CHEM. RES. CHINESE UNIVERSITIES 2012, 28(1), 103—107

——————————— *Corresponding author. E-mail: liuhongsheng@lnu.edu.cn Received May 10, 2011; accepted August 24, 2011. Supported by the National Natural Science Foundation of China(No.30671852), the Open Research Fund Program of the

State Key Laboratory of Virology of China(Nos.2010009, 2007007) and the Research Fund of the Key Laboratory of Department of Education of Liaoning Province, China(No.2009S043).

Screening of Proteins Interacting with Nonstructural 1 Protein of H5N1 Avian Influenza Virus from T7-phage Display Library

ZHU Chun-yu1,2, SUN Ting-ting2, ZHAO Jian1,2, WANG Ning1,2, ZHENG Fang-liang1, AI Hai-xin1, ZHU Jun-feng1,2, WANG Xiao-ying3, ZHU Ying4, WU Jian-guo4 and LIU Hong-sheng1,2*

1. Key Laboratory of Animal Resource and Epidemic Disease Prevention of Liaoning Province, 2. Research Center for Computer Simulating and Information Processing of Bio-macromolecules of

Liaoning Province, Academy of Science, Liaoning University, Shenyang 110036, P. R. China; 3. Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, P. R. China;

4. State Key Laboratory of Virology, Wuhan University, Wuhan 430072, P. R. China

Abstract Avian influenza virus(AIV) nonstructural 1(NS1) gene was amplified by real-time polymerse chain reac-tion(RT-PCR) and inserted into pET28a, then transformed into E. coli BL21(DE3) competent cell. With the induction of isopropyl-β-D-thiogalactoside(IPTG) and the purification of Ni-NTA column, we finally obtained purified NS1 protein. T7-phage display system was used to screen the proteins that interacted with NS1 from lung cell cDNA li-brary. The selected positive clones were identified by DNA sequencing and analyzed by BLAST program in Gene-Bank. Two proteins were obtained as NS1 binding proteins, Homo sapiens nucleolar and coiled-body phosphoprotein 1(NOLC1) and Homo sapiens similar to colon cancer-associated antigen. By co-immunoprecipitation and other me-thods, Homo sapiens NOLC1 was found to interact with the NS1 protein, the results would provide the basis for fur-ther studying biological function of NS1 protein. Keywords T7-phage display; Nonstructural 1(NS1) protein; Interacting protein Article ID 1005-9040(2012)-01-103-05

1 Introduction

Nonstructural 1(NS1) protein is the only nonstructural protein in the A-type influenza virus. It is encoded by the eighth gene which can encode 202―237 amino acids. NS1 protein is a control factor which has a wide range of activity, and it is diversity in different viruses[1]. It can be only found in the cells which are infected by the virus. NS1 protein contri-butes significantly to disease pathogenesis by regulating many viruses and host cell[2]. In the early period of the infection, it mainly appears in the nucleus. But in the late period, it may also appear in the cytoplasm, and it can stimulate the body to produce the antibody of NS1 protein[3]. The dsRNA(double- stranded RNA) in the host cell is the obvious signal of the virus infection and replication, and the immune response to the virus can be induced by it[4,5]. NS1 protein in the A-type influenza virus can be highly expressed in the cells which are infected by the virus, its main function is inhibiting IFN(interferon)- α/β-mediated antiviral activity, promoting the level of expres-sion of virus gene, and inhibiting the manufacture of mRNA in the host cells[6,7]. As the causative agent of the virus, NS1 pro-tein of influenza virus can interact with a variety of host pro-teins in cells, by enhancing the expression of viral proteins or changes in the redistribution of host proteins, thereby enhanc-

ing virus pathogenicity and virulence. Human nucleolar and coiled-body phosphoprotein 1

(NOLC1) is a highly phosphorylated protein and exists in the interphase nuclei nucleolus. It can distribute uniformly into cytoplasm, bind to RNA polymerase I and regulate the tran-scription of rRNA during cell mitotic division, so it plays an important role in the regulation of cell growth, inflammation genesis and hepatoma development.

In recent years, both national and international specialists have researched on the NS1 protein evolution, structure, regu-lation of the pathogenicity of the virus and so on. In our study, screening and identifying the proteins which can interact with NS1 protein, we can further reveal the pathogenicity of A-type influenza virus, as well as provide an important reference about the mechanism of the avian influenza virus.

2 Materials and Methods

2.1 Materials

Avian influenza virus A/chicken/Hubei/327/2004(H5N1) was maintained in Wuhan University(China), pET28a vector and BLT5615 bacteria were purchased from Novagen Inc., E. coli DH5α and E. coli BL21(DE3) were preserved in the laboratory.

