Coronavirus Cell Entry Occurs through the Endo-/ Lysosomal Pathway in a Proteolysis-Dependent Manner Christine Burkard1¤a,Monique H.Verheije1¤b,Oliver Wicht1,Sander I.van Kasteren2,
Frank J.van Kuppeveld1,Bart L.Haagmans3,Lucas Pelkmans4,Peter J.M.Rottier1,Berend Jan Bosch1, Cornelis A.M.de Haan1*
1Virology Division,Department of Infectious Dias and Immunology,Faculty of Veterinary Medicine,Utrecht University,Utrecht,The Netherlands,2Division of Bio-Organic Synthesis,Leiden Institute of Chemistry,Leiden University,Leiden,The Netherlands,3Department of Viroscience,Erasmus MC,Rotterdam,The Netherlands, 4Institute of Molecular Life Sciences,University of Zurich,Zurich,Switzerland
Abstract
Enveloped virus need to fu with a host cell membrane in order to deliver their genome into the host cell.While some virus fu with the plasma membrane,many virus are endocytod prior to fusion.Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion.In the prent study we investigated the entry of coronavirus(CoVs).Using siRNA gene silencing,we found that proteins known to be import
ant for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mou hepatitis coronavirus(MHV).Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur.Nevertheless,MHV was shown to be less nsitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus,which fu in early and late endosomes,respectively.Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteas.Fusion of MHV was verely inhibited by a pan-lysosomal protea inhibitor,while trafficking of MHV to lysosomes and processing by lysosomal proteas was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide.Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteas.In contrast,MERS-CoV,which contains a minimal furin cleavage site just upstream of the fusion peptide,was negatively affected by inhibition of furin,but not of lysosomal proteas.We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an esntial determinant of the intracellular site of fusion.
Citation:Burkard C,Verheije MH,Wicht O,van Kasteren SI,van Kuppeveld FJ,et al.(2014)Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in
a Proteolysis-Dependent Manner.PLoS Pathog10(11):e1004502.doi:10.1371/journal.ppat.1004502
关于读书的征文Editor:Stanley Perlman,University of Iowa,United States of America
Received May26,2014;Accepted October2,2014;Published November6,2014
Copyright:ß2014Burkard et al.This is an open-access article distributed under the terms of the Creative Commons Attribution Licen,which permits unrestricted u,distribution,and reproduction in any medium,provided the original author and source are credited.解密演员表
Data Availability:The authors confirm that all data underlying the findings are fully available without restriction.All relevant data are within the paper and its Supporting Information files.
Funding:This work was supported by the EU7th Framework Programme(Virus Entry,project235649,PJMR)and by a Utrecht University High potential grant to CAMdH.The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.
Competing Interests:The authors have declared that no competing interests exist.
*Email:dehaan@uu.nl
¤a Current address:The Roslin Institute and Royal(Dick)School of Veterinary Studies,University of Edinburgh,Easter Bush,Edinburgh,United Kingdom,¤b Current address:Department of Pathobiology,Division Pathology,Faculty of Veterinary Medicine,Utrecht University,Utrecht,The Netherlands
Introduction
To achieve successful infection enveloped virus need to fu with a host cell membrane to deliver the viral genome into the host cell.Some virus,such as herpes simplex virus,Sendai virus,and human immunodeficiency virus,appear to be capable of direct fusion at the plasma membrane after initial attachment[1–5]. However,the majority of enveloped virus u endocytosis for uptake and transport prior to fusion.Since endocytic cargo may eventually end up in the destructive environment of the lysosome, environmental cues are crucial to trigger viral fusion at the right stage of trafficking.The triggers,which may include a decrea in pH,changes in redox environment,and proteolytic activity[6–8], induce conformational changes in the viral fusion proteins leading to the mer
