Interactome Analysis of NS1 Protein Encoded by Influenza A H7N9

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Interactome analysis of NS1 protein encoded by influenza A H7N9 virus reveals an inhibitory role of NS1 in host mRNA maturation Rei-Lin Kuo, Chi-Jene Chen, Ee-Hong Tam, Chung-Guei Huang, LiHsin Li, Zong-Hua Li, Pei-Chia Su, Hao-Ping Liu, and Chih-Ching Wu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00815 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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Journal of Proteome Research

Interactome analysis of NS1 protein encoded by influenza A H7N9 virus reveals an inhibitory role of NS1 in host mRNA maturation

Rei-Lin Kuoa,b,c,d, Chi-Jene Chene,f, Ee-Hong Tama,d, Chung-Guei Huanga,d,g, Li-Hsin Lia, Zong-Hua Lia, Pei-Chia Sua, Hao-Ping Liuh, and Chih-Ching Wua,d,i,*

a

Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan

b

Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan

c

Division of Asthma, Allergy, and Rheumatology, Department of Pediatrics, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan

d

Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

e

Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan

f

Research Center for Emerging Viruses, China Medical University Hospital, Taichung, Taiwan

g

Department of Laboratory Medicine, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan

h

Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan

i

Department of Otolaryngology-Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan

* Correspondence to Chih-Ching Wu, Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan. Phone: 886-3-2118800, ext. 5093. e-mail: [email protected].

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Abstract Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using co-immunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.

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Key words: cleavage and polyadenylation specificity factor (CPSF); GeLC-MS/MS; influenza A H7N9 virus; interactome; interferon (IFN); immunoprecipitation; nonstructural protein 1 (NS1); mRNA maturation; mRNA polyadenylation; spectral counting-based quantification

Introduction Infections of influenza A virus can result in severe respiratory complications and death. Strains of the virus have been categorized into different subtypes according to the combination of the envelope proteins hemagglutinin (HA) and neuraminidase (NA).1 Pandemics of influenza A virus have affected public health and caused huge economic loss, such the new H1N1 pandemic in 2009.2,3 Since 2013, an H7N9 subtype of avian influenza A virus has been reported to infect humans and has caused severe diseases.4-7 Influenza A virus is a segmented negative-stranded RNA virus. Its infectious viral particle consists of eight genomic RNA segments coated with a viral nucleocapsid protein (NP).8,9 Rearrangement of genomic segments, also called reassortment, can therefore occur in cells infected with different subtypes of the virus. This novel pathogenic H7N9 virus was a reassortant from the H9N2, Eurasian wild bird H7, and

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Eurasian wild bird N9 viruses.6 The shortest segment of the influenza A viral genome is the nonstructural (NS) segment, which generally encodes NS1 and NS2 proteins. The NS1 protein is a small (219-237 amino acids) and multifunctional protein. This protein consists of two domains, an N-terminal double-stranded RNA (dsRNA)-binding domain (composed of 1-73 amino acids) and a C-terminal effector domain.10 NS1 protein plays a crucial role in regulating host innate immune response by different mechanisms. For example, the effector domain of the NS1 protein binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30). This interaction interferes with the host’s antiviral response.11-16 More specifically, the binding of NS1 protein to CPSF30 suppresses polyadenylation of cellular pre-mRNA and reduces production of mature mRNA, including the mRNAs of antiviral proteins.17 In addition, several studies have shown that NS1 protein directly targets host molecules involved in RIG-I activation such as RIG-I and TRIM25 to block activation of type I interferon (IFN).1820

The NS1 protein of an H3N2 influenza virus contains a histone mimic sequence and

can suppress hPAF1C-mediated transcriptional elongation, which is important in antiviral responses.21 The RNA-binding domain of NS1 protein was proposed to sequester the double-stranded RNA that initiates RIG-I signaling.22,23 Nevertheless, the mechanisms that suppress RIG-I activation and IFN-β transcription differ by

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strain.24 Comparing the length and compositions of NS1 proteins encoded by influenza A viruses indicates that the NS1 protein of H7N9 may be different from that of the virus circulating in humans.6,25 In the present study, we applied a proteomic approach to globally identify host proteins that interact with the NS1 protein encoded by the influenza A H7N9 virus. Our results show that the H7N9 NS1 interacts with host proteins involved in pre-mRNA polyadenylation machinery, including cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, though the NS1 protein does not contain the residues, F103 and M106, crucial for stabilizing interactions with CPSF30.17 We also demonstrate that expression of H7N9 NS1, as well as infection by the H7N9 virus, interferes with pre-mRNA polyadenylation in host cells. Our findings may illustrate the dysregulation of antiviral responses during H7N9 virus infections.

Materials and methods Cells, plasmids, transfection and virus infection HEK293T (293T), MDCK and A549 cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C. The

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3×FLAG-tagged H7N9 NS1-expressing plasmid was constructed by inserting the cDNA of NS1 amplified from A/Taiwan/1/2013(H7N9) RNA into a p3×FLAG-MycCMV26 vector (Sigma-Aldrich, St. Louis, MO, USA). The cDNA of the human mitochondrial antiviral-signaling (MAVS) coding sequence was inserted into the KpnI and XhoI sites of a pcDNA3 vector, which contains the polyadenylation signal from the gene of bovine growth hormone (BGH). The cDNA of human CPSF7 coding sequence was cloned into the p3×FLAG-Myc-CMV26 vector (Sigma-Aldrich). The expression plasmid for 3×FLAG-tagged 3D protein of enterovirus 71 (EV71) was provided by Dr. Shin-Ru Shih at Chang Gung University. A firefly luciferase reporter for the IFN-β promoter was provided by Dr. Helene M. Liu at National Taiwan University. Plasmid transfection was performed by a TransIT-LT1 Transfection Reagent (Mirus Bio, Madison, WI, USA) following the manufacturer’s instructions. Transfection

of

siRNA

duplex

against

CPSF7

GCAAUUUCCAGCAGUGCCA[dT][dT]-3’

(sequences:

and

5’5’-

UGGCACUGCUGGAAAUUGC[dT][dT]-3’) and the negative control siRNA duplex (sequences:

5’-UUCUCCGAACGUGUCACGUTT-3’

and

5’-

ACGUGACACGUUCGGAGAATT-3’) was performed with Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific,

NY,

USA) following the

manufacture’s protocol. Wild-type A/Taiwan/1/2013(H7N9) virus was isolated and

6


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provided by the Laboratory of Clinical Virology in Chang Gung Memorial Hospital. Infection with the H7N9 virus was performed in a BSL3 laboratory in Chang Gung Memorial Hospital following the guidelines of the Centers for Disease Control, Taiwan. H7N9 virus was amplified by MDCK cells and titrated by plaque formation assay. A549 cells were infected with the H7N9 virus at a multiplicity of infection (MOI) of 3 for 9 h.

Immunoprecipitation, immunoblotting and antibodies 293T cells transfected with 3×FLAG-tagged H7N9 NS1-expressing plasmid were lysed with a buffer containing 100 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.5% sodium deoxycholate, 1 mM PMSF and 0.5% NP40. An aliquot containing 4 mg proteins of the lysate in a buffer containing 67 mM Tris-HCl (pH 7.5), 183 mM NaCl, 0.17% sodium deoxycholate, 0.33 mM PMSF and 0.17% NP40 was mixed with 40 µl of anti-FLAG-M2 affinity resin (Sigma-Aldrich), and then rotated at 4°C for 12 h. After rotation, the supernatant was removed, and the resin was washed with a wash buffer containing 50 mM Tris-HCl (pH7.5), 150 mM NaCl, and 0.5% NP40. The precipitates were then heated in a sample buffer (containing 62.5 mM Tris-HCl pH6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.05% bromophenol blue), or eluted with 3×FLAG peptide (final concentration 188 ng/µl). Cell lysates or

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immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membranes. After the membranes were blocked with 5% non-fat milk, they were incubated with antibodies against CPSF2 (Sigma-Aldrich), CPSF7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MOV10 (GeneTex, Hsinchu, Taiwan), FLAGM2 (Sigma-Aldrich), MAVS (Enzo Life Sciences, Farmingdale, NY, USA), or β-actin (Sigma-Aldrich). The secondary antibodies, produced from rabbit or mouse, were purchased from GE Healthcare Life Sciences (Taipei, Taiwan).

Reverse-phase LC-MS/MS analysis and protein identification The immunoprecipitates were separated by SDS-PAGE and stained by a Colloidal Blue Staining kit (Thermo Fisher Scientific). Each gel lane was then cut into 20 parts, and each part was sliced into three pieces to provide technical replicates. After in-gel tryptic digestion, the peptides were extracted and identified by reversed-phase LCMS/MS as previous described.26-28 The obtained MS/MS spectra were analyzed using the Mascot algorithm (version 2.1, Matrix Science, MA, USA) against the Swiss-Prot human sequence database (released Mar 16, 2016, selected for Homo sapiens, 20199 entries) of the European Bioinformatics Institute and the search results were further integrated using Scaffold software. The criteria of the protein database search and the process of obtaining search files were previously described.26-28 We used the

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PeptideProphet and ProteinProphet probabilities of Scaffold software (version 3.6.5; Proteome Software, OR, USA) ≥ 0.95 to ensure an overall false discovery rate below 0.5%. Only proteins with at least two unique peptides were retained in this study.

Bioinformatics analysis For the purpose of identifying the associated partners of H7N9 NS1, we compared the protein levels between immunoprecipitation products of the control vector and H7N9 NS1 vector groups with a label-free spectral counting-based quantification as previously described.27,28 Briefly, the spectral number of each protein was obtained with Scaffold software and exported in MS Excel format. The spectral count (SC) of each protein was divided by the SC of total proteins by which the normalized SC of each protein was calculated and used for the fold-change analysis. The fold change was estimated as the ratio of the average of normalized SCs in the H7N9 NS1 group to that in the control group. Due to the lack of identifying all proteins in all replicates, we purposely assigned the SCs of unidentified proteins or missing values in a certain sample as the number of one to avoid overestimating the fold changes and dividing by zero. The biological pathways and processes involved with the H7N9 NS1-interacting proteins were revealed by the Kyoto Encyclopedia of Genes and Genomes (KEGG)

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database

(http://www.genome.jp/kegg/pathway.html)

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and

the

Database

for

Annotation, Visualization, and Integrated Discovery (DAVID, version 6.8, https://david.ncifcrf.gov/),29 respectively. The known and predicted associations between the H7N9 NS1-inteacting partners were analyzed with STRING online software (version 10.5, https://string-db.org/cgi/input.pl). A combined score of confidence ≥ 0.9 was used as the cutoff criterion.30

Revere transcription and real-time PCR Total RNA of cells transfected or infected under the indicated conditions were isolated by TRIzol reagent (Invitrogen, Taipei, Taiwan) following the manufacturer’s instruction. To analyze the pre-mRNA transcribed from the cotransfected MAVS expression plasmid, a reverse transcription reaction was performed by Superscript III reverse transcriptase and a primer complementary to sequences after the poly(A) signal of BGH (primer sequence: 5’-CAGCATGCCTGCTATTGTCTTC-3’), followed by a real-time PCR with SYBR Green reagents and MAVS-specific primers (forward primer

sequence:

5’-CAGGCCACAAGCCGACCGGAAG-3’;

reverse

primer

sequence: 5’-CTAGTGCAGACGCCGCCGGTAC-3’). Polyadenylated mRNA was detected by reverse transcription with an oligo-dT primer and then amplified by realtime PCR as described above. Primers for quantifying HSP70-1A pre-mRNA and

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mRNA by reverse transcription and real-time PCR were designed according to the previous study of Shimizu et al.31 The sequence of the reverse primer for HSP70-1A pre-mRNA was 5’-GGAAACACCTTAGCAGACCT-3’. The primers used for detecting the cDNAs of HSP70-1A pre-mRNA and mRNA by real-time PCR were 5’CCATTGAGGAGGTAGATTAG-3’

(forward)

and

5’-

GGCAAGTTCAGTACTTCACC-3’ (reverse). The levels of aforementioned premRNA and mRNA were normalized with quantification of 18S ribosomal RNA (rRNA) by reverse transcription and real-time PCR. The primer for reverse transcription of 18S rRNA was 5’-CCATCCAATCGGTAGTAGCG-3’. The PCR primers for cDNA of 18S rRNA were 5’-GTAACCCGTTGAACCCCATT-3’ (forward) and 5’-CCATCCAATCGGTAGTAGCG-3’(reverse).

Reporter assay for IFN-β β promoter activity 293T cells were cotransfected with an IFN-β promoter-driven firefly luciferase reporter, a pRL-TK renilla luciferase reporter, the MAVS-expressing plasmid, and either H7N9 NS1-expressing plasmid or empty vector. At 24 h posttransfection, cell lysates were collected and subjected to the Dual-luciferase reporter assay (Promega, Madison, WI, USA) following the manufacture’s protocol. To examine the role of CPSF7 in regulation of IFN-β promoter activity, 293T cells were transfected with

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siRNA against CPSF7 (at final concentration of 66 nM) for 9 h, and then transfected with the aforementioned plasmids. The IFN-β promoter activity was determined at 24 h after plasmid transfection as described previously.

Statistical analysis All statistical data are expressed as mean ± standard deviation (SD). Protein levels or values in the control and H7N9 NS1 groups were compared using the unpaired Student’s t test. Differences between values in two groups with p values < 0.05 were considered statistically significant. All data were processed by SPSS software version 12.0 (SPSS Inc., IL, USA).

Results Identification of the host factors that interact with NS1 protein encoded by influenza A H7N9 virus NS1 proteins encoded by influenza A virus play an important role in regulating host biological processes such as antiviral responses and splicing. To globally explore the functions of the NS1 protein, we previously performed co-immunoprecipitation and GeLC-MS/MS to identify host proteins that interact with the NS1 protein of the

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influenza A H1N1 virus.27 However, the sequence of the NS1 protein encoded by the highly pathogenic H7N9 influenza A virus is different from that of the NS1 protein encoded by the H1N1 virus (Figure S1). The host proteins that interact with NS1 of the H7N9 virus therefore required further identification. To determine the H7N9 NS1associating host proteins, we applied a co-immunoprecipitation approach to pull down proteins that interacted with the overexpressed FLAG-tagged NS1 protein of H7N9 (Figure 1A). To identify protein constituents of the immunoprecipitants, the proteins precipitated from the cells transfected with the H7N9 NS1 or control vectors were detected with a GeLC-MS/MS method (Figure 1B).27 The analysis resulted in identification of 1881 nonredundant proteins with at least two unique peptides (Table S-1 and Figure 1C). Among 1881, 233 (12.39%) and 203 (10.79%) were only detected in the H7N9 NS1 and control groups, respectively (Table S-2 and Figure 1C), whereas 1445 (76.82%) were found in both groups. To ascertain the reproducibility of the proteomic analyses, proteins detected in three replicates were further examined for overlap. In total, 1218 (72.59%) and 1202 (72.94%) proteins were present in the triplicate of the H7N9 NS1 and control groups, respectively (Table S-2 and Figure 1D). Approximately 86% of the proteins were detected in either the duplicate or the triplicate of experiments, whereas around 14% were unique to single replicates (Figure 1D). Furthermore, using a decoy database, the

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false discovery rate (FDR) of peptide identification in each group was evaluated. All FDRs were less than 0.05%. The results collectively demonstrate that the proteome analysis was appropriately conducted.

Spectral counting-based quantification for identification of H7N9 NS1interacting proteins To identify proteins potentially interacted with H7N9 NS1 protein, we performed the spectral counting-based quantification to evaluate the relative amounts of proteins in the immunoprecipitants (Table S-2). The fold change for individual protein was a ratio of the average SC of the protein in the H7N9 NS1 group versus that in the control group. Proteins with fold changes more than two SD above the mean ratio (the fold changes were above 7.728) and observed in three replicates of the H7N9 NS1 group were considered proteins interacted with H7N9 NS1. As shown in Table 1, 36 proteins were observed based on these cutoffs. Twenty-six out of 36 proteins are specifically present in the H7N9 NS1 group, and the relative levels of 10 proteins were elevated in the H7N9 NS1 group compared to the control group. The 67 proteins having the fold changes that were greater than the mean plus one SD (the ratios were larger than 4.611) are also listed in Table S-3 to ensure that the potential interacting partners of H7N9 NS1 are mostly included in the proteome profiling.

