Overexpression and characterization of the 100K protein of Fowl adenovirus-4 as an antiviral target

Accepted Manuscript Title: Overexpression and characterization of the 100K protein of Fowl adenovirus-4 as an antiviral target Authors: Majid Ali Shah, Raheem Ullah, Matteo De March, Muhammad Salahuddin Shah, Fouzia Ismat, Mudasser Habib, Mazhar Iqbal, Silvia Onesti, Moazur Rahman PII: DOI: Reference:

S0168-1702(17)30340-4 http://dx.doi.org/doi:10.1016/j.virusres.2017.06.024 VIRUS 97180

To appear in:

Virus Research

Received date: Revised date: Accepted date:

2-5-2017 26-6-2017 27-6-2017

Please cite this article as: Shah, Majid Ali, Ullah, Raheem, March, Matteo De, Shah, Muhammad Salahuddin, Ismat, Fouzia, Habib, Mudasser, Iqbal, Mazhar, Onesti, Silvia, Rahman, Moazur, Overexpression and characterization of the 100K protein of Fowl adenovirus-4 as an antiviral target.Virus Research http://dx.doi.org/10.1016/j.virusres.2017.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Overexpression and characterization of the 100K protein of Fowl adenovirus-4 as an antiviral target Majid Ali Shaha,b, Raheem Ullaha,b, Matteo De Marchb, Muhammad Salahuddin Shaha,c, Fouzia Ismata, Mudasser Habibc, Mazhar Iqbala, Silvia Onestib, Moazur Rahmana,* a

Drug Discovery and Structural Biology group, Health Biotechnology Division, National

Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. b

Structural Biology Laboratory, Elettra-Sincrotrone Trieste S.C.p.A., Basovizza 34149,

Trieste, Italy c

Vaccine Development group, Animal Sciences Division, NIAB, Faisalabad, Pakistan

*Corresponding author: Moazur Rahman Email: [email protected] Tel: +92 (41) 920-1316 Fax: +92 (41) 920-1322

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Highlights     

High lights Overexpression and high purification yield of the soluble 100K protein was achieved Gel filtration analysis suggest that 100K exist as trimer CD and FT-IR spectroscopy reveal that 100K contains high content of alpha helices Anti 100K antibodies can differentiate vaccinated and FAdV-4 infected chickens

Abstract 100K is an important scaffolding protein of adenoviruses including fowl adenovirus serotype 4 (FAdV-4) that causes inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) in poultry. 100K carries out the trimerization of the major capsid hexon protein of the virus for the generation of new virions inside the target host cells. Despite its critical role for FAdV-4, no structural study, in particular, has been conducted so far. Here, the overexpression of soluble 100K protein was successfully carried out in E. coli using various expression constructs and purification yield of 3 mg per litre culture volume was obtained. Gel filtration chromatography suggested that 100K protein exists in trimeric form. Circular dichroism and Fourier transform infrared spectroscopy clearly reveal that 100K protein folds with a high content of α-helices. The 3-dimentional homology model of the 100K protein, refined with molecular dynamics tools also depicts higher α-helical content within the protein model. Moreover, overexpressed recombinant 100K protein could be used to differentiate vaccinated and FAdV-4 infected chickens on the basis of higher serum anti 100K antibody titres. Our work provides preliminary structural and functional results to study biological role of the 100K protein and for further investigations to develop 100K inhibitors to control IBH-HPS in poultry. Keywords: FAdV-4; Inclusion-body hepatitis-hydropericardium syndrome; 100K protein; 3D model; circular dichroism; infrared spectroscopy 1. Introduction Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of the well-known and widely spread inclusion body hepatitis-hydro pericardium syndrome (IBH-HPS) in poultry (Akhtar, 1994; Shah et al., 2016). Young infected broilers in the age of 3 to 6 weeks are affected by high mortality. Common pathological characteristics are the presence of straw coloured fluid in the pericardial sac (Balamurugan and Kataria, 2004), the enlargement of friable liver with necrotic foci and spots, presence of pale bone marrow and hepatocytes with inclusion bodies in their nuclei (Abe et al., 1998). It has been reported that the severity of the disease increases in birds co-infected with the immunosuppressive agents, like infection bursal disease virus (Mazaheri et al., 1998). Current epidemiological reports show that, in spite of the availability of

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conventional and attenuated vaccines against IBH-HPS, the disease still exists and cause severe losses (Choi et al., 2012; Junnu et al., 2015). FAdV-4 is an icosahedral adenovirus, having 70 to 80 nm diameter and lack envelope. Inside the capsid, a genomic dsDNA encodes 40 proteins of the virus (Shah et al., 2017). The major structural proteins which constitute the 250 capsomeres of the viral capsid are 240 hexon and 12 penton proteins. Hexon are trimeric assembly of three polypeptides chains (330–360 kDa) in conjunction with penton (Hong et al., 2005) that associate to knobbed fibers protruding out of the viral capsid (Ganesh et al., 2002). The mature trimeric hexon can associate with the phospholipids of the target cell membrane bypassing the primary and secondary cellular receptors that constitute the barrier against the viral entry (Balakireva et al., 2003; Yan et al., 2016). Furthermore, they are also important in transporting the viral DNA into the nucleus through the nuclear pore complex (Yan et al., 2016). The maturation of the hexon from the monomer to the trimeric assembly is pivotal for the capsid formation and the completion of viral life cycle inside the host. It is carried out by the non-structural 100K chaperons coded by viral genome during the late phase of cell cycle (Hong et al., 2005; Morin and Boulanger, 1986). It has been experimentally shown in baculovirus system that recombinant hexon can be obtained as folded protein only if co-expressed with 100K chaperon (Hong et al., 2005; Yan et al., 2016). Electron microscopy studies of 100K protein from human adenovirus serotype 2 (AdV-2) shows two terminal globular domains, linked by a rod like connecting domain (Hong et al., 2005). The overall morphology resembled a dumbbell shape. Hexon has been observed to be attached to one of the globular domains of the 100K protein and the Hexon-100K association lead to the formation of homo-trimers of hexon (Hong et al., 2005; Yan et al., 2016). Three units of the 100K protein associate with each of the three separate hexon units to form 800 kDa hexon-100K complex which is transported into the nucleus. The trimeric hexons are unloaded from the complex in the nucleus and 100K protein is released back to the cytoplasm (Yan et al., 2016). Mutations and/or truncations affecting the region between F215 and V420 amino acids in 100K chaperon of human adenovirus serotype 5 (AdV-5) have resulted in the cytoplasmic retention of the both 100K and hexon monomers and failure of the generation of new virions. On the other hand, 100K chaperon containing only the functionally active region from F215 to V420 was able to trimerize and transport the hexon into the nucleus (Koyuncu et al., 2013). Apart from its role in supporting the association of hexon protein for the structural architecture of the virus, 100K also protects the virus from the host defence system. In fact, it stably interacts 3

with granzyme B, a serine protease produced within the granules of natural killer cell and cytotoxic lymphocytes of the host, responsible for the cytotoxicity and apoptosis of virus infected cells. The persistent interaction of the 100K with the Granzyme B suppresses the proteolytic activity and helps in the viral propagation (Andrade et al., 2001). Although there are several studies available for the 100K chaperon from human AdV-2 and AdV-5, structural data, in particular, on the weak homologue from fowl is still missing. To this end, we successfully developed a strategy to overexpress and purify FAdV-4 100K protein from E. coli bacterial cells. Biophysical analysis together with a predicted three dimensional model discovered structural features of the protein. Moreover, using the overexpressed 100K protein an immunoassay, which can be used to distinguish vaccinated and FAdV-4 infected chickens, is presented. 2. Material and Methods 2.1. Genomic FAdV-4 DNA The viral DNA, used in the present study, was obtained from Animal Sciences Division, Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistan. The viral DNA was previously extracted from the liver homogenate of infected birds using phenol chloroform method (Ganesh et al., 2002) during an outbreak of IBH-HPS and its identity was confirmed by PCR (Shah et al., 2011). 2.2. Construction of expression vectors Gene specific primers (Forward: CAA TTC CAT ATG GAA AGC ACC GCC GA and Reverse: GCC GGA ATT CTC AGG TCG ACC ATT CTC TGG GC) were designed using Vector NTI software (Invitrogen) from the sequence of FAdV-4 100K gene retrieved from NCBI GenBank (Acc. No. FR693741.1) and used to amplify 100K gene. The gene was then inserted into corresponding vectors to generate pET28a-PreS-100K and pET28a-TEV-100K (Ullah et al., 2016), using restriction site for Nde I (TTC CAT) and Eco RI (GGA ATT) introduced by forward and reverse primers, respectively. Positive expression clones were identified by colony PCR followed by restriction analysis using Nde I and Eco RI restriction enzymes. PreS represent PreScision or human rhinovirus 3C protease site; whereas TEV represent tobacco etch virus protease site. Finally expression vectors were confirmed by DNA sequencing (Macrogen Inc. Seoul South Korea). 100K gene of FAdV-4, cloned into pET28a vector to generate pET28a-Thrombin-100K (pSMJ-100K) was obtained from Dr. Salah-ud-Din (former PhD student at Health Biotechnology Division, NIBGE) for comparative expression studies (Shah et al., 2011). 2.3. Protein expression and purification 4

