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Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core–NS2 and NS5A of genotypes 1–7

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Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core–NS2 and NS5A of genotypes 1–7
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  Analysis of hepatitis C virus core/NS5A proteinco-localization using novel cell culture systemsexpressing core–NS2 and NS5A of genotypes 1–7 Andrea Galli, Troels K. H. Scheel, Jannick C. Prentoe, Lotte S. Mikkelsen,Judith M. Gottwein and Jens Bukh Correspondence Jens Bukh jbukh@sund.ku.dkReceived 26 March 2013Accepted 24 July 2013 Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and ClinicalResearch Centre, Copenhagen University Hospital, Hvidovre, and Department of InternationalHealth, Immunology and Microbiology, Faculty of Health and Medical Sciences, University ofCopenhagen, Copenhagen, Denmark Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the adventof infectious cell culture systems, the HCV particle assembly and release processes are finallybeing uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets(cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Currentknowledge on assembly and release is primarily based on studies in genotype 2a cell culturesystems; however, given the high genetic heterogeneity of HCV, variations might exist amonggenotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core–NS2 and NS5A from genotypes 1–7, and analysed core and NS5A co-localization in infectedcells. Huh7.5 cells were transfected with RNA of core–NS2/NS5A recombinants and putativeadaptive mutations were analysed by reverse genetics. Adapted core–NS2/NS5A recombinantsproduced infectivity titres of 10 2.5 –10 4.5 f.f.u. ml ” 1 . Co-localization analysis demonstrated that thecore and NS5A proteins from all genotypes co-localized extensively, and there was no significantdifference in protein co-localization among genotypes. In addition, we found that the core andNS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ERassociated at later stages. Finally, we found that different genotypes showed varying levels ofcore/cLD co-localization, with a possible effect on viral assembly/release. In summary, wedeveloped a panel of HCV genotype 1–7 core–NS2/NS5A recombinants producing infectiousvirus, and an immunostaining protocol detecting the core and NS5A proteins from seven differentgenotypes. These systems will allow, for the first time, investigation of core/NS5A interactionsduring assembly and release of HCV particles of all major genotypes. INTRODUCTION Hepatitis C virus (HCV) is a major cause of severe liverdisease such as chronic hepatitis, liver cirrhosis andhepatocellular carcinoma (Alter & Seeff, 2000). Due to itshigh degree of genetic diversity, HCV has been classifiedinto seven major genotypes and several subtypes based onsequence homology (Bukh  et al. , 1993; Simmonds  et al. ,1993, 2005). The virus particle contains a positive-sense ssRNA genome, which encodes a single ORF flanked by 5 9 -and 3 9 -untranslated regions (UTRs). The ORF encodes apolyprotein that is processed by viral and cellular proteasesto produce structural proteins (core, E1 and E2), p7 andnon-structural proteins (NS2, NS3, NS4A/B and NS5A/B)(Gottwein & Bukh, 2008). The entire viral life cycle of HCVcould not be studied  in vitro   until 2005, when the first cellculture system based on the genotype 2a strain JFH1 wasdeveloped (Wakita  et al. , 2005). J6/JFH1 recombinants,produced by replacing the core–NS2 region of JFH1 with thecorresponding region derived from the genotype 2a J6CFisolate (Yanagi  et al. , 1999), produced higher infectivity titres and did not rely on culture adaptation (Gottwein  et al. ,2007; Lindenbach  et al. , 2005; Pietschmann  et al. , 2006).Several other JFH1-based recombinants, carrying the core–NS2 region from the seven major genotypes, were subse-quently developed (Gottwein  et al. , 2007, 2009; Jensen  et al. ,2008; Pedersen  et al. , 2013; Pietschmann  et al. , 2006; Scheel et al. , 2008, 2011a; Yi  et al. , 2007). These constructs havebeen useful for studying the effect of neutralizing antibodiesand antiviral drugs against diverse HCV genotypes, and forfunctional studies of core–NS2 interactions (Gottwein  et al. ,2009, 2011; Griffin  et al. , 2008; Pedersen  et al. , 2013; Prentoe et al. , 2011). In addition, we developed a panel of recombinants carrying the NS5A region from genotypes Two supplementary figures and seven tables are available with theonline version of this paper. Journal of General Virology   (2013),  94,  2221–2235  DOI  10.1099/vir.0.053868-0053868 G 2013 SGM  Printed in Great Britain  2221  1–7 in the J6/JFH1 backbone, with the purpose of analysingthe effect of newly developed NS5A inhibitors on differentstrains of HCV and for functional genotype-specific studiesof NS5A (Scheel  et al. , 2011b, 2012). However, at the onset of this study, efficient culture systems using recombinantscombining strain-specific structural proteins and NS5A wereonly available for genotype 2a isolates (Scheel  et al. , 2011b;Wakita  et al. , 2005; Zhong  et al. , 2005).The availability of genotype 2a HCV cell culture systemshas allowed the study of essential steps of the viralreplication cycle, including viral assembly and release(Bartenschlager  et al. , 2011; Lindenbach, 2013). To produce infectious viruses, newly synthesized HCV gen-omes need to be delivered to the site of assembly, probably the surface of cytoplasmic lipid droplets (cLDs) or thecytosolic side of the endoplasmic reticulum (ER), andpackaged into the nucleocapsid (Appel  et al. , 2008;Miyanari  et al. , 2007; Shavinskaya  et al. , 2007). The coreand NS5A proteins play essential roles in the early stage of virion assembly (Masaki  et al. , 2008) and are among thebetter-understood proteins involved in particle formation.The core protein is the main constituent of the viralnucleocapsid, which packages the genomic RNA forparticle release. NS5A is a phosphoprotein involved inthe formation of the viral replication complex and has beenshown to possess RNA-binding capacity (Huang  et al. ,2005; Tellinghuisen  et al. , 2005), suggesting that it could beinvolved in the transport of viral genomic RNA to theassembly site (Miyanari  et al. , 2007). After synthesis, coreprotein is mobilized to the surface of cLDs, where it isthought to initiate particle assembly (McLauchlan  et al. ,2002; Miyanari  et al. , 2007). The extent of core proteinaccumulation on the surface of cLDs is inversely correlatedwith the efficiency of particle assembly (Boulant  et al. ,2007; Shavinskaya  et al. , 2007), probably reflecting atransient localization of core protein on cLDs prior to itstransfer to the ER where the assembly process can proceed(Boson  et al. , 2011). The core and NS5A proteins co-localize extensively in the cytoplasm and their interaction iscritical for the production of viral particles (Masaki  et al. ,2008; Miyanari  et al. , 2007). Moreover, both localize on thesurface of cLDs and the extent of core/NS5A co-localization on cLDs is also inversely correlated with theefficiency of virion production (Appel  et al. , 2008). NS2 is amajor player in the subsequent stages of assembly (Jirasko et al. , 2008; Ma  et al. , 2011; Popescu  et al. , 2011). Thenascent viral particle subsequently enters the ER lumen,where it associates with E1/E2 through a not fully definedmechanism and interacts with very-low-density lipopro-teins to form lipoviroparticles (Gastaminza  et al. , 2008;Huang  et al. , 2007). Mature virions are then releasedthrough the secretory pathway via the Golgi network.Several aspects of these processes remain unclear, and ourknowledge of the factors implicated and their interaction isincomplete. Moreover, these processes have been only elucidated in experiments with genotype 2a viruses, and thegenetic divergence between HCV isolates could result invariation in the assembly and release pathways amongdifferent genotypes.Here, we developed novel cell culture-adapted recombi-nants with genotype 1–7-specific core–NS2/NS5A in theJFH1 backbone. We demonstrated that core and NS5Aproteins from all recombinants could be visualized by immunofluorescence microscopy, and displayed similarco-localization patterns irrespective of the HCV genotype.The developed culture systems are suitable for investigationof the assembly of HCV particles in the context of differentgenotypes, since they carry isolate-specific versions of mostviral proteins involved in particle production and release. RESULTS Development of HCV recombinants carryinggenotype-specific core–NS2 and NS5A regions Most functional studies of HCV in the context of thecomplete viral life cycle have relied on genotype 2a cellculture systems. To enable genotype-specific studies of interactions between NS5A and the structural HCVproteins for all seven major HCV genotypes, we developedcore–NS2/NS5A recombinants containing the core–NS2and NS5A regions from prototype isolates of genotypes 1a,1b, 3a, 4a, 5a, 6a and 7a (Bukh  et al. , 2010; Gottwein  et al. ,2009; Sakai  et al. , 2007). Thus, we replaced the completeNS5A of the following JFH1-based core–NS2 recombinantsthat also carried culture-adaptive mutations with thecorresponding strain-specific NS5A consensus sequence:H77C/JFH1 V787A,Q1247L  (1a), TN/JFH1 R1408W  (1a), J4/JFH1 F886L,Q1496L  (1b), S52/JFH1 I787S,K1398Q  (3a), ED43/JFH1 T827A,T977S  (4a), SA13/JFH1 A1021G,K1118R   (5a), HK6a/JFH1 F349S,N417T  (6a) and QC69/JFH1 L878P  (7a) (all num-bering is according to the H77 reference polyprotein,GenBank accession no. AF009606) (Gottwein  et al. , 2007,2009; Jensen  et al. , 2008; Scheel  et al. , 2008, 2011a). Such core–NS2/NS5A recombinants could potentially pro-duce higher titres compared with previously developedgenotype-specific core–NS2 and/or NS5A recombinants(Gottwein  et al. , 2009; Scheel  et al. , 2011b), since efficientvirus production could depend on genotype-specificinteractions between NS5A and core. In vitro  -transcribed RNA corresponding to the HCVgenome of core–NS2/NS5A genotype 1–7 recombinantswas transfected into Huh7.5 hepatoma cells in parallelwith RNA of the genotype 2a recombinant, J6/JFH1(Lindenbach  et al. , 2005). One day after transfection, formost recombinants 10–30% of cells expressed HCV coreprotein, as determined by immunostaining. However, forthe ED43(4a), SA13(5a) and QC69(7a) recombinants, only 1–5% of cells were positive (Fig. 1). J6/JFH1 infected mostof the culture after 3–6 days, while the same was observedfor S52(3a), HK6a(6a) and QC69(7a) after 6–12 days, andfor H77C(1a), TN(1a), J4(1b), ED43(4a) and SA13(5a)after 18–39 days (Fig. 1). Only the QC69 recombinant A. Galli and others2222  Journal of General Virology   94  produced infectivity titres above 10 3 f.f.u. ml 2 1 during thefirst 10 days after transfection, although delayed around5 days compared with J6/JFH1 (Fig. 2). Since mutationsadapting the previously developed core–NS2 recombinantscould potentially compensate for interactions with JFH1NS5A and could thus be a disadvantage for core–NS2/NS5Arecombinants with strain-specific NS5A, we tested ED43(4a)and SA13(5a) core–NS2/NS5A without such mutations.However, this led to no spread of infection for ED43(4a) andslower spread of infection for SA13(5a) (Fig. 1). (a)755025100    C  o  r  e  -  p  o  s   i   t   i  v  e   H  u   h   7 .   5  c  e   l   l  s   (   %   ) 00 403632282420 Time p.t. (days)Time p.t. (days)Time p.t. (days)Time p.t. (days)Time p.t. (days)Time p.t. (days)161284 44 (c)755025100    C  o  r  e  -  p  o  s   i   t   i  v  e   H  u   h   7 .   5  c  e   l   l  s   (   %   ) 0(e)755025100    C  o  r  e  -  p  o  s   i   t   i  v  e   H  u   h   7 .   5  c  e   l   l  s   (   %   ) 0(f)755025100    C  o  r  e  -  p  o  s   i   t   i  v  e   H  u   h   7 .   5  c  e   l   l  s   (   %   ) 0(d)755025100    C  o  r  e  -  p  o  s   i   t   i  v  e   H  u   h   7 .   5  c  e   l   l  s   (   %   ) 0(b)755025100    C  o  r  e  -  p  o  s   i   t   i  v  e   H  u   h   7 .   5  c  e   l   l  s   (   %   ) 00 403632282420161284 44 0 403632282420161284 44 0 403632282420161284 440 403632282420161284 44 0 403632282420 J6/JFH1ED43(4a) T827A,T997S  ED43(4a) T827A,T977S,Y1644H,E2267G ED43(4a) (no mutations)J4(1b) F886L,Q1496L TN(1a) R1408W J4(1b) F886L,C1185S,Q1496L TN(1a) I1312V,R1408W J6/JFH1H77(1a) V787A,Q1247L  H77(1a) V787A,C1185S,Q1247L J6/JFH1S52(3a) I787S,K1398Q  S52(3a) I787S,K1398Q,C2419R J6/JFH1HK6a(6a) F349S,N417T  HK6a(6a) F349S,N417T,I2268N J6/JFH1QC69(7a) L878P J6/JFH1SA13(5a) A1021G,K1118R  SA13(5a) A1021G,K1118R,R1978G,C2419R SA13(5a) (no mutations)161284 44 Fig. 1.  Evaluation of HCV antigen-positive Huh7.5 cells after transfection of JFH1-based recombinants with genotype-specificcore–NS2/NS5A. The percentage of positive cells in culture was evaluated by staining with anti-core antibody after transfectionwith RNA transcripts of recombinants of isolates(genotypes): (a) H77C(1a), TN(1a) and J4(1b), (b) S52(3a), (c) ED43(4a), (d)SA13(5a), (e) HK6a(6a) and (f) QC69(7a). Each panel contains data from one or more experiments and includes arepresentative J6/JFH1 control. Selected recombinants were tested in multiple experiments. Numbering of mutations isaccording to the H77 reference polyprotein (GenBank accession no. AF009606). p.t., Post-transfection. Co-localization of HCV core and NS5A of genotypes 1–7http://vir.sgmjournals.org 2223  (a)(c) (d)(b)435 <2.3 J6/JFH1    R  1  4  0  8   W   V   7  8   7  A ,    Q  1  2  4   7   L   F  8  8  6   L ,    Q  1  4  9  6   L   I   7  8   7  S ,     K  1  3  9  8  Q   I   7  8   7  S ,     K  1  3  9  8  Q ,    C  2  4  1  9   R   F  8  8  6   L ,    Q  1  4  9  6   L ,    C  1  1  8   5  S   V   7  8   7  A ,    Q  1  2  4   7   L ,    C  1  1  8   5  S   R  1  4  0  8   W ,     I  1  3  1  2   V TN(1a) J6/JFH1 H77C(1a) J4(1b) S52(3a)J6/JFH1 SA13(5a) QC69(7a)   A  1  0  2  1  G ,     K  1  1  1  8   R   F  3  4  9  S ,     N  4  1   7   T   F  3  4  9  S ,     N  4  1   7   T ,     I  2  2  6  8   N  A  1  0  2  1  G ,     K  1  1  1  8   R ,    R  1  9   7  8  G ,    C  2  4  1  9   R   L  8   7  8   P J6/JFH1 HK6a(6a)    I  n   f  e  c   t   i  v   i   t  y   t   i   t  r  e   [   l  o  g    1   0    (   f .   