【病毒外文文獻】2009 A two-pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1 protein

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1134 VOLUME 16 NUMBER 11 NOVEMBER 2009 nature structural expressed SCoV nsp1 induces host mRNA degradation and suppresses host translation 8 The expressed nsp1 suppresses the host antiviral signaling pathways as well 9 Furthermore nsp1 suppresses host gene expression including type I interferon IFN production by promoting host mRNA degradation and host translation suppres sion in infected cells 10 The nsp1 of a closely related mouse hepatitis virus also suppresses host gene expression and is a viral virulence factor 11 These data suggest that the SCoV nsp1 mediated suppression of host genes is important in the pathogenesis of SARS Accordingly a delineation of the mechanisms of the nsp1 induced suppression of host gene expression is important for providing insight into SCoV pathogenesis at the molecular level We designed the present study to uncover the mechanism of nsp1 induced suppression of host gene expression primarily by using an in vitro system Our data revealed that nsp1 uses a novel two pronged strategy to inhibit host transla tion and gene expression RESULTS Nsp1 suppresses translation in vitro We tested whether nsp1 protein could suppress translation in rabbit reticulocyte lysate RRL We expressed the full length wild type nsp1 protein and its mutant form nsp1 mt carrying K164A and H165A mutations as glutathione S transferase GST tagged fusion proteins in Escherichia coli followed by the removal of the GST tag to gener ate recombinant nsp1 and nsp1 mt proteins respectively nsp1 mt neither suppresses host translation nor promotes host mRNA degra dation in expressing cells and infected cells 10 After incubation of the nsp1 protein with RRL at 4 C for 30 min we added different con centrations of capped and polyadenylated Renilla luciferase mRNA transcripts rLuc RNA to the mixture and incubated the samples in the presence of 35 S methionine for 30 min In control samples in which rLuc RNA was incubated with GST and nsp1 mt proteins rLuc activity and labeled rLuc protein abundance increased with rising mRNA concentrations In contrast the rLuc activity and labeled rLuc protein abundance in the nsp1 containing sample were substantially lower than in the control samples the rLuc activity and the radio labeled rLuc protein abundance in the presence of nsp1 were about 6 8 of the levels observed with GST or nsp1 mt Fig 1a b which clearly demonstrates that nsp1 efficiently inhibited the rLuc protein synthesis from capped rLuc RNA in RRL Next we examined the effect of nsp1 on translation mediated by the internal ribosome entry sites IRES using in vitro synthesized bicistronic mRNAs Ren HCV FF RNA and Ren CrPV FF RNA in which expression of the upstream rLuc open reading frame ORF was mediated by cap dependent translation and the translation of 1 Department of Microbiology and Immunology University of Texas Medical Branch at Galveston Galveston Texas USA 2 Present address Global Centers of Excellence Program Research Institute for Microbial Diseases Osaka University Osaka Japan 3 These authors contributed equally to this work Correspondence should be addressed to S M shmakino utmb edu Received 6 January accepted 21 August published online 18 October 2009 doi 10 1038 nsmb 1680 A two pronged strategy to suppress host protein synthesis by SARS coronavirus Nsp1 protein Wataru Kamitani 1 3 Cheng Huang 1 3 Krishna Narayanan 1 Kumari G Lokugamage 1 all of these encoded proteins carried a C terminal myc His tag At 8 h after transfection we prepared the cell extracts and performed polysome profile analysis Fig 2a c Expression of CAT and nsp1 mt yielded similar polysome profiles whereas nsp1 expression resulted in the accumulation of 80S monosomes along with a substantial reduction in the polysome abundance in accordance with the observation that the expressed nsp1 but not nsp1 mt suppresses host translation 10 Western blot analysis of the resolved gradient fractions showed that the majority of nsp1 but not CAT and nsp1 mt sedimented with the 40S ribosomal subunit Fig 2b Likewise only nsp1 sedimented with the 40S ribosomal sub unit in RRL Fig 2d f To confirm the interaction of nsp1 with the 40S ribosomal subunit we transfected 293 cells with in vitro synthesized mRNAs encoding nsp1 myc His fusion protein CAT myc His protein or nsp1 mt myc His protein and immunoprecipitated the cell extracts with antibody to myc anti myc The immune complexes were washed with 0 5 M KCl a stringent high salt condition in which the 60S ribo somal subunits and the translation initiation factors dissociate from the 40S subunits 14 Consistent with our previous report 10 the expression of nsp1 was substantially lower than that of nsp1 mt or CAT Nevertheless an efficient immunoprecipitation of 18S ribosomal RNA and S6 ribosomal protein core components of the 40S subunit with nsp1 but not with CAT and nsp1 mt Fig 2g i suggested the tight association of nsp1 with the 40S ribosomal subunit Similarly coimmunoprecipitation analysis showed that nsp1 also inter acted with the 40S ribosomal subunit in RRL Supplementary Fig 1 Nsp1 inhibits 80S formation The finding that nsp1 inhibited CrPV IRES driven translation suggested that nsp1 could affect an event or events downstream of the 43S com plex formation that involve the binding of eIF2 GTP Met tRNA ternary complex and other initiation factors such as eIF3 to the 40S subunit To test the effect of nsp1 on 48S complex formation in which the 43S complex binds to the mRNA and 80S monosome assembly we examined by sucrose gradient fractionation the accumulation of these complexes on a radiolabeled mRNA template in the presence of nsp1 To analyze the effect of nsp1 on 80S formation we incubated 32 P labeled rLuc RNA template with nsp1 GST or nsp1 mt in RRL in the presence of cycloheximide CHX CHX treatment inhibits the elongation step but does not affect 80S monosome assembly 15 We used hippuristanol which blocks the function of initiation factor eIF4A and inhibits 48S complex formation as a control 16 CHX treatment induced 80S complex accumu lation and hippuristanol inhibited it Fig 3a Nsp1 but not GST and nsp1 mt suppressed 80S complex formation Fig 3b We tested the effect of nsp1 on 48S complex formation by incubating the samples with GMP PNP a non hydrolyzable analog of GTP and subsequently subjecting them to sucrose gradient centrifugation GMP PNP does not affect 48S complex formation whereas it blocks GTP hydrolysis by eIF2 in eIF2 GTP Met tRNA complexes and inhibits the release of eIF2 and subsequent joining of the 60S ribosomal subunit 17 The 48S complex accumulated in the presence of GMP PNP but not in the presence of hippuristanol Fig 3c Nsp1 nsp1 mt and GST did not suppress the 48S complex formation Fig 3d These data show that nsp1 did not inhibit 48S complex forma tion but it suppressed 60S subunit joining Characterization of the 48S complex by toeprinting analysis Next we performed a toeprinting analysis which defines the positions of stalled ribosomes on the mRNA chain 18 After initial incubation of RRL with nsp1 GST or nsp1 mt in the presence of CHX or a mixture of CHX and GMP PNP we added rLuc RNA that had been prean nealed with a 5 end labeled primer to the samples Subsequently we subjected the samples to primer extension without extracting the 9 a c d e b GST rLuc Protein RNA 10 min GST wt mt GST GST 1 6 1 4 1 2 1 0 0 8 0 6 0 4 0 2 0 fLuc fLuc fLuc rLuc rLuc 0 0 5 1 0 2 0 0 5 1 0 2 0 0 5 1 0 2 0 0 5 1 0 2 0 0 5 1 0 2 0 0 5 1 0 2 0 wt wt mt mt g g GST wt mt GST wt mt 20 min 30 min wt mt rLuc activity 10 8 rLuc RNA transcripts g Firefly luciferase Firefly luciferase Firefly luciferaseRenilla luciferase Renilla luciferase HCV CrPV IRES IRES 8 7 6 5 4 3 2 1 0 m 7 G FF Ren HCV FF Ren CrPV FF m 7 G FF Ren HCV FF Ren CrPV FF fLuc translation m 7 G FF Ren HCV FF Ren CrPV FF 0 01 0 05 0 1 0 25 0 5 1 0 2 0 Figure 1 Effects of nsp1 on cap dependent translation