E. coli BL21-DE3 (Novagen) competent cells were transformed with pET28a-PreS-100K, pET28a-TEV-100K or pET28a-Thrombin-100K plasmids and incubated overnight at 37 °C on LB-agar plates supplemented with kanamycin (30 μg/mL). Kanamycin-resistant colonies were pre-inoculated into LB media and grown at 37 °C and 220 rpm. Next day overnight cell culture was diluted fifty folds with LB media supplemented with kanamycin and cells were grown at 37 °C and 220 rpm. The growth phase of cells was periodically determined using cell density meter (Bio-Wave) until OD600 of cultures raised to 0.5-0.6, whereby protein expression was induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) for 16 hours at 18 °C and 220 rpm. Cells were finally harvested by centrifugation at 4000 rpm for 10 minutes. Cell pellet was lysed in 20 mM Tris-HCl pH 7.9, 300 mM NaCl, 4 mM β-mercaptoethanol (βME) and 1 mM phenylmethylsulfonyl fluoride (PMSF) using cell disrupter (Constant System Ltd.) at 25 Kpsi. The soluble fraction was loaded on a pre-equilibrated Nickel-nitrilotriacetate (Ni-NTA) agarose column and the protein was eluted in 20 mM Tris-HCl pH 7.9, 100 mM NaCl, 150 mM imidazole and 4 mM β-ME. All fractions containing pure protein were pooled, dialysed against buffer (20 mM Tris-HCl and 100 mM NaCl). Concentration of purified protein was quantified using Nanodrop spectrophotometer (Thermo Scientific) and stored at 4 °C. In order to remove the His6-PreS fusion, His6-PreS-100K protein was treated with human rhinovirus 3C protease (HRV 3C) or PreScission protease in 1:25 ratio (protease:protein) in 20 mM Tris HCl pH 7.9, 100 mM NaCl and 2 mM β-ME buffer (Ullah et al., 2016). The successful cleavage was analysed by SDS-PAGE and western blotting. The identity of the native 100K protein was confirmed by mass spectrometry at Taplin Mass spectrometry facility, Harvard Medical School, Boston (https://taplin.med.harvard.edu/). Briefly, trypsin digested peptides of the 100K protein were subjected to electrospray ionization and analysed by LTQ Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Peptides were detected, isolated and fragmented to produce a tandem mass spectrum (MS/MS) of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program, Sequest (Thermo Fisher Scientific, Waltham, MA). All databases include a reversed version of all the sequences and the data was filtered between a one and two percent peptide false discovery rate which is based on using a decoy database of false sequences (Beausoleil et al., 2006). )). Gel filtration (GF) chromatography was performed using Superdex 200 column (GE Healthcare Life Sciences) on ÄKTA system (GE Healthcare Life Sciences) using gel filtration buffer (20 mM Tris-HCl pH 7.9, 100 mM NaCl and 4 mM β-ME). An estimation of the 5

molecular weight was done using a calibration curve, obtained upon running Ferritin, Alodalase, Conalbumin, Ovalbumin, Ribonuclease A and Aprotinin in the same gel filtration buffer through the Superdex 200 GF column. For calibration curve, Kav constant for each standard protein was determined using the equation, Kav = Ve-Vo/Vt-Vo. Here Ve represents elution volume of each standard, Vo (Void volume (8 mL) and Vt (Bed volume (24 mL)). Kav constant was obtained for different standard molecular weight proteins and was plotted against molecular weights of all the standards in linear regression to derive the formula for molecular weight of any protein with unknown molecular weight as described Ln(x) = (y-0.8996) / -0.121. The molecular weight of the 100K protein (x) was calculated using the above equation by putting the Kav (y) of the 100K. 2.4. Western blot analysis Protein samples separated on SDS-PAGE along with pre-stained protein marker (ACTGene, ACT-1DWW24), were transferred to nitrocellulose membrane using semi-dry blotter (Trans blot Semi Dry Bio-Rad system) for 30 minutes at constant voltage of 30 V. The membrane was washed with 3% BSA and 0.05% Tween 20 for 1 hour to block non-specific binding sites and then treated with primary anti-histidine antibodies (dilution 1:3000 in TBST with 3% BSA) for 1 h. The membrane was washed three times with TBST and one time with TBS. Anti-mouse IgG (A3562-Sigma) conjugated with alkaline phosphatase were diluted in 1:10,000 in TBST with 3% BSA to facilitate the binding on the membrane. Finally the membrane was washed four times with TBST. Western blot signals were detected after treating the membrane with bromo-chloro-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) substrate (B5655-Sigma) for 1-2 mints and the reaction was stopped with 2 mM EDTA. 2.5. Fourier Transform Infrared spectroscopy (FT-IR) Pure 100K protein in 20 mM Tris-HCl pH 7.9 buffer containing 100 mM NaCl was concentrated to 2 mg/mL using Millipore Y-50 filter with 50 kDa cut-off. The sample (30 μL) was dried on platinum attenuated total reflectance plate (diamond crystal) to form a dehydrated film. Spectra were obtained in absorption mode using Bruker FT-IR spectrometer with OPUS software in the range of 4000 to 500 cm-1 (Goormaghtigh et al., 1994a) at 1 nm resolution, 256 scans co-addition and Blackman-Harris-3-term apodization. Spectra for buffer alone were also recorded and subtracted from the protein spectra to eliminate the absorption of water at 1645 cm−1 in the amide I region. The protein spectra were manipulated until a flat baseline was obtained in 2000–1770 cm-1 range of spectrum. Spectra in amide I region (1700–1600 cm-1) was smoothened using 2 adjacent points and second derivative was obtained using Origin 6