104 CHEM. RES. CHINESE UNIVERSITIES Vol.28

T7 select human lung cell cDNA library was purchased from Novagen Inc., Ni-NTA His-bind resin was purchased from Novagen Inc., TRIZOL Ls reagent, M-MLV reverse transcrip-tase, RNA OUT and DL-dithiothreitol(DTT) were purchased from Invitrogen; dNTP, DNA Marker DL2000, restriction en-donuclease EcoRI, HindIII, BamHI and T4 DNA ligase were purchased from TAKARA; protein marker was purchased from MBI; pfu DNA polymerase was purchased from Promega; polymerse chain reaction(PCR) product purified(small amount) kit, gel(small amount) kit, plasmid(small amount) extraction kit and isopropyl-β-D-thiogalactoside(IPTG) were purchased from Beijing Tektronix Broad Biological Gene Technology Limited Liability Company(China).

2.2 Methods

2.2.1 Preparation and Purification of Virus Total RNA

The virus was inoculated in 9―10-day-old SPF chick embryo to the proliferation of cysts, influenza virus supernatant cysts of the chick embryo of 250 µL was taken, from which the virus RNA was extracted from the allantoic fluid and dissolved in 40 µL of diethylpyrocarbonate(DEPC) water. 2.2.2 Construction of Recombinant Plasmid pET28a-NS1

Total viral RNA was isolated with TRIZol reagent. The primer was: 5′-AGCAAAAGCAGG-3′. NS1 cDNA clones were amplified by RT(real time)-PCR, forward primer: 5′-AT- GGATCCCAACACTGTGTCGAGCTTTCAGGTAGACTGC- TTTC-3′, reverse primer: 5′-GCGAAGCTTCAAACTTCTGT- CTCAATTGTTCT-3′. PCR product was recovered by Axyprep DNAGel extraction kit, the fragment was sequenced and ana-lyzed by DNASTAR. Vector pET28a and the NS1 gene were doubly digested respectively with BamHI and EcoRI. Recom-binant plasmid pET28a-NS1 was constructed by double en-zyme digestion and sequence analysis. 2.2.3 Expression and Purification of NS1 Protein

Recombinant plasmid pET28a-NS1 was transferred into the expression strain E. coli BL21, positive colonies were screened and cultured to OD600=0.5―0.7(37 °C), with IPTG added to a final concentration of 1 mmol/L, which was cultured for 5 h at 37 °C. Then the expression of NS1 protein was de-tected by SDS-PAGE.

The recombinant bacterium(1 L) was induced and har-vested, solution A(10 mmol/L Tris-HCl, pH=8.0) 5 mL, binding buffer(with 10 mmol/L imidazole) 20―30 mL, lysozyme(100 µg/mL) 1.5 mL, phenylmethanesulfonyl fluoride(PMSF) (1 mmol/L) 30 µL were added in the precipitation and mixed, frozen and thawed twice at –70 °C. The bacterium was dis-rupted on an ultrasonic cell disruption instrument in the ice- bath(power 200―300 W). After centrifugation(12000 r/min, 15 min), the supernatant was preserved. The recombinant protein was purified and recovered according to the manual of Nova-gen Ni-NTA His-bind resins and stored at 4 °C. The recombi-nant protein was dialyzed against double-distilled water overnight at 4 °C. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-

PAGE). 2.2.4 Screening Proteins Interacting with NS1 Pro-tein

(1) Coating the enzyme linked immunosorbent assay (ELISA) plate: the NS1 protein(1―10 µg/mL) was diluted with ddH2O or 1×TBS[10 mmol/L Tris-HCl(pH=8.0)+150 mmol/L NaCl], then it was added in the ELISA plate(100 µL/hole). It was coated at room temperature for 3―4 h or 4 °C overnight. It was washed 3 times with 300 µL of 1×TBS or ddH2O to re-move unconjugated protein, to which the blocking reagent(TBS containing 5% skim milk, 200 µL/hole) was added, and cul-tured at room temperature for 1 h or 4 °C overnight. The plate was blocked and stored at 4 °C.

(2) Screening the phage: the bacterium was cultured till OD600=0.5―0.6, to which IPTG was added to a final concentration of 1 mmol/L, and continued to culture for 30 min. According to the titer of amplified library and the expected number of the phage, the size of the phage library in the first round of screening was calculated. The plate was coated with the phage library according to the result of calculating and placed at room temperature for 30 min or 4 °C overnight, then the plate was washed five times with 1×TBST (200 µL/hole).