f .  u .  m   l   –   1    )   ] 435 <2.3    I  n   f  e  c   t   i  v   i   t  y   t   i   t  r  e   [   l  o  g    1   0    (   f .   f .  u .  m   l   –   1    )   ] 435 <2.3    I  n   f  e  c   t   i  v   i   t  y   t   i   t  r  e   [   l  o  g    1   0    (   f .   f .  u  m   l   –   1    )   ] 4Day 6Day 8Day 3Day 1035 <2.3    I  n   f  e  c   t   i  v   i   t  y   t   i   t  r  e   [   l  o  g    1   0    (   f .   f .  u .  m   l   –   1    )   ] Fig. 2.  Infectivity titres after transfection of Huh7.5 cells with JFH1-based recombinants expressing genotype-specific core–NS2/NS5A. Infectivity was measured in supernatants from the first 10 days after transfection with RNA transcripts ofrecombinants of isolates(genotypes): (a) TN(1a), (b) H77C(1a), J4(1b), S52(3a), (c) SA13(5a), QC69(7a) and (d) HK6a(6a).The ED43 recombinants with and without Y1644H/E2267G mutations spread slowly to the majority of culture cells (22–27 and17 days, respectively), and are therefore not shown. For comparison, J6/JFH1 was included in each separate experiment as acontrol. Coloured boxes indicate the core–NS2 and NS5A isolate of the given recombinant. Numbering of mutations isaccording to the H77 reference polyprotein (GenBank accession no. AF009606). All recombinants were passaged to naı ¨ veHuh7.5 cells to obtain full ORF sequences (Tables S1–S7). The lower limit of detection in the experiments shown was up to10 2.3 f.f.u. ml ” 1 ; titres below this level are shown as  , 2.3 log 10 (f.f.u. ml ” 1 ). Error bars indicate  SEM  of triplicate titredeterminations. A. Galli and others2224  Journal of General Virology   94  After passage to naı¨ve Huh7.5 cells, infectivity titres above10 3 f.f.u. ml 2 1 were observed for all recombinants, exceptfor J4(1b) and ED43(4a). Viral RNA was extracted fromsupernatants with peak HCV infectivity titres and sequencedto obtain the near full-length genomic sequence, includingthe entire ORF. Putative adaptive mutations were identifiedin all recombinants, except QC69, that apparently did notdepend on additional adaptation (Tables S1–S7, availablewith the online version of this paper). Generation of cell-culture-adapted core–NS2/NS5A recombinants by reverse genetic analysis Selected mutations observed for the core–NS2/NS5A recombi-nants were tested in reverse genetic studies for H77C(1a),TN(1a), J4(1b), S52(3a), SA13(5a) and HK6a(6a) (Figs 1 and2). While the SA13(5a) A1021G,K1118R,R1978G,C2419R   mutant imme-diately produced titres comparable to J6/JFH1 in transfectioncultures, the H77C(1a) V787A,C1185S,Q1247L , TN(1a) I1312V,R1408W, S52(3a) I787S,K1398Q,C2419R   and HK6a(6a) F349S,N417T,I2268N mutants produced titres around 10 3 f.f.u. ml 2 1 , andJ4(1b) F886L,C1185S,Q1496L  produced titres around 10 2.5 f.f.u.ml 2 1 (Table 1). Since the ED43(4a) core–NS2/NS5A recom-binant did not adapt to efficient titre production during threeserial passages to naı¨ve cells, mutations identified in recoveredvirus (Table S5) were not tested in reverse genetic studies.We instead inserted the Y1644H and E2267G substitu-tions previously shown to adapt a J6/JFH1-based