software version 7.0 (Wass, 2002) (OriginLab Corporation, USA). Spectra in amide I region was split into discrete bands by non-linear peak fitting using Galactic PeakSolve™ software (version 1.05). Band assignments for interpretation of spectra were done on the basis of previous measurements (Goormaghtigh et al., 1994a; Susi and Byler, 1986). 2.6. Circular Dichroism (CD) Purified 100K protein was dialysed in 10 mM potassium phosphate pH 7.4 buffer for 16 hours at 4 °C. Approximately 300 μL protein sample (0.064 mg/mL) was transferred into a Hellma quartz cuvette of 1 mm path length. Data were recorded both for protein sample and buffer in the range of 185-260 nm using Jasco J-1500 spectropolarimeter with constant nitrogen flushing at 25 °C, speed 1 nm/15 sec, sensitivity 50 mdeg, bandwidth 0.5 nm, resolution 1nm and response time 15 sec. Final CD data were further analysed using DichroWeb Server and Contin-LL (Sreerama and Woody, 2000) following Provencher et al. (Provencher and Gloeckner, 1981). Thermal assays were done to check folding stability at 50 °C and 90 °C: the protein at 0.064 mg/mL was incubated for 10 minutes at the respective temperatures and CD spectra were recorded after slowly cooling the samples to the room temperature. 2.7. Bioinformatics analysis and 3D model generation 100K protein sequence (GenBank ID: CBX24513.1) was submitted to the web based iterative threading server I-TASSER (Zhang, 2008). A sequence-structure alignment against the protein data bank (PDB) was performed to generate protein models for 100K protein; the model with best homologous template was subjected to energy minimization and refined using AMBER99 forcefield (Case et al., 1999) to exclude bad contacts and to optimize molecular interfaces. AMBER99 is available in Molecular Operating Environment (MOE) (Version 2015) (Inc., 2016). The resulting model was further analysed and refined stereo-chemically with Galaxy refinement tool (Heo et al., 2013), while ERRAT (Colovos and Yeates, 1993) and VERIFY_3D (Eisenberg et al., 1997) were finally used for validation of the 100K protein model. PyMol molecular graphic system (DeLano, 2002) was used to produce good quality image of the protein model. 2.8. Antibody production and ELISA One day old chicks were purchased from commercial hatchery (Al-noor Chicks (pvt) Ltd. Faisalabad). Experiments were carried out following the regulations of the Institutional Animal Care and Use Committee (IACUC) of Animal Sciences Division, NIAB, Faisalabad. Chicks were reared for 14 days, after which they were divided into three separate groups, each groups containing three chickens. The purified 100K protein was dialysed against phosphate buffer saline (PBS) and then adjuvanted with highly immunopotent Freund's complete agent 7

(FCA) (Shah et al., 2012) to enhance immunogenicity. Members of group A were subjected to the recombinant 100K protein (100 µg/bird) by intramuscular injection at 14th day of age. Members of groups B and C were also injected intramuscularly with the commercially available vaccine and PBS respectively. Chickens were injected subcutaneously with 500 µL (protein + FCA) at two different sites (250 µL at one site) to reduce the chances of inflammation. Finally, a booster dose of the recombinant protein (100 µg/bird), vaccine and PBS was injected at 28th day adjuanted with FCA into all groups A, B and C. To determine serum antibody titers, blood samples were collected from wing veins at days 1, 14, 28, 35 and 42 from the three groups. The experimental chickens were challenged with pathogenic FAdV4 at 35th day. Enzyme linked immunosorbent assay (ELISA) (Ojkic and Nagy, 2003) was performed to determine the antibody titers against the 100K protein, in treatment and control groups. 100K protein (4 μg) was coated in each well of 96-well ELISA plate as antigen. Serum samples, which were collected at different days, were added to each well as primary antibody at various dilutions in PBS. Alkaline phosphatase conjugated anti-chicken IgG Antibodies (A3562Sigma) were used as secondary antibodies having p-nitrophenyl phosphate substrate (MP Biomedicals). The reaction was stopped using 3 M NaOH and monitored at 405 nm using ELISA microplate reader (Shenzhen heales technology, Co, Ltd). 3. Results 3.1. Strategy for the generation of expression constructs Nucleotide sequence of 100K gene was cloned into pET28a-PreS and pET28a-TEV (Ullah et al., 2016) in reading frame with coding sequences for His6-PreS and His6-TEV at the 5′ end, respectively. Resultant vectors were named as pET28a-PreS-100K and pET28a-TEV-100K and overall strategy is shown in Fig. 1. pET28a-Thrombin-100K vector (pSMJ-100K) containing 100K gene plus nucleotide sequences for His6-Thrombin protease site at the 5′ end was obtained from an earlier study (Shah et al., 2011). 3.1. Overexpression, purification and characterization of FAdV-4 100K The 100K protein from E. coli BL21-DE3 cells harbouring various constructs (pET28a-PreS100K, pET28a-TEV-100K and pET28a-Thrombin-100K) was expressed to produce proportionate quantities for comparative analysis as described in methods section. High level expression of His6-PreS-100K and His6-TEV-100K proteins was obtained compared to His6Thrombin-100K and can be seen at approximately 97 kDa position on SDS-PAGE (Fig. 2). Further, each 100K protein variant was purified by Ni-NTA chromatography (Fig. 2). A higher purification yield amounting to 3.2 and 2.9 mg per litre culture volume was achieved using 8