and IRES mediated translation in RRL a Increasing amounts of rLuc RNA were incubated in RRL in the presence of 1 g of nsp1 wt GST or nsp1 mt mt at 4 C for 30 min the molar ratios of rLuc RNA to nsp1 protein ranged from 1 10 to 1 2 000 Then the samples were incubated for 30 min at 30 C and the rLuc enzymatic activities were measured y axis light units b A mixture of 0 25 g of rLuc RNA and 1 g of nsp1 GST or nsp1 mt was incubated in RRL in the presence of 35 S methionine at 30 C for 10 20 and 30 min Translated products were analyzed on SDS PAGE and detected by autoradiography c Schematic diagram of various RNA transcripts used for d e d Increasing amounts of GST nsp1 or nsp1 mt were incubated with RRL for 10 min at 4 C Then the samples were incubated at 30 C for 30 min with 0 5 g of m 7 G FF RNA Ren HCV FF RNA or Ren CrPV FF RNA in the presence of 35 S methionine Translated products were analyzed on SDS PAGE and detected by autoradiography e For a given RNA group the amount of fLuc protein determined by densitometry in each experimental group was normalized to the amount of the fLuc protein in a control group to which the purified protein was not added For a d averages of three independent experiments error bars s d 1136 VOLUME 16 NUMBER 11 NOVEMBER 2009 nature structural a longer exposure of the CHX treated sample clearly showed the same signals data not shown These data suggest that nsp1 induced the modification of rLuc RNA at several discrete sites which blocked cDNA elongation on these modified RNAs Nsp1 renders the capped mRNA translationally inactive We performed primer extension analyses to determine whether nsp1 indeed induced template RNA modification After incubation of rLuc RNA with GST nsp1 or nsp1 mt in RRL as described above we extracted the RNA and performed primer extension analysis using the same 5 end labeled primer In addition to the full length primer extension product only samples incubated with nsp1 showed sev eral premature primer extension termination products all of which corresponded to those detected in the toeprinting analysis performed in the presence of nsp1 Fig 4b Northern blot analysis of rLuc RNA extracted after incubation with nsp1 in RRL under the same conditions did not show significantly less of the full length rLuc transcript than the control groups Supplementary Fig 2 These data demonstrate that nsp1 induced the modification of capped rLuc RNA at several sites in the 5 region without inducing extensive template RNA degradation Centrifugation 40S d g h i e f a b c CAT 28S 18S wt 28S 18S mt 28S 18S wt 28S 18S mt 28S 18S GST Probe 293 Moc k CA T wt mt 293 Moc k CA T wt mt CA T wt mt 28S 18S 28S 18S WB S6 S6 Relati v e band intensity of 18 S 2 0 1 5 1 0 0 5 0 His CAT nsp1 1 2 3 4 5 6 7 8 9 Fractions Fractions IP anti myc IP anti myc 1 2 3 4 5 6 7 8 9 Fractions Fractions 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Fractions Fractions O D 254 nm O D 254 nm O D 254 nm 40S 40S PolysomesPolysomes Polysomes Centrifugation Centrifugation Centrifugation Centrifugation Centrifugation Figure 2 Binding of nsp1 to the 40S ribosomal subunits a c CAT a nsp1 b or nsp1 mt c RNAs were transfected into 293 cells Cell extracts were prepared 8 h after transfection and subjected to polysome profile analysis top panels CAT nsp1 wt and nsp1 mt mt in each fraction were detected by western blot analysis using antibody to myc middle panels Bottom panels show rRNAs in each fraction d f After incubation of the mixture of 0 25 g of rLuc RNA and 1 g of nsp1 GST or nsp1 mt in RRL for 30 min at 30 C the samples were separated on 10 50 sucrose gradient GST nsp1 wt and nsp1 mt mt in each fraction were detected by western blot analysis using anti GST antibody and anti nsp1 ref 8 antibody The locations of GST d nsp1 e or nsp1 mt f in fractions are shown all top panels The bottom panels show rRNAs g i 293 cells were transfected with CAT RNA CAT nsp1 RNA wt or nsp1 mt RNA mt Cell extracts were prepared 8 h after transfection and subjected to