Influenza neuraminidase is delivered directly to the apical surface of MDCK cell monolayers

AbstractThe aim of this study was to investigate whether influenza neuraminidase travels directly from the Golgi complex to the apical domain of the plasma membrane in virally infected epithelial (MDCK) cell monolayers, or whether it passes transiently through the basolateral domain. Using a new assay for the delivery of neuraminidase to the plasma membrane, we found that the time course of transport of this protein from the Golgi complex to the apical surface of MDCK cell monolayers was very similar to that for influenza haemagglutinin, which is known to be delivered directly to its destination. In addition, a similar time course of neuraminidase transport was found in BHK cells, which are not asymmetric and in which delivery must therefore be direct. Finally, basolateral exposure of MDCK cell monolayers grown on nitro-cellulose filters to an anti-neuraminidase antibody was shown to have no effect on the delivery of active neuraminidase to the apical surface. We conclude from these results that neuraminidase, like haemagglutinin, is delivered directly to the apical surface

Characteristics of a cell-free assay for the delivery of proteins to the plasma membrane

AbstractWe have previously described the reconstitution, in a cell-free system, of the constitutive delivery of a newly synthesized protein, influenza neuraminidase, to the plasma membrane in BHK cells. Here we report some of the characteristics of this in vitro membrane fusion event. We show that fusion requires ATP hydrolysis, and exploit this requirement to distinguish the time-course of fusion from that of neuraminidase action. In addition, we present evidence for the occurrence of multiple fusions between hybrid membrane vesicles

Transport of influenza virus envelope proteins from the Golgi complex to the apical plasma membrane in MDCK cells: pH-Controlled interaction with a cycling receptor is not involved

AbstractIn influenza virus-infected monolayers of the epithelial cell line MDCK the viral envelope proteins, haemagglutinin and neuraminidase, are targetted specifically to the apical surface. In this study we have tested the hypothesis that the polarized delivery of these proteins to the plasma membrane involves the operation of a receptor that cycles between the trans Golgi network and the plasma membrane, binding the proteins at low pH in the former compartment and releasing them at normal extracellular pH in the latter. The hypothesis predicts that apical, but not basolateral, low pH would eventually delay or block delivery of the proteins to the plasma membrane. We found that basolateral low pH in fact had the more profound effect, in line with its greater effect on intracellular pH. We conclude that the hypothesis is not valid, and that low extracellular pH causes its effect on protein transport by changing intracellular pH

Stratified analyses refine association between TLR7 rare variants and severe COVID-19

Summary: Despite extensive global research into genetic predisposition for severe COVID-19, knowledge on the role of rare host genetic variants and their relation to other risk factors remains limited. Here, 52 genes with prior etiological evidence were sequenced in 1,772 severe COVID-19 cases and 5,347 population-based controls from Spain/Italy. Rare deleterious TLR7 variants were present in 2.4% of young (<60 years) cases with no reported clinical risk factors (n = 378), compared to 0.24% of controls (odds ratio [OR] = 12.3, p = 1.27 × 10−10). Incorporation of the results of either functional assays or protein modeling led to a pronounced increase in effect size (ORmax = 46.5, p = 1.74 × 10−15). Association signals for the X-chromosomal gene TLR7 were also detected in the female-only subgroup, suggesting the existence of additional mechanisms beyond X-linked recessive inheritance in males. Additionally, supporting evidence was generated for a contribution to severe COVID-19 of the previously implicated genes IFNAR2, IFIH1, and TBK1. Our results refine the genetic contribution of rare TLR7 variants to severe COVID-19 and strengthen evidence for the etiological relevance of genes in the interferon signaling pathway

Whole-genome sequencing reveals host factors underlying critical COVID-19

Altres ajuts: Department of Health and Social Care (DHSC); Illumina; LifeArc; Medical Research Council (MRC); UKRI; Sepsis Research (the Fiona Elizabeth Agnew Trust); the Intensive Care Society, Wellcome Trust Senior Research Fellowship (223164/Z/21/Z); BBSRC Institute Program Support Grant to the Roslin Institute (BBS/E/D/20002172, BBS/E/D/10002070, BBS/E/D/30002275); UKRI grants (MC_PC_20004, MC_PC_19025, MC_PC_1905, MRNO2995X/1); UK Research and Innovation (MC_PC_20029); the Wellcome PhD training fellowship for clinicians (204979/Z/16/Z); the Edinburgh Clinical Academic Track (ECAT) programme; the National Institute for Health Research, the Wellcome Trust; the MRC; Cancer Research UK; the DHSC; NHS England; the Smilow family; the National Center for Advancing Translational Sciences of the National Institutes of Health (CTSA award number UL1TR001878); the Perelman School of Medicine at the University of Pennsylvania; National Institute on Aging (NIA U01AG009740); the National Institute on Aging (RC2 AG036495, RC4 AG039029); the Common Fund of the Office of the Director of the National Institutes of Health; NCI; NHGRI; NHLBI; NIDA; NIMH; NINDS.Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care or hospitalization after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease