Funktionelle Bedeutung der Protein-Kinase C eta im Infektionszyklus des autonomen Parvovirus Minute Virus of Mice

Das multifunktionale große Nichtstruktur Protein NS1 des autonomem Parvovirus Minute Virus of Mice (MVM) wird posttranslational modifiziert und zumindest teilweise über Phosphorylierung reguliert. Aufgrund seiner enzymatischen Funktionen, wie die ATPase-, Helikase- und Nickase-Aktivitäten ist NS1 essentiell für die Initiierung der viralen Replikation. NS1 ist außerdem in der Lage, heterologe Promotoren zu transregulieren und wirkt zytotoxisch auf die Wirtszelle. Die atypische PKClambda -Isoform phosphoryliert und aktiviert NS1 für Helikase-Funktionen. Diese Aktivierung allein ist jedoch nicht ausreichend, um das virale Protein für die rollende Haarnadelreplikation (rolling circle replication) in vitro zu stimulieren. In der vorliegenden Arbeit konnte eine weitere zelluläre Kinase, PKCeta, identifiziert werden, die NS1 in vitro phosphoryliert und so das virale Polypeptid zusammen mit PKClambda für die rolling circle Replikation stimuliert. Diese Funktion von PKCeta wurde mittels in vivo Analysen bestätigt. Dazu wurde die permissive Maus-Fibroblastenzellinie, A9, stabil mit Flag-Epitop markierten Mutanten transfiziert, die die endogene PKCeta -Aktivität modulieren. Es resultierten A9 Derivat-Zellinien mit konstitutiv aktiver oder Kinase-inaktiver PKCeta. Eine tryptische Phosphopeptid-Analyse von 32P-markiertem NS1 ergab, daß in Gegenwart einer dominant negativen PKCeta-Mutante distinkte Phosphorylierungsereignisse ausblieben. Diese Beobachtung korrelierte mit einer defizienten Synthese an viralen DNA-Replikationsintermediaten der Gesamtzellpopulation im Southern Blotting sowie auf Einzelzellebene mittels BrdU-Inkorporation. Die Ergebnisse belegen damit die Bedeutung der PKCeta -Phosphorylierungen von NS1 für die Stimulierung der parvoviralen Replikation. Interessanterweise löst die MVM-Infektion eine Akkumulierung endogener PKCeta in der nukleären Peripherie aus, was als Zeichen der intrazellulären Aktivierung interpretiert wird. Folgerichtig konnte durch Expression einer Kinase-inaktiven Mutante der PKC-aktivierenden upstream-Kinase PDK-1 diese Translokation von endogener PKCeta nach MVM-Infektion unterbunden werden. Außerdem konnte mittels Fraktionierung gezeigt werden, daß ein Zusammenhang zwischen der PKCeta -Aktivität und der Verteilung der Ezrin Radixin Moesin (ERM) -Proteine in vivo besteht, die mit PKCeta aus einem NS1-aktivierenden zellulären Extrakt über mehrere chromatographische Schritte zusammen aufgereinigt worden waren. Darüberhinaus kolokalisierte Radixin in Immunfluoreszenzanalysen mit den PKCeta-Vollänge-Mutanten an der Plasmamembran. Daraus wurde geschlossen, daß die ERM-Proteine eine Rolle beim Transport von PKCeta zur Plasmamembran spielen könnten. Durch Immunpräzipitation wurde eine spezifische Interaktion zwischen Radixin und der Kinase-inaktiven PKCeta T512A gezeigt, deren funktionelle Relevanz jedoch noch nicht geklärt ist. Da die beobachtete Aktivierung von PKCeta zu Zeitpunkten auftritt, an denen die virale Replikation bereits weit fortgeschritten ist, liegt die Vermutung nahe, daß die Kinase hier möglicherweise weitere, für den viralen Vermehrungszyklus vorteilhafte, Funktionen erfüllt. Es wurde angenommen, daß MVM dieselben Kinase-Aktivitäten, die es zur Initiierung der viralen DNA-Replikation benötigt, auch für die Virus-induzierten morphologischen- und Zytoskelettveränderungen der Wirtszelle ausnutzen könnte. Aus diesem Grund wurden die nach Infektion auftretenden Zytoskelettalterationen anhand von Markerproteinen in der murinen Wirtszelle A9 sowie den Derivaten mit modulierter PKCeta-Aktivität untersucht und miteinander verglichen. So konnten die beobachteten Strukturveränderungen der Wirtszelle erstmals konkret auf Modifikationen der Mikrofilamente sowie Intermediärfilamente zurückgeführt werden. Eine Rolle für PKCeta in diesen Abbau- und Deassemblierungs-vorgängen konnte allerdings nicht gezeigt werden

Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration

The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types

Novel PKCη Is Required To Activate Replicative Functions of the Major Nonstructural Protein NS1 of Minute Virus of Mice

The multifunctional protein NS1 of minute virus of mice (MVMp) is posttranslationally modified and at least in part regulated by phosphorylation. The atypical lambda isoform of protein kinase C (PKCλ) phosphorylates residues T435 and S473 in vitro and in vivo, leading directly to an activation of NS1 helicase function, but it is insufficient to activate NS1 for rolling circle replication. The present study identifies an additional cellular protein kinase phosphorylating and regulating NS1 activities. We show in vitro that the recombinant novel PKCη phosphorylates NS1 and in consequence is able to activate the viral polypeptide in concert with PKCλ for rolling circle replication. Moreover, this role of PKCη was confirmed in vivo. We thereby created stably transfected A9 mouse fibroblasts, a typical MVMp-permissive host cell line with Flag-tagged constitutively active or inactive PKCη mutants, in order to alter the activity of the NS1 regulating kinase. Indeed, tryptic phosphopeptide analyses of metabolically (32)P-labeled NS1 expressed in the presence of a dominant-negative mutant, PKCηDN, showed a lack of distinct NS1 phosphorylation events. This correlates with impaired synthesis of viral DNA replication intermediates, as detected by Southern blotting at the level of the whole cell population and by BrdU incorporation at the single-cell level. Remarkably, MVM infection triggers an accumulation of endogenous PKCη in the nuclear periphery, suggesting that besides being a target for PKCη, parvovirus infections may also affect the regulation of this NS1 regulating kinase. Altogether, our results underline the tight interconnection between PKC-mediated signaling and the parvoviral life cycle

Regulation of Minute Virus of Mice NS1 Replicative Functions by Atypical PKCλ In Vivo

Minute virus of mice NS1 protein is a multifunctional phosphoprotein endowed with a variety of enzymatic and regulatory activities necessary for progeny virus particle production. To regulate all of its different functions in the course of a viral infection, NS1 has been proposed to be modulated by posttranslational modifications, in particular, phosphorylation. Indeed, it was shown that the NS1 phosphorylation pattern is altered during the infectious cycle and that the biochemical profile of the protein is dependent on the phosphorylation state of the polypeptide. Moreover, in vitro approaches have identified members of the protein kinase C (PKC) family, in particular, atypical PKC, as regulators of viral DNA replication through the phosphorylation of NS1 residues T435 and S473, thereby activating the protein for DNA unwinding activities. In order to substantiate these findings in vivo, we produced NS1 in the presence of a dominant-negative PKCλ mutant and characterized the purified protein in vitro. The NS1 protein produced under these conditions was found to be only partially phosphorylated and as a consequence to be deficient for viral DNA replication. However, it could be rescued for this viral function by treatment with recombinant activated PKCλ. Our data clearly demonstrate that NS1 is a target for PKCλ phosphorylation in vivo and that this modification is essential for the helicase activity of the viral polypeptide. In addition, the phosphorylation of NS1 at residues T435 and S473 appeared to occur mainly in the nucleus, providing further evidence for the involvement of PKCλ which, unlike PKCζ, accumulates in the nuclear compartment of infected cells

Ezrin-Radixin-Moesin Family Proteins Are Involved in Parvovirus Replication and Spreading▿