ger of viral and host membranes.Two well-studied virus;influenza A virus(IAV)and vesicular stomatitis virus(VSV),are known to undergo fusion upon exposure to low pH[9–12]. Other enveloped virus,such as respiratory syncytial virus(RSV) and Ebola virus,require proteolytic processing of their viral fusion proteins in the endosomal system for fusion to occur[13–16]. Coronavirus(CoVs)are enveloped,plus-strand RNA virus belonging to the family Coronaviridae in the order Nidovirales. They are capable of infecting a wide variety of mammalian and avian species.In most cas they cau respiratory and/or intestinal tract dia.Human coronavirus(HCoVs)are known as major caus of the common HCoV-229E and HCoV-OC43).However,the emergence of new HCoVs of zoonotic origin has shown the potential of CoVs to cau life-threatening dia in humans as was demonstrated during the 2002/2003SARS-CoV epidemics and more recently for MERS-CoV in the Middle East[17,18].The well-studied mou hepatitis virus(MHV)is often ud as a safe model to study CoV infections.对外宣传
All CoV virions contain a canonical t of four structural proteins.The viral genomic RNA is encapsid
ated by the nucleocapsid protein(N)to form the helical nucleocapsid,which is surrounded by the lipoprotein envelope,containing membrane glycoprotein(M),the small envelope protein(E),as well as the spike glycoprotein(S)(reviewed in[19]).Trimers of the CoV S protein,a type I membrane protein belonging to the class I fusion proteins,form the peplomers that protrude from the virion surface [20].The S protein can be divided into two functional subunits. The amino-terminal S1subunit contains the receptor-binding domain;while the carboxy-terminal S2subunit contains domains required for fusion,including the fusion peptide(FP),heptad repeat domains(HR)HR1and HR2,and the transmembrane (TM)domain.
Various entry routes have been described as being ud by different CoVs for infection of cells.Clathrin-dependent as well as clathrin-and caveolae-independent entry pathways have been reported for SARS-CoV[21,22].Also feline infectious peritonitis virus(FIPV)was suggested to enter via a clathrin-and caveolae-independent endocytic route[23,24].For the HCoV-229E a caveolae-dependent endocytic uptake has been suggested[25]. Although the ability of MHV S proteins to cau cell-cell fusion at a neutral pH was initially interpreted as an indication for fusion of virions at the cell surface,more recent studies indicate the requirement for clathrin-mediated endocytosis for entry of MHV [26–29].However,while some studies report that MHV strain A59is nsitive to lysosomotropic agents that affect endocytosis [26],this is not the ca according to others[27].
Proteolytic cleavage of the CoV S proteins appears to be important for the induction of cell-cell fusion and/or virus entry into host cells.Different cleavage sites have been identified for different CoVs,the importance of which ems to differ for cell-cell and virus-cell fusion.Some CoV S proteins,including that of MHV strain A59,are cleaved at the S1/S2boundary by furin(-like)proteas during transport of the newly asmbled virions through the cretory pathway of the producer cell[30–33]. Inhibition of this S protein cleavage was shown to inhibit cell-cell fusion,but not to affect entry of MHV strain A59into host cells [30,34,35].MHV strain2contains an S protein that is not cleaved at the S1/S2boundary.Interestingly,although MHV strains2 and A59were both reported to enter via clathrin-mediated endocytosis,entry of MHV2but not of MHV A59,was blocked by inhibitors of low-pH activated cathepsin proteas[27,36]. Inhibitors of cathepsin proteas have also been shown to inhibit entry of SARS-CoV and feline CoVs[23,37,38],while treatment of cell-bound virus particles with different proteas was shown to enhance virus entry and/or cell-cell fusion[27,34,39–45].For SARS-CoV and infectious bronchitis virus(IBV),it appears that a proteolytic cleavage of the S protein at a more downstream position than the S1/S2boundary upon receptor binding is of importance for cell entry[40,43,46–49].
