-translational modifications within fibrinaloid microclot complexes distinguish Pre-COVID-19 Postural Orthostatic Tachycardia Syndrome, Long COVID, and Long COVID-POTS and reveal disease-specific molecular pathways

BackgroundPre-COVID-19 Postural orthostatic tachycardia syndrome (PC-POTS), Long COVID, and their overlap (LC-POTS) are chronic post-viral conditions marked by debilitating symptoms despite frequently normal routine laboratory results. We previously identified insoluble fibrinaloid microclot complexes (FMCs) in Long COVID. It is not known whether FMCs are also present in PC-POTS, or whether post-translational modifications (PTMs) within FMC-entrapped proteins contribute to disease mechanisms. MethodsPlatelet-poor plasma from healthy controls, PC-POTS patients (collected prior to the COVID-19 pandemic), Long COVID (without POTS) and LC-POTS patients underwent fluorescence imaging flow cytometry to quantify FMCs. Proteomic analyses were performed on insoluble protein fractions using a double trypsin digestion strategy and data-independent liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differential protein abundance, PTMs, and amyloidogenicity were compared across groups. ResultsMeasured with imaging flowcytometry in objects/mL, higher levels of FMCs were present in disease groups compared to controls, although not statistically significant. Statistically significant differences potentially lay within FMC sizes and composition. Furthermore, despite only a few dysregulated proteins, FMC proteomics revealed extensive and disease-specific peptides with PTM dysregulation across coagulation, immune, and metabolic pathways. Long COVID displayed FMCs with PTMs of coagulation proteins including prominent advanced glycation end-products (AGE)- and oxidation-based modifications of fibrinogen subunits, particularly fibrinogen subunit A (FIBA), resembling diabetic glycation profiles. FMCs in PC-POTS showed fewer fibrinogen PTMs but markedly increased modifications in metabolic proteins, including oxidised apoA1 and apoB, and immune patterns with complement-related proteins (C3, C4A/B, IC1), immunoglobulin G1 (IGG1) and alpha 2 macroglobulin (A2MG). LC-POTS shared coagulation pathology with Long COVID and immune pathology with PC-POTS. Many dysregulated peptides were determined by in silco methods to be highly amyloidogenic, consistent with FMCs as {beta}-sheet-rich aggregates. Protein-level differences were minimal compared with PTM changes. ConclusionsThis study provides the first evidence that post-translational modifications (PTMs) within fibrinaloid microclots complexes (FMCs) uniquely distinguish pre-COVID-19 POTS, Long COVID, and Long COVID-POTS. Because PC-POTS samples were collected before SARS-CoV-2, their PTM patterns reflect intrinsic disease biology, allowing a clear separation from Long COVID-related changes. PTM profiling revealed pro-coagulant fibrinogen modifications in Long COVID and LC-POTS, metabolic-oxidative disruptions in Long COVID and PC-POTS, and immune dysregulation in PC-POTS and LC-POTS. None of these is detectable with routine assays, and all are independent of protein abundance. The consistent presence of amyloidogenic peptides suggests a contribution to microvascular dysfunction. These findings define disease-specific PTM landscapes and support new diagnostic and therapeutic avenues across autonomic and post-viral disorders.

physiology↗

bioRxiv2022

Cleavage of 14-3-3ϵ by the enteroviral 3C protease dampens RIG-I mediated antiviral signaling

Viruses have evolved diverse strategies to evade the host innate immune response and promote infection. The RIG-I-like-receptors RIG-I and MDA5 (RLRs) are antiviral factors that sense viral RNA and signal downstream via mitochondrial antiviral-signaling protein (MAVS) to activate type I interferon (IFN) expression. 14-3-3{varepsilon} is a key component of the RIG-I translocon complex that interacts with MAVS at the mitochondrial membrane; however, the exact role of 14-3-3{varepsilon} in this pathway is not well understood. In this study, we demonstrate that 14-3-3{varepsilon} is a direct substrate of both the poliovirus and coxsackievirus B3 (CVB3) 3C proteases (3Cpro), and that it is cleaved at Q236{downarrow}G237, resulting in the generation of N- and C-terminal fragments of 27.0 and 2.1 kDa, respectively. Expression of the N-terminal cleavage fragment in cells reduces IFNB mRNA production during poly(I:C) stimulation, thus suggesting an antagonistic effect in the presence of the endogenous 14-3-3{varepsilon} protein. The N-terminal 14-3-3{varepsilon} fragment does not interact with RIG-I in co-immunoprecipitation assays, nor can it facilitate RIG-I translocation to the mitochondria. Probing the intrinsically disordered C-terminal region identifies key residues responsible for RIG-I signaling. Finally, overexpression of the N-terminal fragment promotes CVB3 infection and influenza A virus (H1N1) RNA production and reduces IFNB mRNA production during infection. The strategic enterovirus 3Cpro-mediated cleavage of 14-3-3{varepsilon} antagonizes RIG-I signaling by disrupting critical interactions within the RIG-I translocon complex, thus contributing to evasion of the host antiviral response. Author SummaryHost antiviral factors work to sense virus infection through various mechanisms, including a complex signaling pathway known as the RIG-I like receptor (RLR) pathway. This pathway drives the production of antiviral molecules known as interferons, which are necessary to establish an antiviral state in the cellular environment. Key to this antiviral signaling pathway is the small chaperone protein 14-3-3{varepsilon}, which facilitates the delivery of a viral sensor protein, RIG-I, to the mitochondria. In this study, we show that the enteroviral 3C protease cleaves 14-3-3{varepsilon} during infection, rendering it incapable of facilitating this antiviral response. We also find that the cleavage fragment inhibits RIG-I signaling and promotes virus infection. Our findings reveal a novel viral strategy that restricts the antiviral host response and provides insights into the mechanisms underlying 14-3-3{varepsilon} function in RIG-I antiviral signaling.

