【病毒外文文獻(xiàn)】2009 Suppression of Host Gene Expression by nsp1 Proteins of Group 2 Bat Coronaviruses

《【病毒外文文獻(xiàn)】2009 Suppression of Host Gene Expression by nsp1 Proteins of Group 2 Bat Coronaviruses》由會(huì)員分享，可在線閱讀，更多相關(guān)《【病毒外文文獻(xiàn)】2009 Suppression of Host Gene Expression by nsp1 Proteins of Group 2 Bat Coronaviruses（7頁(yè)珍藏版）》請(qǐng)?jiān)谘b配圖網(wǎng)上搜索。

JOURNAL OF VIROLOGY May 2009 p 5282 5288 Vol 83 No 10 0022 538X 09 08 00H110010 doi 10 1128 JVI 02485 08 Copyright 2009 American Society for Microbiology All Rights Reserved Suppression of Host Gene Expression by nsp1 Proteins of Group 2 Bat Coronaviruses H17188 Yukinobu Tohya 1 2 Krishna Narayanan 1 Wataru Kamitani 1 Cheng Huang 1 Kumari Lokugamage 1 and Shinji Makino 1 Department of Microbiology and Immunology The University of Texas Medical Branch at Galveston Galveston Texas 77555 1019 1 and Department of Veterinary Microbiology Graduate School of Agricultural and Life Sciences The University of Tokyo Tokyo 113 8657 Japan 2 Received 3 December 2008 Accepted 19 February 2009 nsp1 protein of severe acute respiratory syndrome coronavirus SARS CoV a group 2b CoV suppresses host gene expression by promoting host mRNA degradation and translation inhibition The present study analyzed the activities of nsp1 proteins from the group 2 bat CoV strains Rm1 133 and HKU9 1 belonging to groups 2b 2c and 2d respectively The host mRNA degradation and translational suppression activities of nsp1 of SARS CoV and Rm1 nsp1 were similar and stronger than the activities of the nsp1 proteins of 133 and HKU9 1 Rm1 nsp1 expression in trans strongly inhibited the induction of type I interferon IFN I and IFN stimulated genes in cells infected with an IFN inducing SARS CoV mutant while 133 and HKU9 1 nsp1 proteins had relatively moderate IFN inhibitory activities The results of our studies suggested a conserved function among nsp1 proteins of SARS CoV and group 2 bat CoVs Severe acute respiratory syndrome coronavirus SARS CoV is the etiological agent of a newly emerged disease SARS which originated in southern China in 2002 and spread to various areas of the world in a 2003 epidemic reviewed in reference 2 Bats are the natural reservoir of a variety of group 1 and 2 CoVs including viruses closely related to SARS CoV SARS like CoVs SLCoVs 11 13 21 23 Considering the potential of bat CoVs as emerging pathogens for humans and animals it is of the utmost importance to characterize these viruses SARS CoV nsp1 is a 180 amino acid protein that is trans lated from the most 5H11032 coding region of the SARS CoV ge nome 19 SARS CoV nsp1 protein induces host mRNA deg radation and translational suppression both in nsp1 expressing cells and in SARS CoV infected cells 9 14 Studies of the nsp1 proteins of mouse hepatitis virus MHV and SARS CoV which belong to CoV groups 2a and 2b respectively suggest that nsp1 plays important roles in the suppression of host innate immune functions and contributes to viral patho genesis 9 14 22 26 These observations and the fact that SARS CoV nsp1 exhibits sequence similarity to the nsp1 pro teins of other group 2 CoVs but not to those of group 1 CoVs 18 led us to hypothesize that nsp1 proteins of the newly identified group 2 bat CoVs have similar functions to suppress host gene expression and block host antiviral immune re sponses The present study explored the activities of nsp1 proteins of group 2 bat CoVs for the suppression of host gene expression At the initiation of our studies group 2 bat CoVs had been further divided tentatively into three subgroups including 2b and the putative subgroups 2c and 2d 24 We have chosen to characterize the nsp1 proteins of three group 2 bat CoV strains Rm1 15 133 20 and HKU9 1 24 belonging to the subgroups 2b 2c and 2d respectively The levels of amino acid sequence identity of Rm1 133 and HKU9 1 nsp1 proteins to SARS CoV nsp1 protein are 92 2 19 7 and 30 9 respec tively The low levels of amino acid sequence homology be tween nsp1 proteins of group 2c and 2d CoVs and SARS CoV nsp1 are comparable to