In the prent study we performed a detailed investigation of the entry of different CoVs.Using siRN
A gene silencing,we found that the prototypic coronavirus MHV strain A59(further referred to as MHV)requires proteins known to be important for late endosomal maturation and endosome-lysosome fusion for efficient infection of cells.By using recombinant MHVs expressing reporter genes as well as by applying a novel,replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV virions to lysosomal compartments and processing of the S protein by lysosomal proteas was required for productive entry to occur. Our results indicate that a cleavage site in the S protein of CoVs immediately upstream of the FP determines the site of fusion.In agreement herewith FIPV,which requires processing by lysosomal proteas,was also shown to depend on trafficking to lysosomes.In contrast,MERS-CoV,which contains a minimal furin-cleavage site connsus quence in the S protein immediately upstream of the FP,was negatively affected by inhibition of furin,but not of lysosomal proteas.
Results
RNAi mediated gene silencing identifies endocytosis-associated proteins to be important in MHV infection
In an automated,high-throughput RNAi screen[50]targeting the druggable genome(approximately7000genes)a number of proteins associated with endocytosis were found to be required for efficient infection of HeLa cells with GFP-expressing MHV.To validate the findings the proteins were subjected to a follow-up analysis using siRNA-mediated gene silencing with oligonucleo-tides from a different supplier than the one ud for the initial RNAi screen(Fig.1A).The follow-up analysis included ACTR2 and ACTR3,two major constituents of the Arp2/3complex which are important for the formation of actin branches and cell surface protrusions,as well as for the motility of veral pathogens inside host cells(reviewed in[51,52]).Also lected were the RAS-related GTP-binding protein family members,RAB7A and RAB7B,which have been shown to be involved in endosomal maturation(reviewed in[53]).RAB7interacts amongst others with members of the homotypic fusion and vacuole protein sorting (HOPS)tethering complex,involved in late endosome to lysosome maturation.The HOPS subunit VPS39(reviewed in[54])was also found to be a strong hit in the siRNA screen and therefore lected.Other proteins included SNX1,involved in retrograde transport of cargo between endosomes and the trans-Golgi network(reviewed in[55]),VCL,inter alia involved in connecting the Arp2/3complex with integrins during actin polymerization (reviewed in[56]),and the Ser/Thr-protein kina PAK1,which is activated by the Rho/Rac/Cdc42family and is implicated in a variety of downstream effects including modulation of the actin cytoskeleton(reviewed in[57]).
Author Summary
Enveloped virus need to fu with a host cell membrane in order to deliver their genome into the host cell.In the prent study we investigated the entry of coronavirus (CoVs).CoVs are important pathogens of animals and man with high zoonotic potential as demonstrated by the emergence of SARS-and MERS-CoVs.Previous studies resulted in apparently conflicting results with respect to CoV cell entry,particularly regarding the fusion-activating requirements of the CoV S protein.By combining cell-biological,infection,and fusion assays we demonstrated that murine hepatitis virus(MHV),a prototypic member of the CoV family,enters cells via clathrin-mediated endocy-tosis.Moreover,although MHV does not depend on a low pH for fusion,the virus was shown to rely on trafficking to lysosomes for proteolytic cleavage of its spike(S)protein and membrane fusion to occur.Bad on the results we predicted and subquently demonstrated that MERS-and feline CoV require cleavage by different proteas and escape the endo/lysosomal system from different com-partments.In conclusion,we elucidated the MHV entry pathway in detail and demonstrate that a proteolytic cleavage site in the S protein of different CoVs is an esntial determinant of the intracellular site of fusion.