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Involvement of H7N9 NS1-associated protein complexes in RNA processing revealed by bioinformatics analysis To determine the biological processes that are most likely affected by the presence of the H7N9 NS1-associated complexes, DAVID was used to annotate the functions of 36 proteins which were described above (Table 1). Enriched biological processes

including

spliceosome-associated

mRNA splicing,

mRNA 3'-end

processing, and mRNA export from the nucleus were shown in Table 2. Moreover, pathway-wise analysis of these proteins using the KEGG database revealed that the proteins probably engage in the spliceosome and the mRNA surveillance pathway (Table 3). We further used the STRING online database to establish a network of protein-protein interaction (PPI) between the 36 proteins, and 85 strong interaction links between individual nodes/proteins were depicted in the PPI network (Figure 2). In line with the results from DAVID and KEGG analyses, one module was identified in STRING analysis that depicted the interactions of polyadenylation specificity factors (CPSFs) with proteins associated with RNA processing, including mRNA 3'end processing and the mRNA surveillance pathway (Figure 2 and Tables 2 and 3).

H7N9 NS1 associates with cellular proteins involved in host polyadenylation

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machinery Among the cellular proteins involved in RNA splicing/processing and unlike the proteins previously identified in H1N1 NS1 interactome analysis, we found that several proteins involved in pre-mRNA polyadenylation such as CPSF2, CPSF7, and PABP2 may associate with H7N9 NS1. According to previous studies, the NS1 protein encoded by seasonal human influenza A H1N1 or H3N2 viruses binds to CPSF30 (also named CPSF4) to interrupt cellular pre-mRNA polyadenylation. Although H7N9 NS1 differs in two important residues, F103L and M106I, that stabilize interactions with CPSF30, the components of the complex involved in premRNA polyadenylation were still identified in the interactome analysis of H7N9 NS1. To validate that the host proteins profiled interacted with NS1, we performed immunoprecipitation of 3×FLAG-tagged H7N9 NS1-transfected cell lysates with controls from either mock cells or empty plasmid-transfected cells and examined whether CPSF2 and CPSF7 was co-purified with H7N9 NS1. Figure 3A shows that CPSF2 and CPSF7 were detected in complex with H7N9 NS1 but were weakly detected in complex with PR8 NS1, which contains S103 and I106 in the stabilizing positions for binding to CPSF30. To confirm that the interaction between H7N9 NS1 and

CPSF2

or

CPSF7

was

specific,

we

performed

transfection

and

immunoprecipitation experiments with a control of 3×FLAG-tagged 3D protein of

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EV71. The result shows that CPSF2 and CPSF7 could only be detected in the H7N9 NS1 but not EV71 3D immunoprecipitates (Figure S-2). Since we have revealed that several host proteins involved in posttranscriptional regulation and gene expression may interact with H7N9 NS1, we further demonstrated by immunoprecipitation that one of the proteins participating in this process, MOV10, interacted with H7N9 NS1 (Figure 3B). These results validated the hypothesis that H7N9 NS1 interacts with cellular factors involved in the mRNA posttranscriptional machineries, including the polyadenylation process.

H7N9 NS1 disturbs host mRNA maturation and inhibits IFN-β β promoter activation We have previously demonstrated that H7N9 NS1 interacts with components from the mRNA polyadenylation complex. We further examined whether the maturation of cellular mRNA was affected by the NS1 protein. Since a previous study had demonstrated that the polyadenylation of HSP70-1A mRNA was inhibited during influenza A virus infection,31 the polyadenylated mRNA of endogenous HSP70-1A was analyzed in NS1-overexpressing 293T cells. The expression of HSP70-1A mRNA was lower in H7N9 NS1-transfected cells than control cells, while the amount of HSP70-1A mRNA was not significantly affected by the NS1 of the PR8 H1N1 virus

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(Figure 4), suggesting that H7N9 NS1 may interfere with cellular mRNA maturation. In addition to determining the expression of endogenous HSP70-1A mRNA, we applied a plasmid that contains a MAVS open reading frame and the polyadenylation signal of BGH as a reporter for mRNA polyadenylation (Figure 5A); 293T cells were cotransfected with a plasmid expressing H7N9 NS1 and the MAVS construct. At 24 h posttransfection, total RNA and cell extracts were collected for analyzing the expression of the pre-mRNA, mRNA, and protein of MAVS. To detect the pre-mRNA transcribed from the reporter plasmid, we performed a reverse transcription reaction with the total RNA and a reverse primer that annealed to the downstream sequence of the BGH polyadenylation and cleavage site (Figure 5A). The generated cDNA was then quantitated by real-time PCR with MAVS-specific primers. As shown in Figure 5B, the pre-mRNA transcribed from the MAVS construct accumulated in cells that had been cotransfected with H7N9 NS1-expressing plasmid. In contrast, the abundance of mature mRNA of MAVS, which contains a poly(A) tail and can be detected by reverse transcription and real-time PCR with oligo-dT and MAVS-specific primers, was lower in the cells co-expressed with H7N9 NS1 than in control cells (Figure 5B). Lower levels of MAVS protein were also detected by immunoblotting with an anti-MAVS antibody (Figure 5C). These results collectively evidenced that H7N9 NS1 interfered with the polyadenylation of cellular pre-mRNA.

