IntroductionProcalcitonin expression is thought to be stimulated by bacteria and suppressed by viruses via interferon signalling. Consequently, during respiratory viral illness, clinicians often interpret elevated procalcitonin as evidence of bacterial coinfection, prompting antibiotic administration. We sought to evaluate the validity of this practice and the underlying assumption that viral infection inhibits procalcitonin synthesis.MethodsWe conducted a retrospective cohort study of patients hospitalised with pure viral infection (n=2075) versus bacterial coinfection (n=179). The ability of procalcitonin to distinguish these groups was assessed. In addition, procalcitonin and interferon gene expression were evaluated in murine and cellular models of influenza infection.ResultsPatients with bacterial coinfection had higher procalcitonin than those with pure viral infection, but also more severe disease and higher mortality (p<0.001). After matching for severity, the specificity of procalcitonin for bacterial coinfection dropped substantially, from 72% to 61%. In fact, receiver operating characteristic curve analysis showed that procalcitonin was a better indicator of multiple indices of severity (eg, organ failures and mortality) than of coinfection. Accordingly, patients with severe viral infection had elevated procalcitonin. In murine and cellular models of influenza infection, procalcitonin was also elevated despite bacteriologic sterility and correlated with markers of severity. Interferon signalling did not abrogate procalcitonin synthesis.DiscussionThese studies reveal that procalcitonin rises during pure viral infection in proportion to disease severity and is not suppressed by interferon signalling, in contrast to prior models of procalcitonin regulation. Applied clinically, our data suggest that procalcitonin represents a better indicator of disease severity than bacterial coinfection during viral respiratory infection.
Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.
Gap junction channels and hemichannels formed by concatenated connexins were analyzed. Monomeric (hCx26, hCx46), homodimeric (hCx46-hCx46, hCx26-hCx26), and heterodimeric (hCx26-hCx46, hCx46-hCx26) constructs, coupled to GFP, were expressed in HeLa cells. Confocal microscopy showed that the tandems formed gap junction plaques with a reduced plaque area compared to monomeric hCx26 or hCx46. Dye transfer experiments showed that concatenation allows metabolic transfer. Expressed in Xenopus oocytes, the inside-out patch-clamp configuration showed single channels with a conductance of about 46 pS and 39 pS for hemichannels composed of hCx46 and hCx26 monomers, respectively, when chloride was replaced by gluconate on both membrane sides. The conductance was reduced for hCx46-hCx46 and hCx26-hCx26 homodimers, probably due to the concatenation. Heteromerized hemichannels, depending on the connexin-order, were characterized by substates at 26 pS and 16 pS for hCx46-hCx26 and 31 pS and 20 pS for hCx26-hCx46. Because of the linker between the connexins, the properties of the formed hemichannels and gap junction channels (e.g., single channel conductance) may not represent the properties of hetero-oligomerized channels. However, should the removal of the linker be successful, this method could be used to analyze the electrical and metabolic selectivity of such channels and the physiological consequences for a tissue.
Connexins (Cx) are proteins that form cell‐to‐cell gap junction channels. A mutation at position 188 in the second extracellular loop (E2) domain of hCx46 has been linked to an autosomal dominant zonular pulverulent cataract. As it is dominantly inherited, it is possible that the mutant variant affects the co‐expressed wild‐type Cx and/or its interaction with other cellular components. Here, we proposed to use concatenated hC x46wt‐ hC x46N188T and hC x46N188T‐ hC x46wt to analyze how hC x46N188T affected co‐expressed hC x46wt to achieve a dominant inheritance. Heterodimer hC x46wt‐ hC x46N188T formed fewer gap junction plaques compared to homodimer hC x46wt‐ hC x46wt, while the hC x46N188T‐ hC x46N188T homodimer formed almost no gap junction plaques. Dye uptake experiments showed that hemichannels of concatenated variants were similar to hemichannels of monomers. Molecular dynamics simulations revealed that for docking, the N188 of a protomer was engaged in hydrogen bonds (HBs) with R180, N189, and D191 of the counterpart protomer of the adjacent hemichannel. T188 suppressed the formation of HBs between protomers. Molecular dynamics simulations of an equimolar hC x46wt/ hC x46N188T gap junction channel revealed a reduced number of HBs between protomers, suggesting reduction of gap junction channels between lens fibers co‐expressing the variants.
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