画元宵Transfection of HeLa cells carrying the receptor for MHV (HeLa-mCC1a cells)with different siRNAs w
as followed by an infection with GFP-expressing MHV (MHV-EGFPM)at low multiplicity of infection (MOI),resulting in approximately 10–15%infected cells under control conditions.After 8h of infection cells were collected and GFP expression by the replication of MHV was analyzed by fluorescence-activated cell sorting (FACS).As controls siRNAs silencing GFP and negative-control siRNAs were ud.A hit from the screen was considered as confirmed when transfection with at least two out three independent siRNAs resulted in significant reduction in MHV-driven GFP expression relative to the negative-control siRNAs.siRNA-mediated gene silencing of ACTR2and ACTR3resulted in reduced infections for all three siRNAs,indicating that actin branching is important for MHV infection (Figure 1A,dark orange).Also the importance of the RAB7A,RAB7B and VPS39proteins,involved in late-endosome and late-endosome to lysosome maturation,for MHV infection could be confirmed (Figure 1A,turquoi and light green).The importance of SNX1,VCL and PAK1for infection of HeLa cells with MHV could not be confirmed (Figure 1A,grey).The latter three genes were not studied any further.To validate our
transfection protocol and confirm the efficacies of the siRNAs at the mRNA level,quantitative RT-PCR analysis was performed.All siRNAs ud reduced the corresponding mRNA levels with 75–95%(Figure 1B).siRNAs targeting RAB7A were shown to inhibit the expression of a RAB7a-fusion protein (Figure S1in Text S1).
炒更是什么意思
To confirm and extend our understanding of the role of endocytosis in MHV entry we subquently lected a number of proteins known to be involved in either caveolae-or clathrin-mediated endocytosis,actin-or microtubule-mediated transport,as well as proteins associated with endosomal vesicles and endosomal maturation,to be screened using the siRNA silenc-ing-approach described above.Again,proteins were considered important for infection with MHV when transfection with at least two out three independent siRNAs resulted in significant reduction in MHV-driven GFP expression relative to the negative-control siRNAs.siRNA-mediated downregulation of proteins involved in caveolae-mediated endocytosis revealed that CAV2,but not the other proteins analyzed are important for infection with MHV (Figure 1C,light blue).Downregulation of most proteins associ-ated with clathrin-mediated endocytosis inhibited MHV
infection,
Figure 1.RNAi-mediated downregulation of endocytosis-associated proteins affects MHV infection.A )
Confirmation of endocytosis-associated hits from druggable genome-wide siRNA screen.Gene silencing was performed using individual transfection of three different siRNAs per gene in HeLa-mCC1a cells.Cells were infected with MHV-EGFPM at MOI =0.5for 8h and analyzed by FACS for cell viability and virus replication.The effect of downregulation of expression on MHV infection was studied for the actin cytoskeleton-associated proteins ACTR2and ACTR3(orange),late endosomal proteins RAB7A and RAB7B (turquoi),HOPS complex sububit VPS39(light green),ER/Golgi cretion-associated protein SNX1,Integrin/Actin-associated protein VCL,and Serine/Threonine-protein kina PAK1(grey).Error bars reprent SEM,n =4.B )Confirmation of siRNA-mediated reduction in mRNA levels.mRNA levels at 72h post transfection were measured by qRT-PCR in comparison to non-transfected cells.Error bars reprent SEM,n =3*3.C )The effect of the RNAi-mediated downregulation of an extended t of endocytosis-associated proteins on MHV infection.Infection of MHV-EGFPM was analyzed after downregulation of proteins associated with caveolae-mediated endocytosis (light blue),clathrin-mediated endocytosis (dark blue),early endosomes (cerulean),actin cytoskeleton (dark orange),microtubule cytoskeleton (orange),late endosomes (turquoi),and late endosome-to-lysosome trafficking (light green)as described above.Error bars reprent SEM,n =3.A,C )Dotted lines show the lower 95%confidence interval of the negative siRNA controls.doi:10.1371/journal.ppat.1004502.g001
春节拜年的由来