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To investigate whether the suppression of pre-mRNA polyadenylation by H7N9 NS1 contributed to the inhibition of type I IFN activation, we cotransfected 293T cells with a firefly luciferase reporter plasmid driven by IFN-β promoter, a MAVSexpressing plasmid for activating the IFN-β promoter, and an H7N9 NS1-expressing construct or an empty vector. At 24 h post-transfection, extracts of the transfected cells were collected to assay the activation of the IFN-β promoter. We detected a lower level of MAVS in the cells cotransfected with H7N9 NS1 expressing plasmid (Figure 6, lane 2 vs. 3) than in the control cells. We consistently found that the expression of H7N9 NS1 inhibited the IFN-β promoter, which is activated by MAVS (Figure 6). The result indicated that H7N9 NS1 reduces the amount of co-expressed MAVS protein and that this event may be involved in the suppression of IFN-β promoter activation.

H7N9 NS1-interacting protein CPSF7 is involved in cellular mRNA maturation and IFN-β β promoter activation To correlate the identified host factors that interact with H7N9 NS1 to the changes of cellular mRNA maturation, 293T cells were cotransfected with H7N9 NS1 and either CPSF7-expressing plasmid or empty vector. Total RNA of the transfected cells was collected and subjected to RT-qPCR for monitoring the change of HSP70-

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1A mRNA. As shown in Figure 7A, the reduction of HSP70-1A caused by H7N9 NS1 was recovered by overexpression of CPSF7. The result demonstrates that the NS1interacting protein CPSF7 is involved in host mRNA maturation. In addition, we further investigated the role of CPSF7 in regulation of IFN-β promoter activity. 293T cells were transfected with siRNA against CPSF7, and then cotransfected with IFN-β promoter reporter, MAVS-expressing plasmid, and either H7N9 NS1-expressing or empty vector. We found that knockdown of CPSF7 expression could further reduce the suppression of IFN-β promoter activity caused by H7N9 NS1 (Figure 7B). The result indicates that CPSF7 plays a role in activation of IFN-β promoter.

Infection by H7N9 virus interferes with the polyadenylation of cellular premRNA To prove that the polyadenylation machinery of host mRNA can be affected during an influenza A H7N9 virus infection, we infected A549 cells with H7N9 virus at an MOI of 3. At 9 h postinfection, total RNA of the infected cells was isolated to analyze the polyadenylation of cellular mRNA. We performed reverse transcription real-time PCR assays as previously described and compared the levels of the premRNA and mRNA of HSP70-1A in mock and infected cells (Figure 8A). As shown in Figure 8B, the HSP70-1A pre-mRNA accumulated during H7N9 virus infection,

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whereas mature HSP70-1A mRNA content was reduced during H7N9 infection. The results demonstrated that H7N9 virus infection interfered with the polyadenylation of host mRNA.

Discussion The influenza A H7N9 virus is highly pathogenic to humans. The NS1 protein of this virus could be an important virulence factor involved in this pathogenicity. In this study, we applied proteomic approaches to identify host proteins that may interact with the NS1 protein. Analysis of the biological processes of the candidate proteins may reveal information regarding the cellular functions targeted by the H7N9 virus. Thirty-six host proteins were identified as interacting partners of H7N9 NS1 (Table 1). Compared to the previous interactome profiling of H1N1 NS1, which demonstrated that the NS1 protein of influenza A/PR8/H1N1 virus interacts with 64 cellular proteins,27 11 cellular proteins (DHX8, IGF2BP2, PABPN1, PIK3R2, PRKRA, SAFB, SAFB2, SNRNP40, STAU1, XAB2, and ZFR) were identified in both H7N9 and H1N1 NS1-associated protein complexes. Pathway analysis demonstrates that the 64 H1N1 NS1-associated proteins are involved in the cellular processes of RNA splicing/processing, cell cycle, and protein folding/targeting.27

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However, the 36 proteins identified in H7N9 NS1-associated complexes mostly participate in RNA maturation and the mRNA surveillance pathway, suggesting that H7N9 NS1 is involved in the inhibition of host mRNA maturation via a mechanism that is different from that of H1N1 NS1. Unlike H3N2 NS1, H7N9 NS1 lacks two relevant interacting sites, F103 and M106, for stabilizing the binding of CPSF3017 (also named CPSF4, which recognizes the polyadenylation signal); however, an interactome analysis demonstrated that H7N9 NS1 still associates with cellular polyadenylation complex. In addition, we validated that H7N9 NS1 interacted with CPSF2 and CPSF7 proteins in the complex. These results indicate that the machinery for host polyadenylation could be tightly regulated by H7N9 NS1. By examining the polyadenylated mRNA transcribed from the endogenous HSP70 gene, its pre-mRNA (a precursor before polyadenylation), and its mature mRNA generated from a reporter plasmid, we found that co-expression of H7N9 NS1 interfered with polyadenylation and limited cellular mRNA and protein expression. The interference with mRNA maturation was also shown in H7N9 virus-infected cells. NS1 protein encoded by influenza A virus plays a critical role in regulating host antiviral responses. Two mechanisms that explain the downregulation of antiviral responses caused by influenza A NS1 protein were elucidated previously. The NS1 protein may inhibit the activation of the IFN-β promoter by binding to molecules in

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the signaling pathway such as RIG-I and TRIM25.18,19 However, NS1 proteins encoded by several subtypes of influenza A viruses circulating in humans binds CPSF30 to globally suppress host mRNA maturation,15 including the mRNAs of antiviral proteins, such as IFN-β. H7N9 NS1 inhibited MAVS-stimulated IFN-β promoter activation in the present study, as expected. Additionally, H7N9 NS1 reduced the level of co-expressed MAVS protein. This result was consistent with the suppression of pre-mRNA polyadenylation caused by H7N9 NS1. Therefore, it is considerable that these two properties possessed by H7N9 NS1 may contribute to the pathogenicity of H7N9 virus infection. In addition to CPSF2 and CPSF7, we also identified CPSF4 (i.e., CPSF30) to be associated with H7N9 NS1. The crystal structure of an NS1 protein from an H3N2 virus showed that the CPSF30 binding pocket in NS1 is formed by amino acid residues K110, I117, I119, Q121, V180, G183, G184, and W187 and that two amino acids outside the binding pocket, F103 and M106, stabilized CPSF30 binding.17 Because H7N9 NS1 possesses M119 and I180 in the binding pocket and L103 and I106 in the stabilizing positions, H7N9 NS1 has been proposed to not efficiently bind CPSF30.32 Nevertheless, our results suggest that H7N9 NS1 still interacts with other components in the CPSF complex to inhibit cellular pre-mRNA polyadenylation. Furthermore, CPSF7 may also be involved in the regulation of alternative