immunoprecipitation using anti myc antibody Extracted RNAs from the immunoprecipitates were separated by agarose gel electrophoresis and rRNAs were detected by northern blot analysis g The immunoprecipitated proteins were examined by western blot analysis using anti S6 antibody 40S subunit specific and anti His antibody for CAT nsp1 and nsp1 mt h Panel I represents the values each of which was obtained by dividing the abundance of the immunoprecipitated 18S rRNA with the immunoprecipitated CAT protein nsp1 protein or nsp1 mt protein in an arbitrary scale 4 000 a b c d 80S 80S 48S 48S CHX CHX GST CHX wt CHX mt GMP PNP GST GMP PNP wt GMP PNP mt GMP PNP GMP PNP hipp Centrifugation Centrifugation Centrifugation Centrifugation CHX hipp 3 000 2 000 1 000 3 000 2 500 2 000 1 500 1 000 500 0 32 P c p m mRNA 32 P c p m mRNA 3 000 2 500 2 000 1 500 1 000 500 0 32 P c p m mRNA 2 500 2 000 1 500 1 000 500 0 32 P c p m mRNA 0 Fractions 1 5 10 15 20 Fractions 1 5 10 15 20 Fractions 1 5 10 15 20 Fractions 1 5 10 15 20 Figure 3 Ribosome binding assays of radiolabeled rLuc RNA Representative data from three independent experiments a rLuc RNA was incubated in RRL in the presence of CHX only or a mixture of CHX and hippuristanol hipp The percentages of the rLuc RNA bound in 80S complexes were for CHX 17 4 CHX hipp 1 0 b rLuc RNA was incubated in RRL with GST nsp1 wt or nsp1 mt mt in the presence of CHX The percentages of the rLuc RNA bound in 80S complexes were for GST 13 3 nsp1 5 4 nsp1 mt 14 8 c rLuc RNA was incubated in RRL in the presence of GMP PNP or a mixture of GMP PNP and hipp The percentages of the rLuc RNA bound in 48S complexes were for GMP PNP 11 7 GMP PNP hipp 2 9 d rLuc RNA was incubated in RRL with GST nsp1 wt or nsp1 mt mt in the presence of GMP PNP The percentages of the rLuc RNA bound in 48S complexes were for GST 13 1 nsp1 12 1 nsp1 mt 12 0 nature structural GST and nsp1 mt served as controls After incubation we analyzed the extracted RNAs on a 5 sequencing gel The amount of intact cap labeled rLuc RNA remaining after incubation with nsp1 was clearly lower than that in the control incubations Fig 5a b demonstrating that the 5 end region of the capped rLuc RNA was removed in the presence of nsp1 Incubation of the 3 end labeled rLuc RNA with nsp1 resulted in the appearance of an extra band that migrated slightly faster than the intact RNA Fig 5a The total radioactivity of the intact 3 end labeled rLuc RNA and the fast migrating RNA band in nsp1 treated samples was simi lar to that of the intact 3 end labeled rLuc RNA in the controls Fig 5b To determine the approximate size of the fast migrating band we incu bated 3 end labeled non polyadenylated rLuc RNA with nsp1 in RRL we used non polyadenylated rLuc RNA because some of the polyadenylated rLuc preparations had mRNA species with shorter poly A tails which interfered with the positive identification of the truncated RNA products in the gel We observed two main fast migrating bands and their sizes were approximately 20 to 40 nt shorter than the full length RNA template Supplementary Fig 3 These data demonstrate that nsp1 induced the removal of the 5 end but not the 3 end from the rLuc RNA Next we tested the translational competence of the rLuc RNA that had undergone the nsp1 induced modification After incubation of the rLuc RNA in RRL in the presence of nsp1 GST or nsp1 mt we added the same amount of CAT RNA to each sample as an internal control RNA extracted the RNAs and performed a second in vitro translation reac tion without adding nsp1 GST or nsp1 mt We performed this analysis to examine the translational competence of RNAs that were extracted from the first in vitro translation reaction As expected nsp1 suppressed the translation of rLuc RNA in the initial translation Fig 5c d The rLuc RNA that had undergone the nsp1 mediated modification was not efficiently translated into rLuc protein Fig 5c d which demonstrated that the modified rLuc RNA was translationally inactive Nsp1 induces RNA cleavage in