Antiviral Innate Immune Response Interferes with the Formation of Replication-Associated Membrane Structures Induced by a Positive-Strand RNA Virus

Infection with nidoviruses like corona- and arteriviruses induces a reticulovesicular network of interconnected endoplasmic reticulum (ER)-derived double-membrane vesicles (DMVs) and other membrane structures. This network is thought to accommodate the viral replication machinery and protect it from innate immune detection. We hypothesized that the innate immune response has tools to counteract the formation of these virus-induced replication organelles in order to inhibit virus replication. Here we have investigated the effect of type I interferon (IFN) treatment on the formation of arterivirus-induced membrane structures. Our approach involved ectopic expression of arterivirus nonstructural proteins nsp2 and nsp3, which induce DMV formation in the absence of other viral triggers of the interferon response, such as replicating viral RNA. Thus, this setup can be used to identify immune effectors that specifically target the (formation of) virus-induced membrane structures. Using large-scale electron microscopy mosaic maps, we found that IFN-β treatment significantly reduced the formation of the membrane structures. Strikingly, we also observed abundant stretches of double-membrane sheets (a proposed intermediate of DMV formation) in IFN-β-treated samples, suggesting the disruption of DMV biogenesis. Three interferon-stimulated gene products, two of which have been reported to target the hepatitis C virus replication structures, were tested for their possible involvement, but none of them affected membrane structure formation. Our study reveals the existence of a previously unknown innate immune mechanism that antagonizes the viral hijacking of host membranes. It also provides a solid basis for further research into the poorly understood interactions between the innate immune system and virus-induced replication structures

Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication

Microscopic imaging and technolog

Escaping Host Factor PI4KB Inhibition : Enterovirus Genomic RNA Replication in the Absence of Replication Organelles

Enteroviruses reorganize cellular endomembranes into replication organelles (ROs) for genome replication. Although enterovirus replication depends on phosphatidylinositol 4-kinase type IIIβ (PI4KB), its role, and that of its product, phosphatidylinositol 4-phosphate (PI4P), is only partially understood. Exploiting a mutant coxsackievirus resistant to PI4KB inhibition, we show that PI4KB activity has distinct functions both in proteolytic processing of the viral polyprotein and in RO biogenesis. The escape mutation rectifies a proteolytic processing defect imposed by PI4KB inhibition, pointing to a possible escape mechanism. Remarkably, under PI4KB inhibition, the mutant virus could replicate its genome in the absence of ROs, using instead the Golgi apparatus. This impaired RO biogenesis provided an opportunity to investigate the proposed role of ROs in shielding enteroviral RNA from cellular sensors. Neither accelerated sensing of viral RNA nor enhanced innate immune responses was observed. Together, our findings challenge the notion that ROs are indispensable for enterovirus genome replication and immune evasion

Escaping Host Factor PI4KB Inhibition : Enterovirus Genomic RNA Replication in the Absence of Replication Organelles

Enteroviruses reorganize cellular endomembranes into replication organelles (ROs) for genome replication. Although enterovirus replication depends on phosphatidylinositol 4-kinase type IIIβ (PI4KB), its role, and that of its product, phosphatidylinositol 4-phosphate (PI4P), is only partially understood. Exploiting a mutant coxsackievirus resistant to PI4KB inhibition, we show that PI4KB activity has distinct functions both in proteolytic processing of the viral polyprotein and in RO biogenesis. The escape mutation rectifies a proteolytic processing defect imposed by PI4KB inhibition, pointing to a possible escape mechanism. Remarkably, under PI4KB inhibition, the mutant virus could replicate its genome in the absence of ROs, using instead the Golgi apparatus. This impaired RO biogenesis provided an opportunity to investigate the proposed role of ROs in shielding enteroviral RNA from cellular sensors. Neither accelerated sensing of viral RNA nor enhanced innate immune responses was observed. Together, our findings challenge the notion that ROs are indispensable for enterovirus genome replication and immune evasion

Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions

A major cause of respiratory failure during influenza A virus (IAV) infection is damage to the epithelial-endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid, erythrocytes and inflammatory cells. To date, the exact roles of pulmonary epithelial and endothelial cells in this process remain unclear. Here, we used an in vitro co-culture model to understand how IAV damages the pulmonary epithelial-endothelial barrier. Human epithelial cells were seeded on the upper half of a transwell membrane while human endothelial cells were seeded on the lower half. These cells were then grown in co-culture and IAV was added to the upper chamber. We showed that the addition of IAV (H1N1 and H5N1 subtypes) resulted in significant barrier damage. Interestingly, we found that, while endothelial cells mounted a pro-inflammatory/pro-coagulant response to a viral infection in the adjacent epithelial cells, damage to the alveolar epithelial-endothelial barrier occurred independently of endothelial cells. Rather, barrier damage was associated with disruption of tight junctions amongst epithelial cells, and specifically with loss of tight junction protein claudin-4. Taken together, these data suggest that maintaining epithelial cell integrity is key in reducing pulmonary oedema during IAV infection

Diabetic Nephropathy Alters the Distribution of Circulating Angiogenic MicroRNAs Among Extracellular Vesicles, HDL, and Ago-2

Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate [eGFR] >30 mL/min/1.73 m2), and patients with DN (eGFR <30 mL/min/1.73 m2). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132)

Erratum to: Diabetic nephropathy alters the distribution of circulating angiogenic micrornas among extracellular vesicles, hdl, and ago-2, (Diabetes 2019, 68, 2287-2300)

In the article cited above, “Amsterdam University Medical Center, Amsterdam” was mistakenly displayed for affiliations 1 and 2. The institution and city for both should have read “Leiden University Medical Center, Leiden.” The editors apologize for the errors. The online version of the article (https://doi.org/10.2337/db18-1360) has been updated to correct this