pET28-PreS-100K and pET28-TEV-100K, respectively, compared to 0.02 mg per litre for pET28-Thrombin-100K expression vector. Among the different expression constructs, the purified protein yield from pET28-PreS-100K, containing the HRV 3C protease target site, was highest, so this construct was used in further studies. To enhance soluble expression and yield of the purified 100K protein from pET28-PreS100K/BL21 (DE3), inducer concentration (IPTG) and post-induction temperature conditions were optimized. Various IPTG concentrations (0.25 mM, 0.5 mM, 0.75 mM and 1 mM) tested had no significant effect and approximately similar quantities of the purified protein (3.3 mg per litre culture volume) were obtained at different concentrations of IPTG except the lowest IPTG concentration which produced 2.9 mg per litre (Fig. 1S). Whereas in case of temperature optimization, higher yield (3.8 mg per litre) was achieved at the post induction temperature 20 °C and 28 °C compared to 37 °C (Fig. 2S). The higher cleavage rate shown by HRV 3C protease compared to TEV protease (Ullah et al., 2016), allow us to successfully cleave the His6-PreS fusion from His6-PreS-100K and to obtain a native 100K protein. The cleavage was confirmed by SDS-PAGE electrophoresis (Fig. 3A) and western blotting (Fig. 3B). Further, mass spectrometry confirmed the identity of the purified native 100K by probing identification of several peptide fragments (obtained by trypsin digestion) corresponding to 100K protein sequence of FAdV-4 (Fig. 3C). After a final gel filtration chromatography step, we were able to obtain the native pure homogenous protein as shown in Fig. 3SA and 3SB, which was used for further structural and immunoassay studies. Since 100K protein elutes as 292 kDa protein (Fig. 4A and 4B), we suggested the existence of a trimeric form. 3.2. Experimental data indicate a global α-helix structure of FAdV-100K Both FT-IR and CD spectroscopy techniques revealed that the 100K protein folds into regular structure composed mainly by α-helices. To provide quantitative estimate of secondary structure content of the protein, hydrated film of the 100K protein was investigated by FT-IR. The measured amide I region of the spectrum (Fig. 5A and 5B) was reproduced by fit of eleven components, dominated by those indicative of α-helix (Fig. 5C) (Arrondo et al., 1993; Goormaghtigh et al., 1994a; Goormaghtigh et al., 1994b). Quantitative analysis of the spectrum predicted the presence of 41% α-helix and 28% β-turns plus unordered regions (Table 3.1). CD spectra of the protein showed characteristic minima around 208 and 220 nm and a maximum between 190 and 195 nm (Fig. 6A) (Kelly et al., 2005). α-helical content (41%) estimated by the analysis of the CD spectrum (Fig. 6B and Table 3.1) agreed well with that determined from FT-IR spectra whereas content of β-turns and unordered regions (54%) are 9

different. This discrepancy, particularly in case of β-turns and unordered contents, might be due to different algorithms used for analysis of FT-IR and CD data. Further, thermal stability of the 100K protein was analysed by measuring CD ellipticity at various temperatures 25 °C, 50 °C and 90 °C. CD spectra revealed that at higher temperatures (50 °C and 90 °C), a decrease in amplitude of the molar residual ellipticity occurred which is indicative of the fact that the 100K protein is thermo-labile (Fig. 6C). 3.3. Three-dimensional model of the 100K protein The three dimensional structure of the 100K protein was obtained after submitting the protein sequence into I-TASSER web server, which produced 5 models for 100K sequence, using 10 different templates threading alignment. The model with highest confidence-score -1.83 and TM-score (Template modelling score) 0.5 was selected (TM-score ≥ 0.4 means that two structures are significantly similar) (Zhou and Skolnick, 2012). The refinement of the model resulted in the improvement of the stereo-chemical properties and the percentage of the Rama favoured/allowed residues increased up to 97% from initial 88.5% while the number of outliers was reduced to 3% from 11.5%. ERRAT and Verify_3D scores were also improved after refinement and there values were found 88.70% and 70.19% as compared to the initial values 80.53% and 58.02%, respectively. A final refined image of the protein model was produced as shown in Fig. 7. The model showed a higher proportion of α-helical structure (49%) along with coiled loops and turns which constituted the remaining 51% of the protein model. Similar proportion of α-helical content was obtained by FT-IR and CD spectroscopies (Table 3.1) which may validate the model. 3.4. Comparative immune response against 100K protein and vaccine Immune responses against recombinant 100K protein and inactivated vaccine were compared in chickens. Antibody titers were determined in sera using ELISA as described in material and methods. It was found that significantly high serum antibody titres were present against the 100K protein in chickens (group A), as compared with commercially available inactivated vaccine (group B) and control group which was injected with PBS (group C) (Fig. 8). It was also revealed that after experimental infection of FAdV-4 (at 35th day), antibody titers against recombinant 100K protein were rapidly increased (at 45th day) in birds of all groups who recovered from infection. Whereas before experimental infection, serum antibody titers against recombinant 100K protein of vaccinated group (B) and control group (C) were almost same and significantly less compared to group (A) (Fig. 8). So, it is suggested that ELISA against the recombinant 100K protein could be used to develop strategies for differentiating infected from vaccinated animals (DIVA). For DIVA purpose ELISAs can be developed against non10