including DNM1,DNM2,CLTC,and DAB2.siRNA-mediated silencing of EPS15or AAK1,accessory factors of clathrin-mediated endocytosis,did not affect MHV replication(Figure1C,dark blue). Silencing of early endosome-associated genes(EEA1,RAB5A, RAB5B,and RAB5C;Figure1C,cerulean)each decread replication-mediated GFP expression.While downregulation of MYO6,involved in actin-bad motility,did not influence MHV infection(Figure1C,dark orange),our results indicate that the microtubule-associated motility proteins DYNC1H1and DYNC2H1are important for infection with MHV(Figure1C, orange).Silencing of NSF,required for transport from early to late endosomes[58],or of the HOPS subunits VPS11and VPS41, which are involved in late endosome to lysosome maturation (Reviewed in[54]),all resulted in verely reduced MHV infection (Figure1C,turquoi and light green,respectively). Endocytosis-affecting agents indicate clathrin-mediated endocytosis and endosome maturation to be important in MHV infection
To further explore the endocytic route and factors involved in MHV infection we determined the effect of inhibitors on MHV infection.HeLa-mCC1a cells were treated with endocytosis-affecting agents for30min and then infected with lucifera-expressing MHV(MHV-EFLM;[59])in prence of the inhibitors,after which the inhibitors were kept prent until cell lysis.When cells were inoculated with
MHV-EFLM in the abnce of inhibitors,the inhibitors were added to the cells at2h post infection(hpi)to asss effects of inhibitors on post-entry steps.At
7hpi cells were lyd and firefly lucifera expression levels were determined.节能量
Infection in the prence of the solvents dimethyl sulfoxide (DMSO)and methanol(MeOH),as well as the known inhibitors of MHV RNA synthesis Brefeldin A(BrefA,inhibitor of GBF1)[60] and MG132(proteasome inhibitor,probably also affects MHV entry;[61])were included as controls.MHV infection was not affected by addition of the solvents,whereas both MG132and BrefA verely decread lucifera expression regardless of the time of addition.Inhibition of endosome maturation with ammonium chloride(NH4Cl),Bafilomycin A1(BafA1),or Chloroquine(Chloq)verely diminished lucifera expression when the inhibitors were added prior to infection.Much smaller effects were obrved when the drugs were added at2hpi, indicating that the inhibitors mainly affect MHV entry(Figure2, deep sky blue).Similar effects were obrved with known inhibitors of clathrin-mediated endocytosis;Chlorpromazine(Chlopro), Monensin(Mon),Dynasore,and Dyngo-4A(Dyngo).All the compounds strongly decread MHV replication-mediated lucif-era expression when added early but not when added at2hpi (Figure2,dark blue).The actin-and macropinocytosis-affecting drug EIPA,which inhibits the Na+/H+exchanger NHE1,led to reduced lucif
era expression both when added prior to and after entry of MHV at2hpi.Actin cytoskeleton altering drugs Latrunculin A(LatA),Jasplakinolide(Jasp),Cytochalasin B (CytoB),and Cytochalasin D(CytoD),or the inducer of microtubule depolymerization Nocodazole(Noc)only decread MHV infection when added early,indicating a role for the actin and microtubule cytoskeleton in entry but not RNA replication (Figure2,dark orange and orange).Likewi U18666A,a cholesterol transport-affecting agent,which also prevents matura-tion of late endosomes[62],had a strong inhibitory effect on MHV infection when added early(Figure2,turquoi).Collec-tively,the results indicate an important role for clathrin-mediated uptake and for endosome-and endosome-to-lysosome maturation for MHV infection.Clathrin-mediated endocytosis and late endosomal factors are required for MHV fusion
The time-of-addition experiments with the different inhibitors indicated that particularly the entry step of the MHV infection cycle is negatively affected by perturbation of clathrin-mediated endocytosis or of endosome maturation.However,assays bad on reporter gene expression driven by virus replication do not allow discrimination between virus entry and RNA replication when analyzing siRNAs or agents that also affect RNA synthesis.To unequivocally demonstrate the importance of clathrin-mediated endocytosis and endosome maturation for MHV entry,we therefore made u of a f
usion assay we recently developed[63]. The assay is bad on minimal complementation of defective b-galactosida(b-galactosida D M15)with the short a-peptide [64].MHV-a N,a recombinant MHV containing an N protein tagged with the a-peptide(a N),is ud to infect D M152fragment expressing target cells.Upon fusion of the virion with a host cell membrane a N is relead into the cytoplasm resulting in complementation of the defective b-galactosida thereby recon-stituting a functional enzyme.Conversion of the non-fluorescent substrate fluorescein-di-b-D-galactopyranoside(FDG)by b-galac-tosida into green fluorophores fluorescein(FIC)can be measured by FACS or fluorescence microscopy(Figure S2in Text S1).