the levels of amino acid sequence homology between nsp1 proteins of the group 2a CoVs in cluding MHV and bovine CoV and SARS CoV nsp1 the degrees of amino acid sequence identity of MHV nsp1 and bovine CoV nsp1 to SARS CoV nsp1 are 20 6 and 17 3 respectively 18 Figure 1A shows the phylogenetic relation ships of the group 2 CoV nsp1 proteins including the group 2 bat CoV nsp1 proteins analyzed in this study To examine the effects of group 2 bat CoV nsp1 proteins on reporter gene expression cDNAs were synthesized by Bio Ba sic Inc according to the nsp1 amino acid sequences of Rm1 180 amino acids 15 133 195 amino acids 20 and HKU9 1 175 amino acids 24 with the appropriate codon modifications for optimized translations in human cells The plasmids pCAGGS Rm1 pCAGGS 133 and pCAGGS HKU9 1 were constructed by inserting the Rm1 nsp1 open reading frame ORF the 133 nsp1 ORF and the HKU9 1 nsp1 ORF respectively into pCAGGS MCS each with a sequence encod ing a C terminal myc tag As controls the parental plasmid pCAGGS pCAGGS Nsp1 WT encoding the SARS CoV nsp1 protein 9 and pCAGGS Nsp1 mt encoding a mutant form of SARS CoV nsp1 SCoVnsp1 mt containing alanines in place of the positively charged amino acids K164 and H165 in nsp1 of SARS CoV 14 were used expressed SARS CoV nsp1 protein but not SCoVnsp1 mt protein suppresses host gene expression 14 293 cells grown in 24 well plates were cotransfected in trip licate with 0 1 H9262g of pRL SV40 in which the Renilla luciferase Corresponding author Mailing address Department of Microbi ology and Immunology The University of Texas Medical Branch at Galveston Galveston TX 77555 1019 Phone 409 772 2323 Fax 409 772 5065 E mail shmakino utmb edu H17188 Published ahead of print on 4 March 2009 5282 on March 7 2015 by University of Michigan Library http jvi asm org Downloaded from RLuc gene was cloned downstream of the simian virus 40 promoter 9 and 0 5 H9262g of the pCAGGS based nsp1 expres sion plasmids described above by using TransIT 293 reagent Mirus At 20 h posttransfection cell extracts were prepared Consistent with the findings of our previous studies 9 14 Western blot analysis showed efficient accumulation of SCoVnsp1 mt protein and low accumulation levels of SARS CoV nsp1 protein Fig 2A HKU9 1 nsp1 protein accumu FIG 1 Unrooted phylogenetic tree of CoV nsp1 proteins and amino acid sequence variations among group 2b CoV nsp1 proteins A The unrooted phylogenetic tree of CoV nsp1 proteins was constructed by the neighbor joining method and the Treeview program and based on 100 bootstrapped data sets The names of virus strains with nsp1 proteins that were analyzed in this study are underlined The virus strains used in the phylogenetic analysis are as follows Urbani human isolate of SARS CoV Rm1 and 279 bat isolates from R macrotis horseshoe bat Rf1 bat isolate from R ferrumequinum Rp3 bat isolate from R pearsoni 133 and HKU4 1 bat isolates from Tylonycteris pachypus lesser bamboo bat HKU5 5 bat isolate from Pipistrellus abramus Japanese pipistrelle HKU9 1 HKU9 2 HKU9 3 and HKU9 4 bat isolates from Rousettus leschenaulti Leschenault s rousette MHV strain A59 and feline infectious peritonitis virus FIPV strain WSU 79 1146 The amino acid aa lengths of nsp1 proteins of group 2b 2c and 2d CoVs are indicated in parentheses The scale bar indicates the estimated number of substitutions per 10 amino acids B The full length SARS CoV Urbani nsp1 amino acid sequence is represented Only the amino acid residues that differ between SARS CoV nsp1 and other group 2b CoV nsp1 proteins are shown In addition to the group 2b CoV amino acid sequences used for the phylogenetic tree those of GZ02 human isolate of SARS CoV and HKU3 1 bat isolate from R sinicus were compared The total number of amino acid differences in each nsp1 protein compared with SARS CoV nsp1 is indicated in parentheses The number at the end of each line of the SARS CoV Urbani nsp1 sequence indicates the amino acid position VOL 83 2009 NOTES 5283 on March 7 2015 by University of Michigan Library http jvi asm org Downloaded