The propagation of autonomous parvoviruses is strongly dependent on the phosphorylation of the major nonstructural protein NS1 by members of the protein kinase C (PKC) family. Minute virus of mice (MVM) replication is accompanied by changes in the overall phosphorylation pattern of NS1, which is newly modified at consensus PKC sites. These changes result, at least in part, from the ability of MVM to modulate the PDK-1/PKC pathway, leading to activation and redistribution of both PDK-1 and PKCη. We show that proteins of the ezrin-radixin-moesin (ERM) family are essential for virus propagation and spreading through their functions as adaptors for PKCη. MVM infection led to redistribution of radixin and moesin in the cell, resulting in increased colocalization of these proteins with PKCη. Radixin was found to control the PKCη-driven phosphorylation of NS1 and newly synthesized capsids in vivo. Conversely, radixin phosphorylation and activation were driven by the NS1/CKIIα complex. Altogether, these data argue for ERM proteins being both targets and modulators of parvovirus infection

Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion

In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality

Long-term safety and efficacy of patisiran for hereditary transthyretin-mediated amyloidosis with polyneuropathy: 12-month results of an open-label extension study

© 2020 Elsevier Ltd. All rights reserved.Background: Hereditary transthyretin-mediated amyloidosis is a rare, inherited, progressive disease caused by mutations in the transthyretin (TTR) gene. We assessed the safety and efficacy of long-term treatment with patisiran, an RNA interference therapeutic that inhibits TTR production, in patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy. Methods: This multicentre, open-label extension (OLE) trial enrolled patients at 43 hospitals or clinical centres in 19 countries as of Sept 24, 2018. Patients were eligible if they had completed the phase 3 APOLLO or phase 2 OLE parent studies and tolerated the study drug. Eligible patients from APOLLO (patisiran and placebo groups) and the phase 2 OLE (patisiran group) studies enrolled in this global OLE trial and received patisiran 0·3 mg/kg by intravenous infusion every 3 weeks with plans to continue to do so for up to 5 years. Efficacy assessments included measures of polyneuropathy (modified Neuropathy Impairment Score +7 [mNIS+7]), quality of life, autonomic symptoms, nutritional status, disability, ambulation status, motor function, and cardiac stress, with analysis by study groups (APOLLO-placebo, APOLLO-patisiran, phase 2 OLE patisiran) based on allocation in the parent trial. The global OLE is ongoing with no new enrolment, and current findings are based on the interim analysis of the patients who had completed 12-month efficacy assessments as of the data cutoff. Safety analyses included all patients who received one or more dose of patisiran up to the data cutoff. This study is registered with ClinicalTrials.gov, NCT02510261. Findings: Between July 13, 2015, and Aug 21, 2017, of 212 eligible patients, 211 were enrolled: 137 patients from the APOLLO-patisiran group, 49 from the APOLLO-placebo group, and 25 from the phase 2 OLE patisiran group. At the data cutoff on Sept 24, 2018, 126 (92%) of 137 patients from the APOLLO-patisiran group, 38 (78%) of 49 from the APOLLO-placebo group, and 25 (100%) of 25 from the phase 2 OLE patisiran group had completed 12-month assessments. At 12 months, improvements in mNIS+7 with patisiran were sustained from parent study baseline with treatment in the global OLE (APOLLO-patisiran mean change -4·0, 95 % CI -7·7 to -0·3; phase 2 OLE patisiran -4·7, -11·9 to 2·4). Mean mNIS+7 score improved from global OLE enrolment in the APOLLO-placebo group (mean change from global OLE enrolment -1·4, 95% CI -6·2 to 3·5). Overall, 204 (97%) of 211 patients reported adverse events, 82 (39%) reported serious adverse events, and there were 23 (11%) deaths. Serious adverse events were more frequent in the APOLLO-placebo group (28 [57%] of 49) than in the APOLLO-patisiran (48 [35%] of 137) or phase 2 OLE patisiran (six [24%] of 25) groups. The most common treatment-related adverse event was mild or moderate infusion-related reactions. The frequency of deaths in the global OLE was higher in the APOLLO-placebo group (13 [27%] of 49), who had a higher disease burden than the APOLLO-patisiran (ten [7%] of 137) and phase 2 OLE patisiran (0 of 25) groups. Interpretation: In this interim 12-month analysis of the ongoing global OLE study, patisiran appeared to maintain efficacy with an acceptable safety profile in patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy. Continued long-term follow-up will be important for the overall assessment of safety and efficacy with patisiran.info:eu-repo/semantics/publishedVersio