To analyze the effect of RNAi-mediated gene silencing on fusion,HeLa cells expressing the MHV receptor and the D M152 fragment(HeLa-mCC1a-D M15cells)were transfected with individual siRNAs and inoculated with MHV-a N at72h post transfection.Before infection cells were pre-loaded with FDG by hypotonic shock.After100min incubation of cells with virus at 37u C,cells were collected and the amount of FIC generated as a results of enzyme complementation analyzed by FACS.The fusion assay showed that silencing of neither CAV1nor CAV2affected MHV fusion(Figure3A,light blue),even though reduction of CAV2was shown to affect MHV infection(Figure1C).However, downregulation of clathrin-mediated endocytosis associated
pro-Figure 2.Endocytosis affecting agents indicate clathrin-mediated endocytosis and endosome ma
turation to be important in MHV infection.HeLa-mCC1a cells,inoculated with MHV-EGFPM at MOI=0.5,were treated with the different inhibitors from30min prior to8h post inoculation(0–8h)or from2–8h post inoculation(2–8h;hatched bars):ammonium chloride(NH4Cl), Bafilomycin A1(BafA1),Chloroquine(Chloq),Chlorpromazine(Chlopro), Monensin(Mon),Dynasore,Dyngo-4A,EIPA,Latrunculin A(LatA), Jasplakinolide(Jasp),Cytochalasin B(CytoB),Cytochalasin D(DytoD), Nocodazole(Noc),MG132,Brefeldin A(BrefA),as well as solvents dimethyl sulfoxide(DMSO)and methanol(MeOH).Infection was determined by FACS and displayed relative to the infection level obrved in mock-treated cells(UNTR).Error bars reprent SEM,n=3. doi:10.1371/journal.ppat.1004502.g002
teins DNM2and CLTC lead to strongly decread fusion,as did the lack of early endosome-associated factors RAB5B and RAB5C (Figure 3A,dark blue and cerulean,respectively).Fusion was also affected by RNAi-mediated reduction of actin cytoskeleton-associated proteins ACTR2and ACTR3(Figure 3A,dark orange),proteins known to be involved in late endosome (RAB7A,RAB7B)and late endosome-to-lysosome maturation (VPS11,VPS39,and VPS41)(Figure 3A,turquoi and light green).
The importance of clathrin-mediated endocytosis and endo-some maturation for MHV fusion was co
nfirmed by analysis of endocytosis-affecting agents using the fusion assay.After pre-loading with FDG,cells were pre-treated with the inhibitors for 30min at 37u C,after which cells were inoculated with MHV-a N
in the prence of the agents,and analyzed by FACS as described above.As controls we included protein synthesis inhibitor cycloheximide (CHX),MHV fusion inhibitor peptide HR2(HR2,[20]),MG132and BrefA.Fusion of MHV was not affected by the solvents or CHX,the latter confirming that this assay is independent of RNA replication and protein synthesis.MHV fusion was barely affected by replication inhibitor BrefA,whereas MG132had a clear negative effect,in agreement with the conclusion drawn previously that MG132inhibits entry of MHV as well as RNA synthesis [61].Inhibition of endosomal maturation by NH 4Cl,BafA1and Chloq (Figure 3B,deep sky blue)or of clathrin-mediated endocytosis by Chlopro,Mon,and Dynasore (Figure 3B,dark blue)verely inhibited MHV fusion.Distur-bance of the actin cytoskeleton by EIPA or by LatA,CytoB,or CytoD reduced fusion by 75–80%(Figure 3B,dark orange),while interference with microtubule polymerization by Noc had a smaller effect (Figure 3B,orange).Late endosomal maturation arrest caud by U18666A reduced fusion to approximately 10%(Figure 3B,turquoi).In conclusion,the replication-independent fusion assay confirmed the importance of clathrin-mediated endocytosis and of endosome maturatio
n for entry of MHV.The data indicate that late endosome-to-lysosome maturation is required for efficient entry and fusion.