from lated efficiently yet to a slightly lower level than the SCoVnsp1 mt protein The level of accumulation of 133 nsp1 protein was lower than that of the HKU9 1 nsp1 protein but higher than that of the Rm1 nsp1 protein These data sug gested that like SARS CoV nsp1 protein Rm1 and 133 nsp1 proteins suppressed their own gene expression A reporter assay using the cell samples lysed in RLuc lysis buffer Promega showed that the expression of SARS CoV nsp1 protein but not that of SCoVnsp1 mt protein suppressed RLuc activity Fig 2B these data were consistent with the results in our previous report 9 14 Rm1 nsp1 protein inhib ited RLuc activity as efficiently as SARS CoV nsp1 protein while 133 and HKU9 1 nsp1 proteins had relatively moderate suppressive activities Fig 2B The abundance of RLuc mRNA was very low in cells expressing SARS CoV nsp1 pro tein but not in those expressing SCoVnsp1 mt protein Fig 2C Rm1 protein suppressed the accumulation of RLuc mRNA as efficiently as SARS CoV nsp1 protein while the reduction in the abundance of RLuc mRNA in cells expressing 133 or HKU9 1 nsp1 protein was less drastic These data clearly showed that the three group 2 bat CoV nsp1 proteins suppressed reporter gene expression and reporter gene mRNA accumulation with different efficiencies To know whether the group 2 bat CoV nsp1 proteins pro moted host mRNA degradation subconfluent 293 cells were transfected independently with one of six capped and poly adenylated RNA transcripts each encoding Rm1 nsp1 133 nsp1 HKU9 1 nsp1 SARS CoV nsp1 SCoVnsp1 mt or chlor amphenicol acetyltransferase CAT protein by using TransIT mRNA Mirus all of these proteins had a C terminal myc tag At 1 h posttransfection the cells were incubated in the absence or presence of 4 H9262g ml of actinomycin D ActD Sigma which blocks new RNA synthesis and facilitates the analysis of the fate of preexisting cellular mRNAs this concentration of ActD severely blocks host RNA synthesis 9 If the expression of bat CoV nsp1 proteins resulted in a reduction in the abundance of host endogenous mRNAs in the presence of ActD then the data would suggest that the expression of the bat nsp1 proteins promotes host mRNA degradation Similar to the plasmid transfection experiments Fig 2A these RNA transfection assays revealed that the expression levels of SARS CoV nsp1 Rm1 nsp1 and 133 nsp1 proteins were significantly lower than those of CAT SCoVnsp1 mt and HKU9 1 nsp1 proteins Fig 3A ActD treatment had little effect on the abundance of endogenous glyceraldehyde 3 phosphate dehydrogenase GAPDH H9252 actin and cyclophilin mRNAs in the cells ex pressing CAT protein Fig 3B These results were not sur prising because these mRNAs have long half lives due to which ActD treatment for less than 10 h often has a minimal effect on their abundance 1 3 12 17 25 The levels of these endogenous mRNAs in SARS CoV nsp1 expressing cells and Rm1 nsp1 expressing cells were significantly lower than those in other samples both in the presence and in the absence of ActD Fig 3B The expression of 133 and HKU9 1 nsp1 proteins induced a moderate reduction in H9252 actin mRNA lev els and the abundance of GAPDH and cyclophilin mRNAs in the cells expressing HKU9 1 nsp1 protein was lower than that in cells expressing CAT or SCoVnsp1 mt protein ActD treat ment did not alter these trends In the absence of ActD 133 nsp1 expression had little effect on the abundance of cyclophi lin mRNA yet it moderately suppressed GAPDH mRNA ac cumulation We have repeated these Northern blot analyses at least three times and obtained similar results These data strongly suggested that the expressed Rm1 nsp1 promoted the FIG 2 Effects of expression of bat CoV nsp1 on reporter gene expression 293 cells were independently cotransfected with pRL SV40 and one of the following plasmids pCAGGS empty vector EV pCAGGS Nsp1 WT WT pCAGGS Nsp1 mt mt pCAGGS Rm1 Rm1 pCAGGS 133 133 or pCAGGS HKU9 1 HKU9 1 At 20 h posttransfection cell extracts were prepared A Western blot analysis using anti myc monoclonal antibody Upstate and anti