structural viral proteins (NSP), as immune system of host is exposed to NSPs when viral replication occurs (Sørensen et al., 1998). 100K is a late phase NSP protein in the life cycle of FAdV and assists in the generation of new virions (Koyuncu et al., 2013). It is understandable that high antibodies titres against 100K may indicate viral infection and replication. Whereas the vaccinated birds likely to have low antibody titres against NSP because of low rate of viral replication. Likewise, in this study, serum antibody titers against 100K protein were at basal level in vaccinated group (B) and unvaccinated control group (C) prior to infection at 35 th day. While 10 days post infection at 45th day, the antibody titers against 100K protein were increased rapidly in all groups including vaccinated and unvaccinated control groups which confirm the above mentioned statement about post-infection increase in antibodies against NSP. 4. Discussion FAdV-4 is the causative agent of IBH-HPS, one of the major prevalent diseases in poultry. Poultry industry, worldwide suffers huge economic losses due to FAdV-4 infections and epidemics arise every year due to inefficacy of the currently available vaccines against the virus (Junnu et al., 2015). Structural studies of the vital proteins of infectious virus provide basis for the development of antiviral drugs for the control and inhibition of infectivity by the virus (Ehebauer and Wilmanns, 2011). Electron Microscopy data revealed that 100K chaperon from human AdV-2 and AdV-5 assists in folding and self-association of the hexon protein for capsid formation and nuclear transport, after co-expression of 100K-hexon protein in baculovirus expression system (Hong et al., 2005). However, structural and functional data is lacking for 100K protein corresponding to pathogenic adenovirus serotypes in avian hosts including fowl adenoviruses (e.g., FAdV-4), causing disease outbreaks in poultry. Also 100K genes of AdV2 and AdV-5 have little sequence similarity (< 40 %) with the corresponding genes present in avian adenoviruses. Sufficient quantities of the highly purified protein are required to characterize the 100K protein both structurally and functionally. Previous attempts to express 100K of FAdV-4 were successful to a very little extent and metal-ion affinity chromatography yielded a meagre quantity of the protein sparsely visible after SDS-PAGE analysis (Shah et al., 2016). In this study, we set out to produce the protein using alternate expression strategies using E. coli expression system and the conditions were optimized for overexpression and achieving higher yield of purified protein (~3 mg per litre culture volume) in soluble form, sufficient for performing structural and functional studies. Gel filtration chromatography revealed the trimeric nature of the 100K protein. The native size of the 100K protein was found to be 292 11

kDa which was three times higher than the theoretical mass of the protein (97 kDa). This showed that 100K monomers possess certain interactive regions within the protein for selfassociation to form mature protein. Previous studies using 100K from human AdV-5 revealed that 100K-hexon association and trimerization of the hexon can be carried out by a truncated 100K protein containing only the conserved 215-420 amino acid region (Koyuncu et al., 2013). In a recent study, it was suggested that only full length 100K protein could interact and lead to self-assembly of the hexon protein for trimerization and generation of new virions inside the host (Yan et al., 2016). This contradiction urges the need of the structural studies and development of 3-dimensional model for the 100K protein as well as crystallization studies to determine the 100K structure, which will be now possible pertaining to availability of the homogeneous and highly purified 100K protein of FAdV-4. There is no structure available either for 100K protein or any of its close homologue in protein data bank (PDB). Therefore, we used web based I-TASSER bioinformatics tool as well as biophysical techniques for mining into the structure of the 100K protein. 100K protein possesses 19.9% identity and 31% similarity with the assembly polypeptide 2 protein whose structure is already available PDB (PDB ID: 4UQI.A). The protein helps as plasma membrane adapter to recruit and polymerize clathrin to carry out clathrin mediated endocytosis of cell surface proteins (Kelly et al., 2014). High proportion of homology is associated with the N-Terminal regions of both proteins. At C-terminal, 100K possess homology (20.1% identity and 32.4% similarity) with tRNA (met) cytidine acetyl-transferase (PDB ID: 2ZPA), whose structure was solved to study its RNA helicase module responsible for the modification of a tRNA anticodon; to reveal its role in carrying out efficient translation process in E. coli (Chimnaronk et al., 2009). Hypothetical model of the 100K showed highest proportions of α-helical structures connected by loops sequences. Biophysical spectroscopy techniques i.e., FT-IR and CD also confirmed highest proportions of α-helical structures and coiled loop sequences present within the protein. The immunogenicity of the 100K protein was determined in chickens.. Higher quantities of antibodies were detected against the purified protein, which can be used for the development of rapid ELISA based methods for the diagnosis of FAdV-4 infections in poultry. Recombinant protein-based ELISA is a rapid diagnosis method for the infectious diseases in large flocks and have been practiced for different poultry diseases in past using the protein as antigen for detecting antibody activity against the pathogen inside the host. The recombinant protein-based ELISA has enhanced specificity, sensitivity and accuracy as the recombinant protein antigen is immuno-dominant and unlike whole cell preparations (Sahle and Burgess, 2002), commonly