Live-cell microscopy confirms co-localization,co-tracking and fusion of MHV in endosomal compartments
To confirm the importance of endocytic uptake and the association of MHV with endosomal compartments we performed live-cell confocal microscopy.To this end,sucro density gradient-purified MHV virus was covalently labeled with the low-pH resistant dye DyLight 488(MHV-DL488).HeLa-mCC1a cells were transfected with plasmids to express monomeric RFP (mRFP)fusion proteins of RAB5,RAB7,or LAMP1.At 24h post transfection,MHV-DL488was bound to cells at 4u C for 90min.Inoculation medium was replaced by warm medium containing trypan blue,which immediately shifts the emission spectrum of surface bound particles rendering them undetectable in the 505–530nm channel unless they get endocytod [65].Cells were imaged using a spinning-disc confocal microscope acquiring z-stacks in 30s intervals over 10min time frames from 10–70min post warming.Only low-level RFP fusion protein expressing cells were lected for analysis.Interestingly,MHV particles newly appeared even 60min post warming,in agreement with the notion that MHV enters in an unsynchronized manner (unpub-lished results).Co-localization and
co-trafficking of virus with endosomal compartments was assd by detecting virus particles bad on size and intensity (green channel)and by measuring the underlying intensity in the red channel (endosomal vesicles).MHV virions were found to co-localize with all three endosomal compartments (Fig.4A).Whereas newly entering/appearing particles were always co-localizing with RAB5molecules,they only associated with RAB7and LAMP1containing vesicles at later time points.
To asss the association of MHV with endosomal vesicles during the entry process more extensively,we manually tracked the virus particles in the green channel and independently tracked the endosomal vesicles in the red channel in x/y and z-direction.A virion was categorized as associating with a certain endosomal marker only if this co-localization was obrved over at least four quential 30s interval images.When the initial co-localization was lost,but the virion did not disappear,this virion was classified as associating/dissociating.Complete disappearance of a virus particle (including in other z-stacks)while immediately previously co-localizing with an endosomal marker was categorized as
a
Figure 3.Clathrin-mediated endocytosis and late endosome-to-lysosome trafficking is required for MHV fusion.A )Fusion assay upon siRNA-mediated gene silencing.Three different siRNAs per gene were transfected individually into HeLa-mCC1a-D M15.72h post transfection,cells were pre-loaded with FDG by hypotonic shock.MHV-a N was allowed to bind to the cells on ice at MOI =20for 90min.100min post warming to 37u C,cells were collected and analyzed by FACS.Fusion was determined relative to the number of FIC-positive cells obrved upon mock treatment of infected cells (UNTR).Error bars reprent SEM,n =3.B )Fusion of MHV upon treatment of cells with different inhibitors was studied as in A.Cells were pretreated with ammonium chloride (NH4Cl),Bafilomycin A1(BafA1),Chloroquine (Chloq),Chlorpromazine (Chlopro),Monensin (Mon),Dynasore,Dyngo-4A,EIPA,Latrunculin A,(LatA),Jasplakinolide (Jasp),Cytochala-sin B (CytoB),Cytochalasin D (DytoD),Nocodazole (Noc),U18666A,MG132,Brefelding A (BrefA),as well as with the solvents dimethyl sulfoxide (DMSO)and methanol (MeOH),protein synthesis inhibitor cyclohexamide (CHX),and MHV fusion inhibitor HR2peptide (HR2)for 30min at 37u C.The inhibitors were kept prent during binding of MHV-a N to cells and during warming to 37u C cells for 100min.Fusion was determined relative to the number of FIC-positive cells after mock treatment (UNTR).Error bars reprent SEM,n =3.doi:10.1371/journal.ppat.1004502.g003