H9252 actin anti body Santa Cruz Biotechnology demonstrated the accumulation of expressed nsp1 proteins and H9252 actin respectively The data are repre sentative of results from three independent experiments B RLuc activities were measured and represented as the average of results from three independent experiments Activities are expressed in rela tive light units C The abundance of expressed RLuc RNA tran scripts from pRL SV40 was examined by Northern blot analysis using an RLuc probe The data are representative of results from three independent experiments The abundance of 28S and 18S rRNAs in each sample is also shown 5284 NOTES J VIROL on March 7 2015 by University of Michigan Library http jvi asm org Downloaded from degradation of endogenous mRNAs as efficiently as expressed SARS CoV nsp1 whereas the expressed 133 and HKU9 1 nsp1 proteins had less prominent activities to promote the degradation of these host mRNAs Next the effect of bat CoV nsp1 expression on host protein synthesis was examined 293 cells were independently trans fected with one of the six RNA transcripts as described in the legend to Fig 3A The cells were either treated with ActD at 1 h posttransfection or left untreated and radiolabeled with 35 S methionine from 6 5 to 7 h after the ActD addition After radiolabeling the cell extracts were prepared and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis The amounts of radioactivity in the selected regions of the gel were determined by using a Storm 860 PhosphorImager Mo lecular Dynamics As expected SARS CoV nsp1 protein ex pression but not SCoVnsp1 mt expression strongly inhibited host protein synthesis Fig 3C The expression of Rm1 133 and HKU9 1 nsp1 proteins also suppressed host protein syn thesis Fig 3C The amounts of radiolabeled proteins in SARS CoV nsp1 expressing cells and in Rm1 nsp1 expressing cells were lower than those in cells expressing 133 nsp1 protein or HKU9 1 nsp1 protein Fig 3C Colloid Coomassie blue staining of the gel confirmed the similar loading amounts of proteins in the gel Fig 3D These data revealed that the Rm1 133 and HKU9 1 nsp1 proteins efficiently inhibited host protein synthesis and also highlighted a difference in the levels of host translational suppression activity among the group 2 bat CoV nsp1 proteins The data presented above led us to hypothesize that the group 2 bat CoV nsp1 proteins also inhibit the expression of genes that are involved in host innate immune functions in infected cells We tested this hypothesis by evaluating the ef fects of group 2 bat CoV nsp1 protein expression on the in duction of beta interferon IFN H9252 and two IFN stimulated genes ISGs ISG15 and ISG56 in the cells infected with the SARS CoV nsp1 mutant SARS CoV mt which induces high levels of IFN H9252 and ISG proteins in infected 293 ACE2 cells 14 the SARS CoV mt nsp1 gene and the SCoVnsp1 mt gene have the same mutation 14 We speculated that host cells probably use the same pattern recognition receptors 16 to recognize unidentified pathogen associated molecular patterns FIG 3 Effect of bat CoV nsp1 expression on host mRNA stability and protein synthesis 293 cells were independently transfected with in vitro synthesized RNA transcripts encoding CAT CAT SARS CoV nsp1 WT SCoVnsp1 mt mt Rm1 nsp1 Rm1 133 nsp1 133 or HKU9 1 nsp1 HKU9 1 protein One hour after RNA trans fection cells were incubated in the absence of ActD ActD H11002 or the presence of ActD ActD H11001 A At 8 h posttransfection total proteins were extracted Western blot analysis was performed using anti myc or anti H9252 actin antibodies B Total RNAs were extracted at 8 h post transfection and used for Northern blot analysis with riboprobes spe cific for GAPDH H9252 actin and cyclophilin The abundance of 28S and 18S rRNAs is shown at the bottom C and D Cells were radiolabeled with 20 H9262Ci ml of 35 S methionine from 6 5 to 7 h after the ActD addition Equivalent amounts of intracellular proteins were analyzed by sodium dodecyl sulfate 12 5 polyacrylamide gel electrophoresis C The gel was exposed to X