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used in commercially available kits (Chaka et al., 2013), recombinant protein based ELISA has less non-specific moieties associated (Das and Kumar, 2015). Both structural and nonstructural recombinant proteins have been used as antigen for the diagnosis of diseases like infectious bursal disease virus (Wang et al., 2008) and Newcastle disease (Das and Kumar, 2015), respectively, in poultry with higher efficiency as compared to standard haemagglutinin inhibition assay (Das and Kumar, 2015). Particularly our results showed the use of nonstructural proteins for the production of antibodies against the purified 100K protein in chickens which could be used to differentiate infected from vaccinated animals (DIVA) (Sørensen et al., 1998). Together, overexpression of the 100K protein and high yield of the purified folded protein as analysed by FT-IR and CD will allow to determine 3-dimensional structure of 100K alone and with its partners such as hexon to decipher the point of interactions of 100K with the hexon protein. Inhibition of such interactions by rationally designed antiviral drugs will lead to development of new virus inhibitors needed in the poultry industry. Moreover, ELISA assay developed using the overexpressed 100K protein can be used to distinguish vaccinated and chickens infected with FAdV-4. Competing interest The authors declare that they have no competing interest. Acknowledgements We thank I. Hussain, LUMS University, Pakistan, for providing access to record IR spectra. This work was supported by the Higher Education Commission (HEC) of Pakistan (grant No. 20-2138 (awarded to M. Rahman) and, HEC Ph.D. studentship and ICTP-IAEA STEP fellowship (awarded to Shah MA). The authors declare that they have no conflict of interest.

13

Figures Captions Fig. 1. Cloning strategy for generation of pET28-PreS-100K and pET28-TEV-100K plasmids containing PreS (HRV 3C) and TEV protease site, respectively. Fig. 2. Expression and western blot analyses of 100K in E. coli strain BL21-DE3 using various expression vectors for comparative expression studies. 100K protein expressed in E. coli strain BL21-DE3 harboring pET28-PreS-100K [A], pET28-TEV-100K [B] and pET28-Thrombin100K (pSMJ-100K) vector [C]. Protein expression was carried out using 1 mM IPTG as inducer and post induction temperature of 20 °C for 16 hours. The protein was purified using Ni-NTA affinity chromatography. Lane 1, 2 and 3 in each panel shows unbound proteins fractions, pellet of the expressed cells and supernatant after cells lysis, respectively. Lane 4 & 5 shows the wash fractions after washing the Ni-NTA columns at 30 mM imidazole. The pure protein obtained after elution from the Ni-NTA columns with 150 mM imidazole has been shown in lane 6-9. The mobility of marker proteins of known molecular mass are shown on the left. [D] Panel shows western blot results of the oligohistidine tagged 100K protein expressed in pET28-PreS-100K (Lane 1), pET28-TEV-100K (Lane 2) and pET28-Thrombin-100K (Lane 3), respectively. Smaller quantity of His6-PreS-100K and His6-TEV-100K was run on SDSPAGE compared to His6-Thrombin-100K for better western blot results. Fig. 3. Removal of oligo-histidine tag and confirmation of the native 100K protein using western blotting and mass spectrometry analysis. [A] PreS or HRV 3C protease treated His 6PreS-100K protein was run on the SDS-PAGE along with the untreated His6-PreS-100K as control. Lane 1 shows uncut His6-PreS-100K protein while lane 2 shows 100K protein after the removal of the His6-tag. [B] shows western blot results of His6-PreS-100K protein (lane 1′) and 100K protein, after the removal of His6-tag (lane 2′). [C] Mass spectrometry analysis of the pure 100K protein after removal of His6-tag. MS analysis identified various peptide fragments corresponding to the 100K protein after trypsin digestion shown in increasing mass order. The identification of peptides was done through the use of a high mass accuracy scan to determine the mass of the intact peptide and the fragmentation pattern (MS/MS). The data was then filtered using a reverse database to a very low false discovery rate (1 to 2%) to generate authentic results. Fig. 4. Oligomerization analysis of the 100K protein by gel filtration chromatography. Gel filtration chromatography was performed using Superdex 200 10/300 GL column for molecular weight standards. [A] Kav versus Mw plot calibration curves were obtained using known Mw standards in appropriate volume (100 µl). Respective elution peaks were determined for ferritin (440 kDa, 10.1 mL), aldolase (158 kDa, 12.04 mL), conalbumin (75 kDa, 13.7 mL), ovalbumin 14