recombinantwith ED43 NS5A (Scheel  et al. , 2011b). TheED43(4a) T827A,T977S,Y1644H,E2267G  recombinant produced titresaround 10 2.5 f.f.u. ml 2 1 (Table 1) 22 days after transfection.After passage of the final genotype 1–7 core–NS2/NS5Arecombinants to naı¨ve Huh7.5 cells, peak HCV RNA titreswere around 10 7 IU ml 2 1 (Table 1). Peak HCV infectivity titres were around 10 4 f.f.u. ml 2 1 for S52(3a), SA13(5a)and QC69(7a), 10 3.5 f.f.u. ml 2 1 for H77C(1a) andHK6a(6a), and below 10 3 f.f.u. ml 2 1 for TN(1a), J4(1b)and ED43(4a) (Table 1). No additional mutations wereidentified in the recovered viruses by sequencing of thecomplete ORF, except for the TN(1a) and ED43(4a)(Tables S1–S7). Thus, we developed a genotype-specificpanel of core–NS2/NS5A recombinants producing infec-tious virus, which could be useful for functional studies.Except for the SA13(5a) and QC69(7a) recombinants, virusproduction was lower for these recombinants comparedwith previously developed J6/JFH1-based NS5A recombi-nants (Scheel  et al. , 2011b), and JFH1-based core–NS2recombinants (Gottwein  et al. , 2009; Scheel  et al. , 2011a). Validation of labelling efficiency of core andNS5A proteins from HCV genotypes 1–7 ininfected Huh7.5 cells Properevaluationofco-localizationofcoreandNS5Afromthedifferent HCV recombinants relies on comparable efficiency of protein labelling and signal intensity across genotypes.Therefore, we initially performed single-staining of core andNS5A from Huh7.5 cells infected with each core–NS2/NS5A recombinant to verify reactivity of primary anti-bodies against different genotypes. Supernatants containingpassaged H77C(1a) V787A,C1185S,Q1247L , TN(1a) I1312V,R1408W ,J4(1b) F886L,C1185S,Q1496L , J6/JFH1, S52(3a) I787S,K1398Q,C2419R  ,ED43(4a) T827A,T977S,Y1644H,E2267G , SA13(5a) A1021G,K1118R  , R1978G,C2419R  ,HK6a(6a) F349S,N417T,I2268N  and QC69(7a) L878P  viruses (Table1) were used to infect naı¨ve Huh7.5 cells. Cells were fixed at48 h post-infection and stained using primary antibodies Table 1.  Characteristics of culture-adapted core–NS2/NS5A HCV recombinants Core–NS2/NS5A Engineered mutations* Peak titres D  Pearson’scoefficient d Genotype Isolate Transfection Viral passagelog  10 (f.f.u. ml ” 1 ) log  10 (f.f.u. ml ” 1 ) log  10 (IU ml ” 1 ) 1a H77C V787A,  C1185S , Q1247L 2.9 3.5 7.5 0.82TN I1312V, R1408W 2.7 2.5 6.8 0.771b J4 F886L,  C1185S , Q1496L 2.5 1.8 7.3 0.732a J6/JFH1§ 4.2 5.0 7.6 0.773a S52 I787S, K1398Q,  C2419R  3.5 3.8 7.7 0.804a ED43 T827A, T977S,  Y1644H ,  E2267G  2.6 2.3 7.2 0.795a SA13 A1021G, K1118R,  R1978G ,  C2419R  4.5 3.8 7.1 0.786a HK6a F349S, N417T,  I2268N  3.0 3.3 7.1 0.757a QC69 L878P 3.4 4.1 7.7 0.76*Mutations are numbered according to the H77 reference polyprotein (GenBank accession no. AF009606). Sequencing results of virus recoveredfrom supernatants are shown in Tables S1–S7. Mutations introduced in this study are shown in bold. D Representative infectivity (f.f.u.) and RNA (IU) titres observed in supernatants from transfection and passages to naı¨ve cells are indicated. d Pearson’s coefficients represent mean values calculated using image stacks from three to five infected cells per sample, after image alignment andthree-dimensional (3D) deconvolution.§J6/JFH1 carries the core–NS2 region from J6CF and the NS5A gene from JFH1. Titre data shown are from Gottwein  et al.  (2009). Co-localization of HCV core and NS5A of genotypes 1–7http://vir.sgmjournals.org 2225
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