(3) Eluting the specific binding phage competitively: T7 elution buffer and 1% sodium dodecyl sulfate(SDS) were added in the holes(200 µL/hole), and cultured at room temperature for 10―20 min, 10 µL of the eluted phage was taken for the de-termination of the titer, and the rest was used for amplification.

(4) Amplification: the eluted phage was added in the competent cell, cultured at 37 °C for 1―3 h, the lysate of bac-terium was centrifugated(8000 r/min, 10 min). The supernatant and polyethylene glycol(PEG)/NaCl(13%) were added in a small tube and mixed overnight at 4 °C and centrifugated(8000 r/min, 20 min). The precipitation was dissolved with TBS buf-fer, centrifugated (12000 r/min, 5 min) again, 10 µL of the su-pernatant was taken for the determination of the titer, the rest was stored as the backup at 4 °C. The cycle of combina-tion-elution-amplification was performed repeatedly, the eluted phage titer was stabilized at a certain number level. After the fifth cycle, the positive plaques were selected at random for phage lysis, and PCR was performed. The primers were as the following: 5′-GGAGCTGTCGTATTCCAGTC-3′(forward) and 5′-AACCCCTCAAGACCCGTTTA-3′(reverse). The result of amplification was identified with 1% agarose gel electrophore-sis.

(5) DNA sequencing and homology search: the sequence (PCR product) was analyzed by the software DNAMAN and NCBI. 2.2.5 Immunoprecipitation Demonstrated Protein Interaction Between NS1 and NOLC1

(1) Constructions of pCMV-NS1 and pCMV-NOLC1: NS1 protein fragment and the vector pCMV-Tag 2B were doubly digested by BamHI and EcoRI, respectively. Then they were ligated by T4 DNA ligase, at 16 °C overnight. Recombi-nant plasmid pCMV-NS1 was constructed and detected by double enzyme digestion and sequence analysis. Phage(clone

No.1 ZHU Chun-yu et al. 105

No.6) was selected and NOLC1 fragment was amplified. NOLC1 fragment and vector pCMV-Tag 2C were digested by BamHI and HindIII, respectively. Then they were ligated by T4 DNA ligase, at 16 °C overnight. Recombinant plasmid pCMV- NOLC1 was constructed and detected by double enzyme diges-tion and sequence analysis .

(2) Immunoprecipitation and Western blot: the 293t cells were cultured. A density of 40%―60% transfection cells was available. The cells on 100 mm plate[(2―5)×10–7] were har-vested and washed with phosphate buffer solution(PBS, 1 mL) and centrifugated(2800 r/min, 5 min). The cell was suspended with radio immunoprecipitation assay(RIPA) and incubated for 10 min at 4 °C, ultraphonic-broken, and centrifugated (10000 r/min, 4 °C, 10 min). The supernatant and negative IgG(1.0 µg) and protein A/G PLUS-Agarose(20 µL) were mixed and incu-bated for 30 min at 4 °C. Then the antibody of NS1 protein1(10 µL) was added to the mixture and incubated for 1 h at 4 °C. After centrifugation(2500 r/min, 4 °C, 5 min), the immunopre-cipitation was harvested and resuspended with 40 µL of 1×SDS buffer. Positive bands were detected by SDS-PAGE and West-ern blot. 2.2.6 Mammalian Two-hybrid System Demonstrated Protein Interactions Between NS1 and NOLC1

The constructions of pVP16-NS1 and pM-NOLC1: phage (clone No.6) was selected and the recombinant plasmid pCMV-NS1 was used as template, NS1 gene and NOLC1 gene were amplified respectively. NS1 gene primers: 5′-ATGGAT- CCCAACACTGTGTCGAGCTTTCAGGTAGACTGCTTTC- 3′(forward) and 5′-GCGAAGCTTCAAACTTCTGTCTCAA- TTGTTCT-3′(reverse); NOLC1 gene primers: 5′-TAGGATC- CGTATGGCGGACGCCGGCATTCGC-3′(forward) and 5′- GCTCTAGATCACTCGCTGTCAAACTTAAT-3′(reverse).

NS1 gene and the vector pVP16 were doubly digested by BamHI and Hind III, respectively, the NOLC1 gene and the vector pM were digested by BamHI and HindIII, respectively. The target DNA fragments and the vector were ligated by T4 DNA ligase at 16 °C overnight. The recombinant plasmids pVP16-NS1 and pM-NOLC1 were constructed and detected by double enzyme digestion and sequence analysis.

