key: cord-329403-jzrlywfe
authors: Teo, Su Hui Catherine; Wu, Jian-Ping; Mok, Chee-Keng; Tan, Yee-Joo
title: A NS1-binding monoclonal antibody interacts with two residues that are highly conserved in seasonal as well as newly emerged influenza A virus
date: 2019-03-06
journal: Pathog Dis
DOI: 10.1093/femspd/ftz012
sha: 
doc_id: 329403
cord_uid: jzrlywfe

The non-structural protein 1 (NS1) of influenza A virus (IAV) is a multifunctional protein that antagonizes host antiviral responses, modulating virus pathogenesis. As such, it serves as a good target for research and diagnostic assay development. In this study, we have generated a novel monoclonal antibody (mAb) 19H9 and epitope mapping revealed that two residues, P85 and Y89, of NS1 are essential for interacting with this mAb. Furthermore, residues P85 and Y89 are found to be highly conserved across different IAV subtypes, namely seasonal H1N1 and H3N2, as well as the highly pathogenic H5N1 and H5N6 avian strains. Indeed, mAb 19H9 exhibits broad cross-reactivity with IAV strains of different subtypes. The binding of mAb 19H9 to residue Y89 was further confirmed by the abrogation of interaction between NS1 and p85β. Additionally, mAb 19H9 also detected NS1 proteins expressed in IAV-infected cells, showing NS1 intracellular localization in the cytoplasm and nucleolus. To our knowledge, mAb 19H9 is the first murine mAb to bind at the juxtaposition between the N-terminal RNA-binding domain and C-terminal effector domain of NS1. It could serve as a useful research tool for studying the conformational plasticity and dynamic changes in NS1.

Influenza A virus (IAV) belongs to the Orthomyxoviridae family and is composed of eight single-stranded, negative-sense RNA segments. Of these, the NS segment is the smallest and it encodes for the non-structural protein 1 (NS1) and nuclear export protein (NEP, also known as NS2). NS1 protein of IAV is a potent antagonist of the cellular antiviral interferon (IFN) response. Although it is not incorporated into the virus, it exerts multiple functions through the interaction with a large number of cellular components either in the cytoplasm or the nucleus. NS1 is able to inactivate the host antiviral interferon responses through a variety of mechanisms to facilitate replication during infection (Kochs, Garcia-Sastre and Martinez-Sobrido 2007) . Although strain dependent, the general functions of NS1 include inhibiting the antiviral effects of 2'-5'-oligo (A) synthetase (OAS)/RNase L and protein kinase R (PKR) (Li et al. 2006 ); activating phosphoinositide 3kinase (PI3K) (Hale et al. 2006) ; inhibiting host gene expression by interacting with components of nuclear transport (Melen et al. 2007 ) and blocking mRNA export (Fortes, Beloso and Ortin 1994) . Moreover, NS1 is also involved in suppressing adaptive immunity by attenuating human dendritic cell maturation and the capacity of dendritic cells to induce T-cell response (Fernandez-Sesma et al. 2006) .

The NS1 protein is composed of 230-237 amino acids depending on the specific strain (Hale et al. 2008) . NS1 has two wellcharacterized functional domains, namely, N-terminal RNAbinding domain (RBD) and C-terminal effector domain (ED). The RBD is composed of the first 73 amino acids of NS1, followed by the linker region (LR) at residues 74-88, and residues 88-202 constitute the ED (Hale 2014) . NS1(RBD) contains the residues critical for the double stranded RNA (dsRNA) binding. NS1(ED) is responsible for interaction with various cellular proteins including PKR (Lu et al. 1995) , the 30-kDa subunit of cleavage polyadenylation specificity factor 30 (CPSF30) (Nemeroff et al. 1998; Noah, Twu and Krug 2003) , and some PDZ domain ligand proteins (Jackson et al. 2008) .