(44 kDa, 14.33 mL), ribonuclease A (13.7 kDa, 17.22 mL), aprtinin (6.5 kDa, 18.53 mL). Kav value for each standard was calculated as the partition coefficient relative to the void and bed volumes of the column. [B] represents analysis of the 100K protein by gel filtration chromatography using Superdex 200 10/300 GL column. The elution volume of the protein was 10.7 mL. Apparent molecular weight of 100K is 292 kDa as calculated from the equation: y = -0121 ln(x) + 0.8996. Fig. 5. Analysis of secondary structure of the 100K protein by FT-IR spectroscopy. [A] FTIR spectra of hydrated film of PBS buffer (red dotted line) and the 100K protein (black continues line) recorded at a concentration of 2 mg per mL. [B] The 100K protein spectrum obtained by subtraction from PBS buffer spectrum. [C] Top, secondary derivative and bottom, Amide I region of the FT-IR spectrum of a hydrated film of 100K and bands are obtained by deconvolution. The dotted grey line shows the curve fitted using component bands. Fig. 6. Analysis of secondary structure and thermal stability of the 100K protein by CD spectroscopy. [A] CD spectra of the 100K protein (continuous line) and potassium phosphate buffer (dotted line). [B] The 100K protein spectra subtracted from buffer. [C] Thermal stability analysis of the 100K protein. The protein was heated up to 90 °C and 50 °C followed by cooling to 25 °C and CD measurements were recorded for observing changes in overall protein structural conformation. The change in the ellipticity denotes the thermal lability of the protein at higher temperatures. Fig. 7. Hypothetical model of the 100K protein of FAdV-4. Hypothetical model of the 100K protein was generated using I-TASSER bioinformatics tool and refined using AMBER99 (Case et al., 1999) and Galaxy refinement tool (Heo, Park, & Seok, 2013). Fig. 8. Antibody titers determined in serum after immunization of different groups of chickens with the 100K protein, live vaccine and control (PBS) by ELISA. Blood serum samples were collected at different days post immunization.

15

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Table 3.1 Comparative secondary structure analysis: table summarizes percentages of α-helix, β-sheet and loop structures for the 100K protein experimentally obtained by FT-IR and CD spectroscopy and predicted from the computes 3D model. α-helices (%)

β-turns & unordered regions (%)

FT-IRa

40

28

CDb

41

54

I-TASSERc

49

51

a

Band assignments for interpretation of spectra shown in Fig. 5C were made on the basis of previous measurements (Goormaghtigh et al., 1994a; Goormaghtigh et al., 1994b).bCD spectrum was analyzed using DichroWeb server (Whitmore and Wallace, 2004; Whitmore and Wallace, 2008) developed for structure analysis program as discussed in “Material and methods” c I-TASSER, Iterative Threading Assembly Refinement server, used for the structural and functional prediction of a target protein sequence (Zhang, 2008)

19

Fig. 1.

20

Fig. 2.

KDa

[A] M 1 2 3 4 5 6 7 8 9

[B] 1 2 3 4 5

6 7 8 9

[C] 1 2 3

4 5 6 7 8 9

[D] 1

2 3

100 70 50

30

21

Fig. 3. [A] M

[B] 1

2

1′

[C] 2′

245

Amino Acid Position

135 100

500-511

1203.38

640-653

1533.74

AIVDVLMDGDR LTPELWANAYLDK

75

712-720

2051.20

DPDTGEVLTPQPDLQAGAAR

247-267

2072.25

QGVTVDDGLGDEVSPITELK

389-407

2112.42

AQEVLHHTFHHGFVALIR

388-407

2240.60

KAQEVLHHTFHHGFVALIR

657-675

2337.54

DYHPFEVVHLPQHEEAFSR

245-267

2386.64

WKQGVTVDDGLGDEVSPITELK

708-732

2498.73

GVYKDPDTGEVLTPQPDLQAGAAR

411-436

2814.18

VNLSNYATFHGITYNDPLNNCMLAK

63

48

Mass

Sequence

22

Fig. 4.

23

Fig. 5.

turns β-sheet 1684.0 1689.8

α-helix

turns turns

1525

1600

1675

1750

(cm-1)

0.12

Absorbance

random α-helix

Amide II

Wavenumber [B]

1647.8 1653.6 1660.8 1669.5 1673.9

0 1450

β-sheet

0.03

1617.4

Absorbance

Amide I 0.06

1636.2

Subtracted= Protein - Buffer

β-sheet β-sheet β-sheet

[C] 0.09

1624.2 1629

[A]

Protein Buffer

0.09

0.06 0.05 0.04

0.06

0.03

0.03

0.02 0.01

0 1450

1525

1600

1675

Wavenumber (cm-1)

1750

1600

1625

1650

1675

1700

Wavenumber (cm-1)

24

Fig. 6.

[B]

[A] 10

CD ellipticity (mdeg)

100K

Buffer

5

0 -5 -10

100K-Buffer

5 0

-5

-10

185

205

265

245

225

185

Wavelength (nm)

205

225

245

265

Wavelength (nm) [C]

CD ellipticity (mdeg)

CD ellipticity (mdeg)

10

10

25 ⁰C

50 ⁰C

90 ⁰C

5 0 -5 -10 185

245 225 205 Wavelength (nm)

265

25

Fig. 7.

26

Fig. 8.

PBS

Vaccinated

100K Protein 1.5

Absorbance at 405 nm

1.2

0.9

0.6

0.3

0 0

21

28

35

42

45

Days post immunization

27