Lipidosome-mediated transfection of mammalian cells and the analysis of luciferase activies: HeLa cells were cultured in a 24-well plate at 37 °C; when the cells’ density reached 80%― 90%, HeLa cells were transfecte. After transfection, the cells were cultured for 4―6 h, the culture medium was discarded and the complete medium was added in the 24-well plate. After transfection for 48 h, the transfected cells were analyzed on the dual-luciferase® reporter assay system(Promega). Luciferase activity of each sample was detected on a Turner BioSystems TD-20/20 fluorometer(Turner Designs, Sunnyvale, CA). The transfection analysis of the recombinant plasmid was performed with three times parallelly.

3 Results

3.1 RT-PCR Amplification

RT-PCR amplification products of NS1 protein by agarose

gel electrophoresis were consistented with the expected size of about 690 bp band(Fig.1).

Fig.1 Agarose gel electrophoresis analysis of NS1 protein RT-PCR

Lane M: DL2000 marker; lane 1: NS1 PCR product.

3.2 Enzyme Digestion of Recombinant Plasmid

Recombinant plasmid pET28a-NS1 was double-digested by the restriction endonuclease BamHI and EcoRI to about 5400 bp and 690 bp fragments consistent with the expected results(Fig.2).

Fig.2 Identification of recombinant plasmid pET28a-NS1 Lane M: DL2000 marker; lane 1: pET28a-NS1 digested by BamHI; lane 2: pET28a-NS1 doubly digested by BamHI and EcoRI.

3.3 Expression and Purification of Fusion Protein

IPTG induced by recombinant expression strain BL21 (DE3) bacterium was ultrasound-broken at 4 °C, 12000 r/min centrifugation for 15 min, separated from the supernatant and precipitated. Specific amplification band was detected by pro-tein electrophoresis, and the size was approximate to 28000. The NS1 protein was purified by the Ni-NTA resin purification system. Significant bands were observed by SDS-PAGE gel electrophoresis(Fig.3). The purity was up to 90%, meeting the needs of the hybrid protein so as to avoid interference.

Fig.3 SDS-PAGE analysis of NS1 expression protein and purified protein

Lane M: protein marker(SM0441); lane 1: NS1 protein precipitation in-duced by IPTG(5 h); lane 2: NS1 protein supernatant induced by IPTG(5 h); lane 3: NS1 protein precipitation without IPTG; lane 4: NS1 protein super-natant without IPTG; lane 5: purified NS1 protein.

106 CHEM. RES. CHINESE UNIVERSITIES Vol.28

3.4 Determination of Titer of Binding Phage

From Table 1, it can be seen that with the increase in the number of screening, the ratio of input-output results tends to

Table 1 Input and output phages after different cycles of screen by NS1

Screening number Input Output Ratio of input to output1 8.73×109 1.5×104 5.82×105 2 1.04×109 9.4×104 1.10×104 3 3.68×109 1.2×106 3.06×103 4 4.60×109 1.0×106 4.60×103 5 9.40×109 7.5×106 1.25×103

stabilization, indicating that after the third round of screening, the enrichment of phage has become saturated. The majority of phage could combine with NS1 protein.

3.5 PCR Amplification of Target Gene, Sequence Comparison and Homology Analysis

Twenty-four single phage clones were selected from 248 plaques randomly. The insert fragments were amplified by PCR with specific primer. The results show that most of PCR ampli-fication fragments are about 750 bp(Fig.4). PCR products were recovered and the sequencing results were submitted to the NCBI software to perform homology search and comparison.

Fig.4 Agarose gel analysis of cDNA insert fragment Lane M: DL2000 marker; lanes 1―24: the cDNA insert fragment selected randomly among 248 samples.

No.6 clone sequence result was shown as following: CG- ATTCAGCGGAGGTGCAGCCCCTTCCAAGCCAGCCTCT- GCAAAGAAAGGAAAGGCTGAGAGCAGCAACAGTTCT- TCTTCTGATGACTCCAGTGAGGAAGAGGAAGAGAAG- CTCAAGGGCAAGGGCTCTCCAAGACCACAAGCCCCC- AAGGCCAATGGCACCTCTGCACTGACTGCCCAGAAT- GGAAAAGAAGCTAAGAACAGTGAGGAGGAGGAAGA- AGAAAAGAAAAAGGCGGCAGTGGTAGTTTCCAAATC- AGGTTCATTAAAGAAGCGGAAGCAGAATGAGGCTGC- CAAGGAGGCAGAGACTCCTCAGGCCAAGAAGATAA- AGCTTCAGACCCCTAACACATTTCCAAAAAGGAAGA- AAGGAGAAAAAAGGGCATCATCCCCATTCCGAAGGG- TCAGGGAGGAGGAAATTGAGGTGGATTCACGAGTTG- CGGACAACTCCTTTGATGCCAAGCGAGGTGCAGCCG- GAGACTGGGAAGCTTGCGGCCGCACTCGAGTAACTA- GTTAACCCCTTGGGGCCTCTAAACGGGTCTGGGAAG- GGGTTA.