microbiology↗

bioRxiv2022

Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation

Viruses have evolved mechanisms to modulate cellular pathways to facilitate infection. One such pathway is the formation of stress granules (SG), which are ribonucleoprotein complexes that assemble during translation inhibition following cellular stress. Inhibition of SG assembly has been observed under numerous virus infections across species, suggesting a conserved fundamental viral strategy. However, the significance of SG modulation during virus infection is not fully understood. The 1A protein encoded by the model dicistrovirus, Cricket Paralysis Virus (CrPV), is a multifunctional protein that can bind to and degrade Ago-2 in an E3 ubiquitin ligase-dependent manner to block the antiviral RNA interference pathway and inhibit SG formation. Moreover, the R146 residue of 1A is necessary for SG inhibition and CrPV infection in both Drosophila S2 cells and adult flies. Here, we uncoupled CrPV-1As functions and provide insight into its underlying mechanism for SG inhibition. CrPV-1A mediated inhibition of SGs requires the E3 ubiquitin-ligase binding domain and the R146 residue, but not the Ago-2 binding domain. Wild-type but not mutant CrPV-1A R146A localizes to the nuclear membrane which correlates with nuclear enrichment of poly(A)+ RNA. Transcriptome changes in CrPV-infected cells are dependent on the R146 residue. Finally, Nup358/RanBP2 is targeted and degraded in CrPV-infected cells in an R146-dependent manner and the depletion of Nup358 blocks SG formation. We propose that CrPV utilizes a multiprong strategy whereby the CrPV-1A protein interferes with a nuclear event that contributes to SG inhibition in order to promote infection. AUTHOR SUMMARYViruses often inhibit a cellular stress response that leads to the accumulation of RNA and protein condensates called stress granules. How this occurs and why this would benefit virus infection are not fully understood. Here, we reveal a viral protein that can block stress granules and identify a key amino acid residue in the protein that inactivates this function. We demonstrate that this viral protein has multiple functions to modulate nuclear events including mRNA export and transcription to regulate stress granule formation. We identify a key host protein that is important for viral protein mediate stress granule inhibition, thus providing mechanistic insights. This study reveals a novel viral strategy in modulating stress granule formation to promote virus infection.

microbiology↗

bioRxiv2018

Spatiotemporal dynamics of river viruses, bacteria and microeukaryotes

Freshwater is an essential resource of increasing value, as clean water sources diminish. Microorganisms in rivers, a major source of renewable freshwater, are significant due to their role in drinking water safety, signalling environmental contamination1, and driving global nutrient cycles2,3. However, a foundational understanding of microbial communities in rivers is lacking4, especially temporally and for viruses5-7. No studies to date have examined the composition of the free-floating river virome over time, and explanations of the underlying causes of spatial and temporal changes in riverine microbial composition, especially for viruses, remain unexplored. Here, we report relationships among riverine microbial communities and their environment across time, space, and superkingdoms (viruses, bacteria, and microeukaryotes), using metagenomics and marker-based microbiome analysis methods. We found that many superkingdom pairs were synchronous and had consistent shifts with sudden environmental change. However, synchrony strength, and relationships with environmental conditions, varied across space and superkingdoms. Variable relationships were observed with seasonal indicators and chemical conditions previously found to be predictive of bacterial community composition4,8-10, emphasizing the complexity of riverine ecosystems and raising questions around the generalisability of single-site and bacteria-only studies. In this first study of riverine viromes over time, DNA viral communities were stably distinct between sites, suggesting the similarity in riverine bacteria across significant geographic distances10-12 does not extend to viruses, and synchrony was surprisingly observed between DNA and RNA viromes. This work provides foundational data for riverine microbial dynamics in the context of environmental and chemical conditions and illustrates how a bacteria-only or single-site approach would lead to an incorrect description of microbial dynamics. We show how more holistic microbial community analysis, including viruses, is necessary to gain a more accurate and deeper understanding of microbial community dynamics.

microbiology↗