23



polyadenylation in mammalian cells.33,34 It is unclear whether the selection of polyadenylation sites in mammalian cells could have a role in mRNA stability or function. Nevertheless, the impact of the interaction between H7N9 NS1 and CPSF7 in alternative polyadenylation must be further determined. In comparison to previous interactome analyses of H1N1 NS1 from the influenza A PR8 strain,27 host proteins that are involved in RNA splicing such as HNRNPH3 and SNRNP40 were also identified as H7N9 NS1-interacting proteins. However, we did not identify CPSFs in the H1N1 NS1-associated complex.27 This result was consistent with previous findings demonstrating that NS1 protein encoded by influenza A virus PR8 strain does not interact with CPSF30. Although influenza A H7N9 virus was originally transmitted from avians, the virus could have the ability to block host pre-mRNA polyadenylation in mammalian cells. The virus may use the strategy to reduce host antiviral responses to support replication in humans.

Acknowledgements We thank Dr. Helene M. Liu for reagents, and Dr. Robert M. Krug and Dr. KuoMing Lee for helpful discussion. This work was supported by grants to Chih-Ching Wu from the Ministry of Science and Technology (MOST), Taiwan (105-2320-B-182-

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003, 105-2320-B-182-025, and 106-2320-B-182-021) and the Chang Gung Memorial Hospital (CGMH), Taiwan (CLRPD190016, CLRPD190017, and BMRPC77) and by grants to Rei-Lin Kuo from the MOST, Taiwan (103-2321-B-182-003 and 106-2320B-182-024-MY3) and the CGMH (CMRPD1E0441~43 and BMRPC09).

SUPPORTING INFORMATION The following files are available free of charge at ACS website http://pubs.acs.org: Table S-1. List of proteins identified in immunoprecipitation product of control vector (Table S-1-1, -2, and -3) and H7N9 NS1 (Table S-1-4, -5, and -6). Table S-2. Spectral counting-based identification of proteins co-immunoprecipitating with H7N9 NS1. Table S-3. List of the 67 proteins with the fold changes larger than the mean plus one SD. Figure S-1. Sequence comparison of NS1 proteins encoded by influenza A virus Puerto Rico/8/34/H1N1 and Taiwan/1/2013/H7N9 strains. Figure S-2. Confirmation of the specificity for interactions of H7N9 NS1 with CPSF2 and CPSF7.

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Figure legends Figure 1. Identification of cellular proteins that interact with H7N9 NS1. Cells (293T) were transfected with a control vector (vector control) and a plasmid that expresses

3×FLAG-tagged

H7N9

NS1

(3×FLAG

NS1/H7N9).

At

24

h

posttransfection, lysates of the transfected cells were collected and subjected to immunoprecipitation with anti-FLAG resin. (A) The FLAG-tagged NS1 protein in the 29



lysates and precipitates was detected by western blot with anti-FLAG and anti-NS1 antibodies. (B) The precipitated proteins were separated by SDS-PAGE and then stained by a Colloidal Blue Staining kit. (C) Venn diagrams show overlaps between the proteins identified in the control and the H7N9 NS1 groups. (D) Venn diagrams display overlaps between the proteins identified in the three replicates. The total numbers of identified proteins are listed in brackets.

Figure 2. Protein-protein interaction (PPI) network of the proteins identified to co-immunoprecipitate with H7N9 NS1. A PPI network of the 36 proteins listed in Table 1 was constructed using the STRING v10 database (http://string-db.org/), depicting 85 interaction links between individual nodes/proteins (solid lines). One module was identified in STRING analysis that depicted the interactions of CPSFs with proteins involved in RNA processing, including mRNA 3'-end processing and the mRNA surveillance pathway.

Figure 3. Validation of the interactions of H7N9 NS1 with CPSF2, CPSF7, and MOV10. Lysates of 293T cells that were transfected with a control vector and a plasmid that expresses either 3×FLAG-tagged H7N9 NS1 or 3×FLAG-tagged PR8 NS1 protein were collected and subjected to immunoprecipitation with anti-FLAG

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resin. The CPSF2, CPSF7 (A) and MOV10 (B) in the lysates and IP precipitates were detected by western blot with anti-CPSF2, anti-CPSF7, and anti-MOV10 antibodies.

Figure 4. Analysis of cellular polyadenylated mRNA in cells overexpressing H7N9 NS1. Cells (293T) were transfected with a control vector and a plasmid that expressed either untagged H7N9 NS1 or PR8/H1N1 NS1 protein. At 24 h posttransfection, total RNA of the transfected cells was collected and subjected to reverse transcription with an oligo-dT primer and a real-time PCR with primers for specifically detecting polyadenylated mRNA of HSP70-1A (A). NS1 protein expression in the transfected cells was monitored by immunoblotting with anti-NS1 antiserum (B). **, p value < 0.01; n.s., not significant.