an IRES driven mRNA template In addition to cap dependent translation nsp1 also suppressed transla tion mediated by IRES elements derived from HCV and CrPV Fig 1 To address the question whether the nsp1 induced template RNA modifi cation is specific for cap dependent RNA template we tested whether nsp1 induced the modification of IRES in RNA templates carrying HCV CrPV and encephalomyocarditis virus EMCV derived IRES elements We incubated the RNA transcripts Ren HCV FF Ren CrPV FF and Ren EMCV FF a bicistronic RNA carrying EMCV IRES between the upstream rLuc ORF and the downstream fLuc ORF Fig 6a in RRL in the presence of nsp1 GST or nsp1 mt After incubation we extracted the RNAs and analyzed them by northern blot Fig 6 Nsp1 did not induce any cleavage of Ren HCV FF and Ren CrPV FF Fig 6b and Fig 6c G a b RN A A U G star t site 1 to 3 GST wt mt GST wt mt A T CHX 5 end TP AUG Ribosome paused along 5 UTR CHX GMP PNP C 1 2 3 4 5 6 7 G A U G star t site 1 to 3 GST wt mt GST wt mt RN A A T CHX 5 end CHX GMP PNP C 1 2 3 4 5 6 7 Figure 4 Toeprinting and primer extension analyses a Toeprinting Samples shown in lanes 2 4 and 5 7 were incubated with CHX and a mixture of CHX and GMP PNP respectively and toeprinting analysis was performed in the presence of GST lanes 2 and 5 nsp1 lanes 3 and 6 or nsp1 mt lanes 4 and 7 Lane 1 the primer extension products using the rLuc RNA that was hybridized with a 5 end labeled primer 5 end the primer extension product that was extended to the 5 end of rLuc RNA UTR untranslated region TP AUG a correctly positioned toeprint Arrowhead and asterisk represent a signal generated by the joining of the 60S subunits to the 40S complex positioned at the AUG codon A dideoxynucleotide sequence of the rLuc gene generated with the same primer was run in parallel left four rows Translation initiation codon is shown at the left side of the gel b Primer extension analysis RRL was incubated with GST lanes 1 and 4 nsp1 lanes 2 and 5 or nsp1 mt lanes 3 and 6 in the presence of CHX lanes 1 3 or a mixture of CHX and GMP PNP lanes 4 6 for 5 min at 30 C Then rLuc RNA was added to each sample and the samples were incubated for another 10 min at 30 C The RNAs were extracted and subjected to primer extension analysis Lane 7 the primer extension products using the rLuc RNA that was hybridized with a 5 end labeled primer Cap labeled GST rLuc a b c d 28S 18S 1 2 Relati v e band intensity rLuc RN A 1 0 0 8 0 6 0 4 0 2 0 1 2 1 4 rLuc translation 1 0 0 8 0 6 0 4 0 2 0 wt mt GST wt mt GST wt mt GST rLuc rLuc CAT 1st 2nd 1st 2st wt mt GST wt mt 3 labeled Cap labeled 3 labeled Figure 5 Nsp1 induced modification of rLuc RNA in RRL a Cap labeled rLuc RNA and the 3 end labeled rLuc RNA were incubated with GST nsp1 wt or nsp1 mt mt in RRL for 15 min at 30 C in the presence of CHX and GMP PNP RNAs were extracted resolved on a sequencing gel and visualized by autoradiography Asterisk a signal that migrated slightly faster than the full length rLuc in the sample incubated with nsp1 The 28S and 18S rRNAs detected by ethidium bromide staining are shown as loading controls b Densitometric determination of rLuc RNA band intensities in a normalized to the value of the GST incubated sample For the 3 labeled rLuc RNA in the nsp1 sample graph shows a sum of the signal intensities of the full length rLuc RNA band and the band marked by the asterisk in a c RRL was incubated with GST nsp1 wt or nsp1 mt mt for 10 min at 4 C Then rLuc RNA was added and samples incubated at 30 C for 20 min in the presence of 35 S methionine 1st translation Aliquots were analyzed by SDS PAGE After addition of the same amount of CAT RNA to each sample RNAs were extracted and translated using fresh RRL in the presence of 35 S methionine 2nd translation and the samples were analyzed on a 12 5 SDS PAGE gel d Quantitation of rLuc protein abundances in the 1st and 2nd translation reactions in experiment in c For b d averages of three independent experiments error bars s d 1138 VOLUME 16 NUMBER 11 NOVEMBER