Given that NS1 is highly expressed in infected cells and plays multiple functions, we have previously produced monoclonal antibodies (mAbs) binding to NS1 as tools for research and diagnostic assay development. Two of them were found to bind to NS1(RBD) and are able to bind different subtypes of IAV (Tan et al. 2010) . In this study, we further generated and characterized another mAb, named as 19H9, which binds to two highly conserved residues P85 and Y89 in NS1.

A549, 293T and MDCK cells were purchased from American Type Culture Collection (Manassas, VA, USA). A549 cells were cultured in Minimum Essential Medium (Gibco). 293T and MDCK cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen). Both media were supplemented with 10% fetal bovine serum (Hyclone), penicillin (10 000 units/ml)streptomycin (10 mg/ml) solution (Sigma-Aldrich). All cell lines were maintained at 37 • C with 5% CO 2 .

The NS1 fragment (residues 85-215) of A/Puerto Rico/8/1934(H1N1) was PCR amplified from a full-length NS1 gene (Accession NO.: ABD77680.1) and cloned into the pGEX-6P-1 vector (GE Healthcare). The GST-tagged wild-type and mutant NS1(ED) proteins were expressed and purified as described previously (Tan et al. 2010) . The purified fusion proteins were dialyzed against phosphate-buffered saline (PBS) and the final concentration was determined using the Coomassie Plus protein assay reagent (Thermo Scientific). The purified proteins were concentrated to 3 mg/ml in a Centrprep-10 (Amicon) and stored in −20 • C for subsequent assays.

Purified GST-NS1-85-215AA protein was used to immunize mice and generate hybridomas as described previously (Oh et al. 2010) . MAb isotype was determined using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche). The mAb was purified using a HiTrap protein G HP affinity column (GE Healthcare) and was stored at −80 • C. The purity of the mAb was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis. The concentration of the purified mAb was determined using the Coomassie Plus protein assay reagent (Thermo Scientific).

For immunofluorescence analysis, MDCK cells on glass coverslips were fixed at 24 hr post-infection (hpi) in either ice-cold methanol for 10 min at −20 • C or in 4% paraformaldehyde for 10 min at room temperature (RT) followed by permeabilization with 0.5% Triton X-100 (Sigma) for 5 min. Fixed cells were then blocked with PBS containing 10% fetal bovine serum for 30 min at RT. Cells were immunolabeled for 1 hr at RT with mAb 19H9 or mouse anti-nucleoprotein (NP; Millipore) and 45 min with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen) together with DAPI (Sigma), and mounting in ProLong R Gold Antifade Mountant (Molecular Probes). Images were acquired with Olympus CKX53 microscope using Olympus LCAch N 20x/0.40 iPC objective lens and Olympus DP27 color camera with Olympus cellSens software. Each channel was collected separately, with images at 1024 × 1024 pixels.

293T cells were seeded onto 6-well plates 24 hr prior to the transient transfection using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. NS1-expressing plasmids of different subtypes (H1N1, H3N2, H5N1 and H5N6) were used in this study. In addition, a series of N and C-terminal truncations and amino acid substitution mutants of H1N1-NS1 were generated by PCR using High Fidelity polymerase (Roche) and cloned into pXJ40-Myc expression vector. These mutant proteins were expressed by transient transfection. About 48 hr posttransfection, cells were harvested, spun down by centrifugation and washed with cold PBS twice. Cells were then resuspended in RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP40, 0.5% deoxycholic acid, 0.005% SDS and 1 mM phenylmethylsulfonyl fluoride] and subjected to freeze-thaw cycle 5 times. Clear supernatant containing the protein of interest was obtained by spinning down the cell lysate at 13 000 rpm at 4 • C to remove the cell debris and further analyzed by Western blot analysis. Commercial antibodies against p85β (Santa Cruz), GST (Santa Cruz), GAPDH (Santa Cruz), Myc (Santa Cruz) and NP (Millipore) were used. A home-made rabbit anti-NS1 polyclonal antibody (Wu et al. 2016 ) as well as the mAb 19H9 generated above were also used.