fusion event(Figures S3and S4in Text S1).When a viral particle co-localized with endosomal compartments but did neither dissociate nor fade during the10min acquisition period it was classified as non-fusing.With this quantification method we analyzed75–100virions in total for each of the endosomal compartment types studied.The fraction of virions not fusing during the acquisition period was consistently found to be at around10–15%.We obrved that all of the entering MHV particles initially co-localized with RAB5-positive early endosomal vesicles and that most virions dissociated(were no longer co-localized)after4–6min.Notably,it appeared that in the events the RAB5marker faded rather than moved away.Only a very small percentage of virions were categorized as fusing while in early endosomes.The number of fusion events was much higher for virions co-localizing with RAB7or LAMP1(Figure4B), indicating that most virions fu in late endosomes or lysosomes. MHV infection depends on endosomal maturation
Our results so far indicate that most virions enter cells after having accesd late endosomes/lysosomes.We hypothesized that the compartments provide the environmental cues required for productive virus-cell fusion.In order to analyze to what extent the low pH in the endosomal system is required for entry of MHV,we analyzed the inhibition of MHV entry at different concentrations of BafA1.While high concentrations of BafA1(as ud for the results shown in Fig.2an
d3)affect endosomal maturation,at low concentrations this inhibitor of vacuolar-type H+-ATPa only elevates the pH of endosomal compartments but does not affect endosomal trafficking per [66].We made u of that property and tested the nsitivity of MHV to BafA1side by side with the control virus VSV and IAV.VSV has been described to fu at pH6.2in early and/or late endosomes[9,11,12,67–69],while IAV has been shown to fu in late endosomes at an even lower pH[9,10,70].HeLa or HeLa-mCC1a cells were pretreated with increasing concentrations of BafA1for30min prior to infection with reporter gene expressing virus:VSV(VSV D G/FLuc-G*; [71,72]),IAV(IAV-RLuc;[73]),or MHV(MHV-EFLM). Lucifera expression levels indicated that infection of cells with VSV and IAV is much more affected by BafA1,with an IC50 values of0.80and0.63nM,respectively,compared to MHV, which displays a three to four fold higher IC50of 2.34nM (Figure5A).
Our results thus indicate that MHV is much less affected by perturbation of the endosomal pH than VSV and IAV. Nevertheless RNAi-mediated silencing of HOPS subunits and treatment of cells with U1866A indicates that late endosome-to-lysosome maturation is required for efficient entry.To
confirm Figure 4.Live-cell microscopy demonstrates co-localization
and co-tracking of MHV with endosomal vesicles and fusion of
MHV in the vesicles.HeLa-mCC1a cells transfected with plasmids
encoding RAB5-mRFP,RAB7-mRFP,or dsRed-LAMP1were inoculated
with DyLight488-labeled MHV.Live cell imaging was performed to
track internalized particles.A)Examples of MHV particles co-localizing
with RAB5-,RAB7-,and LAMP1-positive endosomal vesicles.Size bars
indicate0.2m M B)Virus particles that could be tracked were classified
as‘fusing’(Fusing)‘associating/dissociating’(Assoc/Dissoc),or‘non-
fusing’(Non-fusing)as described in the Materials and Methods ction.
doi:10.1371/journal.ppat.1004502.g004
Figure5.MHV infection depends on endosomal maturation.A)
HeLa-mCC1a cells were pretreated with increasing concentrations of
Bafilomycin A1(BafA1)for30min and subquently infected with
lucifera expressing MHV,VSV,or IAV in the prence of BafA1.
Infection levels were determined by assaying the lucifera activity in
cell lysates relative to lysates of infected cells that had been mock
treated.Error bars reprent SEM,n=3*3.B)Haploid cells(HAP1),
haploid cells lacking VPS33A(H1-D V33)or VPS33A-lacking haploid cells
retransfected with FLAG-tagged VLP33A(H1-D V33-fV33)were infected
with lucifera expressing MHV,VSV,or IAV.Cells were lyd at7h
(MHV and VSV)or16h post infection.Infection is displayed relative to
virus-driven lucifera expression levels in HAP1cells.Error bars
reprent SEM,n=3*3.
doi:10.1371/journal.ppat.1004502.g005
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