Figure 5. Examination of mRNA polyadenylation processing in cells that overexpress H7N9 NS1. (A) Strategy and primer design for quantifying pre-mRNA and polyadenylated mRNA transcribed from a reporter plasmid containing a MAVS ORF and a BGH polyadenylation signal. (B) Cells (293T) cotransfected with the MAVS reporter plasmid and either a control vector or a plasmid that expresses H7N9 NS1. Total RNA of the transfected cells was collected and subjected to reverse transcription and real-time PCR to examine pre-mRNA and mRNA transcribed from

31



the MAVS reporter plasmid. *, p value < 0.05; **, p value < 0.01.

Figure 6. Determination of the role of H7N9 NS1 in regulation of IFN-β β promoter activation. Cells (293T) cotransfected with IFN-β promoter luciferase reporter, a MAVS-expressing plasmid and either a control vector or a plasmid that expresses H7N9 NS1. Lysates of the transfected cells were collected to examine IFN-β promoter activity and protein expression from the transfected plasmids. ***, p value < 0.001.

Figure 7. Determination of the role of CPSF7 in H7N9 NS1-mediated suppression of polyadenylation and IFN-β β promoter activation. (A) 293T cells were cotransfected with a plasmid expressing H7N9 NS1 and either 3×FLAG-tagged CPSF7-expressing plasmid or empty vector for 24 h. Cellular total RNA was collected and subjected to RT-qPCR to detect mRNA of HSP70-1A. (B) 293T cells were transfected with siRNA against CPSF7 for 9 h, and then transfected with IFN-β promoter luciferase reporter, a MAVS-expressing plasmid and either a control vector or a plasmid that expresses H7N9 NS1 for 24 h. Lysates were collected for determining lucifierase activity. The experiments were independently duplicated. The data are representative one of the experiments. *, p value < 0.05; **, p value < 0.01.

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Figure 8. Examination of pre-mRNA accumulation in influenza A H7N9 virusinfected cells. (A) Primer design strategy for quantifying the pre-mRNA and polyadenylated mRNA of HSP70-1A. (B) Cells (A549) were infected with influenza A Taiwan/1/2013/H7N9 virus at an MOI of 3. At 9 h postinfection, HSP70 pre-mRNA and mRNA were quantified by reverse transcription real-time PCR. *, p value < 0.05; ***, p value < 0.001.

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Page 34 of 45

Table 1. Spectral counting-based identification of proteins co-immunoprecipitating with H7N9 NS1 Spectral counts (SCs) in replicates 1/2/3 NS1/VC Protein name (accession number, gene name) ratioa Vector control H7N9 NS1 (VC) (NS1) Interferon-inducible double-stranded RNA-dependent protein kinase activator A (PRKRA_HUMAN, PRKRA) Scaffold attachment factor B1 (SAFB1_HUMAN, SAFB) Phosphatidylinositol 3-kinase regulatory subunit beta (P85B_HUMAN, PIK3R2) Scaffold attachment factor B2 (SAFB2_HUMAN, SAFB2) Insulin-like growth factor 2 mRNA-binding protein 2 (IF2B2_HUMAN, IGF2BP2) Heterogeneous nuclear ribonucleoprotein D-like (HNRDL_HUMAN, HNRNPDL) Pre-mRNA-splicing factor SYF1 (SYF1_HUMAN, XAB2) Spermatid perinuclear RNA-binding protein (STRBP_HUMAN, STRBP) YTH domain-containing protein 1 (YTDC1_HUMAN, YTHDC1) Putative helicase MOV-10 (MOV10_HUMAN, MOV10) Polyadenylate-binding protein 2 (PABP2_HUMAN, PABPN1) ELAV-like protein 2 (ELAV2_HUMAN, ELAVL2) Zinc finger RNA-binding protein (ZFR_HUMAN, ZFR) Crooked neck-like protein 1 (CRNL1_HUMAN, CRNKL1) Serine/arginine-rich splicing factor 7 (SRSF7_HUMAN, SRSF7) Cleavage and polyadenylation specificity factor subunit 2 (CPSF2_HUMAN, CPSF2) Serine/arginine-rich splicing factor 10 (SRS10_HUMAN, SRSF10) ATP-dependent RNA helicase DHX8 (DHX8_HUMAN, DHX8)

34


0/0/0

71 / 65 / 55

65.023

0/0/0 0/0/0 0/0/0 0/2/0 0/0/0 0/0/0 0/0/0 0/0/0 0/2/5 0/0/0 0/0/0 0/3/2 0/2/0 0/0/0 0/0/0 0/0/0 0/0/0

63 / 54 / 44 35 / 37 / 34 29 / 29 / 30 39 / 37 / 45 31 / 31 / 23 32 / 27 / 25 27 / 25 / 27 26 / 24 / 25 75 / 75 / 73 27 / 22 / 22 23 / 22 / 15 38 / 46 / 32 24 / 20 / 27 19 / 16 / 13 14 / 13 / 20 12 / 15 / 19 16 / 17 / 11

54.772 36.141 30.010 28.951 28.938 28.592 26.932 25.562 24.294 24.170 20.412 17.287 16.981 16.327 16.056 15.729 14.977