ray film Phosphorimager analysis was performed to determine the level of host protein synthesis and the numbers below the lanes represent the percentages of radioactivity relative to that for cells transfected with CAT RNA of CAT The boxes indicate the regions of the gel used for phosphorimager analysis Representative data from two independent experiments are shown D The gel was stained with colloid Coomassie blue VOL 83 2009 NOTES 5285 on March 7 2015 by University of Michigan Library http jvi asm org Downloaded from of replicating SARS CoV and group 2 bat CoVs and trigger the signaling pathways that lead to type I IFN and ISG induc tion If nsp1 proteins of the group 2 bat CoVs suppress the induction of IFN H9252 and ISGs in SARS CoV mt infected cells then it is reasonable to extrapolate these results and suggest that the proteins possess the activity to suppress the expression of the IFN H9252 gene and ISGs in bat CoV infected cells 293 ACE2 cells 9 were either mock infected or infected with SARS CoV mt 14 One hour after infection cells were transfected with capped and polyadenylated RNA transcripts encoding Rm1 nsp1 133 nsp1 HKU9 1 nsp1 SARS CoV nsp1 SCoVnsp1 mt or CAT The total intracellular RNAs and proteins were extracted at 18 h postinfection h p i while supernatants were collected at 43 h p i to assay the production of biologically active IFN I The amounts of the expressed SARS CoV Rm1 and 133 nsp1 proteins were significantly lower than those of expressed CAT protein or SCoVnsp1 mt and HKU9 1 nsp1 proteins Fig 4A top panel These results which were similar to the data shown in Fig 3A suggested that the virally encoded SCoVnsp1 mt protein did not alter the nsp1 expression profile in cells transfected with RNA tran scripts and that the differences in the abundance of the ex pressed nsp1 proteins probably reflected the activity of each nsp1 to suppress its own expression The abundance of virally encoded SCoVnsp1 mt protein and that of H9252 actin were similar in all the samples Fig 4A middle and bottom panels Anti nsp1 antibody detected the myc tagged SARS CoV nsp1 pro tein and SCoVnsp1 mt protein as expected Fig 4A middle panel while it also recognized Rm1 nsp1 protein Fig 4A middle panel Efficient viral mRNA accumulation occurred in all the samples yet for an unknown reason the abundance of viral mRNAs was slightly lower in the cells expressing SCoVnsp1 mt 133 nsp1 or HKU9 1 nsp1 than in those ex pressing CAT SARS CoV nsp1 or Rm1 nsp1 Fig 4B IFN H9252 ISG15 and ISG56 mRNAs were not detected in the mock infected cells transfected with RNA transcripts Fig 4C odd numbered lanes whereas these mRNAs accumulated in the SARS CoV mt infected cells transfected with the CAT RNA transcript Fig 4C lane 2 demonstrating that SARS CoV mt replication but not transfection with RNA transcripts induced the accumulation of IFN H9252 ISG15 and ISG56 mRNAs SARS CoV nsp1 expression efficiently suppressed the accumulation of IFN H9252 ISG15 and ISG56 mRNAs Fig 4C lane 4 These data suggested that the efficiencies of RNA transfection in 293 ACE2 cells were high and also reinforced our previous finding that SARS CoV nsp1 plays a critical role in the inhibition of the expression of innate immune response genes 14 The expression of SCoVnsp1 mt protein did not prevent the accumulation of IFN H9252 ISG15 and ISG56 mRNAs Fig 4C lane 6 Expressed Rm1 nsp1 protein sup pressed the accumulation of IFN H9252 ISG15 and ISG56 mRNAs as efficiently as SARS CoV nsp1 protein Fig 4C lane 8 while expressed 133 and HKU9 1 nsp1 proteins showed moderate activities to suppress the accumulation of these mRNAs ISG15 protein accumulated efficiently in SARS CoV mt infected cells expressing CAT protein or SCoVnsp1 mt protein but not in those expressing SARS CoV nsp1 or Rm1 nsp1 Fig 4D Low levels of ISG15 protein accumulation in SARS CoV mt infected cells expressing 133 or HKU9 1 nsp1 protein were found Overall the abundance profile of the ISG15 protein roughly correlated with those of ISG15 mRNAs A biological assay of secreted IFN I showed signi