BLAST results show that positive clones with the Gen-Bank in Homo sapiens NOLC1 amino acid homology reached to 99%.

3.6 Immunoprecipitation Demonstrated Protein Interaction Between NS1 and NOLC1

In order to further verify the interaction between NOLC1 and NS1 protein, the NS1 protein antibody binding beads were used to precipitate the complex that interacted with NS1 protein. Then the flag antibody was used to detect the complex. It can

Fig.5 In vitro co-immunoprecipitation of NS1 and NOLC1 293t cell lysate transfected with pcmv-NS1 and pcmv-NOLC1(lane 1), pcmv-NS1(lane 2) and pcmv-NOLC1(lane 3).

be seen from Fig.5, both NS1-flag and NOLC1-flag were ex-pressed in lane 1 with a strip of NS1 protein(28000) and NOLC1 protein(42000) bands, so it was proved that there were protein interactions between NS1 and NOLC1. In lane 3, be-cause only NS1-flag protein was expressed, there was no preci-pitation of any strip.

3.7 Mammalian Two-hybrid System Demon-strated Protein Interaction Between NS1 and NOLC1

The identifications of plasmids pVP16-NS1 and pM- NOLC1 were shown in Figs.6 and 7, respectively. Because of

Fig.6 Identification of plasmid pVP16-NS1 Lane M: DL2000 marker; lane 1: pVP16-NS1 digested by BamHI; lane 2: pVP16-NS1 digested by BamHI + HindIII.

Fig.7 Identification of plasmid pM-NOLC1 Lane M: marker λDNA/HindIII; lane 1: digested by BamHI/HindIII.

phage display system being not an eukaryotic expression sys-tem, many non-specific protein interactions were screened[8,9]. In order to verify the specific binding of NS1 protein and

No.1 ZHU Chun-yu et al. 107

NOLC1 protein in mammalian cells, we used mammalian two-hybrid system. It can be seen from Fig.8 that the positive control(Fig.8a) and negative control(Fig.8b) are consistent with the experimental requirements. Luciferase value(Fig.8e) of testing samples was significantly higher than basal value(Fig.8c and d). The results show that the NS1 protein specifically binds NOLC1 protein in mammalian cells.

Fig.8 Luciferase activities of mammalian two-hybrid systems

a. Positive control pM-P53 and pVP16-T; b. negative control pM and pVP16; c. substance control pM and pVP16-NS1; d. substance control pM-NOLC1 and pVP16; e. sample pM-NOLC1 with pVP16-NS1.

4 Discussion As a powerful screening tool for protein interaction, phage

display technology has successfully selected a variety of pro-teins, such as antigens, RNA-binding proteins, receptors and the interactive proteins in the transduction pathway[10―12]. In this study, we screened proteins reacting with NS1 protein from the human lung cell cDNA library by T7 phage display tech-nique. Before the screening, we optimized the induction condi-tions to avoid the interference of the hybrid protein. Five cycles of screening were performed until the extent of phage enrich-ment became saturated in order to increase the specificity of screening.

The screened protein, human nuclecular phosphoprotein NOLC1, is a highly phosphorylated protein[13]. It was found in cell nucleolus at interphase. When cells are at mitosis, NOLC1 particles are uniformly distributed in the cytoplasm, which interact with RNA Ι polymerase and participate in the occur-rence of nucleolus, regulate the transcription of rRNA gene. It plays an important role in the regulation of cell growth, the inflammation, the occurrence and the development of liver cancer[14].

The finding of pre-selected protein suggests that NS1 pro-tein may interact with these cellular proteins. Thereby it results in more complex biological functions: on the one hand, it may stimulate immune suppression, on the other hand, the abnormal expression of it may play an important role in the regulation of the pathogenicity and toxicity. It may be the key to the high death rate caused by H5N1 avian influenza virus. At present,

the researches about the interaction of NS1 protein and the cellular protein are relatively rare[15―17]. The results not only expanded the scope of NS1 protein, but also fully demonstrated the feasibility of T7 phage display system screening. It is a simple and effective means that can be successfully applied in the protein screening. It is expected to play an important role in human function genomics and proteomics research[18―20]. References

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