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Double-stranded RNA-binding protein Staufen homolog 2 (STAU2_HUMAN, STAU2) 0/0/0 18 / 13 / 13 14.964 Y-box-binding protein 3 (YBOX3_HUMAN, YBX3) 0/0/0 11 / 11 / 15 12.638 RNA-binding protein Musashi homolog 2 (MSI2H_HUMAN, MSI2) 0/0/0 12 / 12 / 13 12.621 SNW domain-containing protein 1 (SNW1_HUMAN, SNW1) 0/0/0 14 / 10 / 11 11.909 U5 small nuclear ribonucleoprotein 40 kDa protein (SNR40_HUMAN, SNRNP40) 0/0/0 11 / 15 / 8 11.585 Putative RNA-binding protein Luc7-like 2 (LC7L2_HUMAN, LUC7L2) 2/2/0 25 / 20 / 17 11.399 Peptidyl-prolyl cis-trans isomerase-like 1 (PPIL1_HUMAN, PPIL1) 0/0/0 10 / 14 / 7 10.562 Double-stranded RNA-binding protein Staufen homolog 1 (STAU1_HUMAN, STAU1) 4/3/2 35 / 38 / 29 10.021 Protein BUD31 homolog (BUD31_HUMAN, BUD31) 0/0/0 7 /10 / 11 9.575 Protein PRRC2A (PRC2A_HUMAN, PRRC2A) 0/0/0 8 / 13 / 7 9.552 Transformer-2 protein homolog beta (TRA2B_HUMAN, TRA2B) 3/0/0 21 / 18 / 11 9.425 Cleavage and polyadenylation specificity factor subunit 7 (CPSF7_HUMAN, CPSF7) 0/0/0 9/9/8 8.860 Myosin regulatory light chain 12A (ML12A_HUMAN, MYL12A) 0/0/0 10 / 8 / 8 8.850 Ribosome-binding protein 1 (RRBP1_HUMAN, RRBP1) 0/0/0 10 / 9 / 6 8.500 Transformer-2 protein homolog alpha (TRA2A_HUMAN, TRA2A) 5/0/0 22 / 21 / 17 7.982 KH domain-containing, RNA-binding, signal transduction-associated protein 1 0/0/0 7/8/8 7.850 (KHDR1_HUMAN, KHDRBS1) Heterogeneous nuclear ribonucleoproteins A2/B1 (ROA2_HUMAN, HNRNPA2B1) 25 / 31 / 22 229 / 225 / 231 7.735 Zinc finger CCHC domain-containing protein 3 (ZCHC3_HUMAN, ZCCHC3) 0/3/2 20 / 21 / 11 7.732 a The value was obtained by the mean normalized SC of H7N9 NS1 vector (NS1) divided by that of control vector (VC). Proteins with ratios larger than the mean plus two SD (the ratios were above 7.728) and detected in more than two replicates of the NS1 group are defined as NS1 interacting partners.

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Table 2. Enrichment analysis of biological processes for H7N9 NS1-interacting proteins Biological processa Identified proteins involved in process p value mRNA splicing, via SRSF10, CPSF2, DHX8, TRA2A, 5.99 × 10-20 spliceosome PABPN1, CPSF7, SRSF7, PPIL1, TRA2B, XAB2, CRNKL1, BUD31, HNRNPA2B1, ELAV2, SNRNP40, SNW1 Negative regulation of mRNA TRA2B, SRSF10, HNRNPA2B1, SRSF7 9.84 × 10-6 splicing, via spliceosome RNA processing CRNKL1, DHX8, SNRNP40, PABPN1, 4.25 × 10-5 HNRNPDL mRNA 3'-end processing CPSF2, PABPN1, CPSF7, SRSF7 1.39 × 10-4 mRNA export from nucleus SRSF10, CPSF2, HNRNPA2B1, SRSF7 1.07 × 10-3 a The Database for Annotation, Visualization, and Integrated Discovery (DAVID) version 6.8 was applied to functionally annotate enriched proteins, using the annotation category GOTERM_BP_DIRECT. Processes with at least four protein members and p values less than 0.01 are considered significant.

Table 3. Pathway analysis of the proteins interacted with the H7N9 NS1 protein Term in KEGG pathwaya

Identified proteins involved in pathway

Spliceosome

TRA2B, XAB2, SRSF10, CRNKL1, BUD31, DHX8, SNRNP40, TRA2A, SNW1, SRSF7, PPIL1

1.91 × 10-13

mRNA surveillance pathway

CPSF2, MSI2, PABPN1, CPSF7

1.56 × 10-4

a

p value

The DAVID version 6.8 was applied to functionally annotate enriched proteins. The knowledge base used was the KEGG pathway database. Pathways with at least four protein members and p values less than 0.01 are considered significant.

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For TOC only Table of Contents graphic (TOC)

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Figure 1. Identification of cellular proteins that interact with H7N9 NS1. 254x338mm (600 x 600 DPI)


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Figure 2. Protein-protein interaction (PPI) network of the proteins identified to co-immunoprecipitate with H7N9 NS1. 254x338mm (600 x 600 DPI)



Figure 3. Validation of the interactions of H7N9 NS1 with CPSF2, CPSF7, and MOV10. 254x338mm (600 x 600 DPI)


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Figure 4. Analysis of cellular polyadenylated mRNA in cells overexpressing H7N9 NS1. 254x338mm (600 x 600 DPI)



Figure 5. Examination of mRNA polyadenylation processing in cells that overexpress H7N9 NS1. 254x338mm (600 x 600 DPI)


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Figure 6. Determination of the role of H7N9 NS1 in regulation of IFN-β promoter activation. 254x338mm (600 x 600 DPI)



Figure 7. Determination of the role of CPSF7 in H7N9 NS1-mediated suppression of polyadenylation and IFNβ promoter activation. 254x338mm (600 x 600 DPI)


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Figure 8. Examination of pre-mRNA accumulation in influenza A H7N9 virus-infected cells. 254x338mm (600 x 600 DPI)


Interactome Analysis of NS1 Protein Encoded by Influenza A H7N9

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