A total of 25 μg of total cell lysates were resolved using electrophoresis on SDS-PAGE gels and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked in 5% skimmed milk in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 hr at RT and incubated with primary antibodies at 4 • C overnight. After the membrane was washed with TBST, it was incubated with a HRP-conjugated secondary antibody (Pierce) at RT for 1 hr. The membrane was then washed and full-length NS1 transfected cell lysates were subjected to IP with mAb 19H9 and protein A agarose beads followed by detection of immunoprecipitated proteins using rabbit anti-Myc antibody in Western blot. Masses of molecular weight markers are indicated on the left in kDa. (C), MDCK cells were mock-or infected with rgPR8 H1N1 at an MOI of 1 for 24 hr, fixed and processed for immunofluorescence. The subcellular localization of NS1 and NP was detected by mAb 19H9 and mouse anti-NP mAb, respectively. Images were taken at magnification of 20x. with TBST again and detected with enhanced chemiluminescence substrate (Pierce) using ChemiDoc TM MP Imaging System (Bio-Rad). For loading controls, the membrane was re-probed with rabbit anti-GAPDH (Santa Cruz Biotechnology) antibody overnight at 4 • C, followed by secondary antibody and addition of enhanced chemiluminescence substrate (Pierce) for protein detection.

For IP, mAb 19H9 was used to pull down full-length NS1, NS1(RBD) and NS1(ED) in the cell lysates for 1 hr at 4 • C, followed by the addition of Protein A beads (Roche) and then incubation at 4 • C overnight. The beads were washed with RIPA buffer 3 times before subjected to Western blot analysis. Rabbit anti-Myc antibody was used as primary antibody to detect the presence of NS1 protein and goat anti-rabbit HRP-conjugated antibody (Pierce) was used as secondary antibody, followed by the 

Recombinant viruses were generated with pHW2000 reverse genetic system as described previously (Hoffmann et al. 2000) . Residue 85 of NS1 was changed from P to A and residue 89 was changed from Y to F or A by PCR mutagenesis and the resulting DNA were inserted into pHW2000 vector. This plasmid was co-transfected with another seven pHW2000 plasmids encoding the other PR8 genomic RNAs into 293T cells. Two days post-transfection, culture supernatant was collected and used to infect MDCK cells. When cytopathic effect was visibly detected, culture supernatant was collected and used to infect naïve MDCK cells. Individual plaque was then amplified and viral titer was determined by plaque assay.

About 90% confluent MDCK cells were adsorbed with serially diluted supernatants containing viruses for 1 hr at 37 • C. The medium was discarded and the cells were rinsed using PBS. The cells were overlaid with 2 ml of DMEM supplemented by 0.3% agar and 2 μg/ml TPCK-trypsin (Thermo Scientific). After incubation at 37 • C for 2 days, the cells were fixed using 10% formalin for 1 hr and stained using 0.1% crystal violet solution.

About 5 μg GST-tagged NS1(ED) fusion proteins were mixed gently with 40 μl of 50% slurry of glutathione Sepharose 4B beads for 2 hr at 4 • C. Beads were washed three times with binding buffer [20 mM Tris (pH 7.4), 0.1 mM EDTA and 100 mM NaCl] followed by incubation with 100 μg total lysates containing overexpressed p85β protein together with a serial dilution of mAb 19H9 or control antibody at 4 • C overnight. The control antibody is a murine mAb 1A9 which binds to the spike protein of Severe Acute Respiratory Syndrome Coronavirus (Lip et al. 2006; Ng et al. 2014 ). The p85β protein was overexpressed in 293T cells via transient transfection as described above and the p85β expression plasmid (pCMV6-XL6-p85β) was purchased (Origene). Subsequently, beads were sedimented by centrifugation, washed three times with the washing buffer [0.5% NP40, 0.1 mM EDTA, 20 mM Tris, pH 7.4, 300 mM NaCl]. Laemmli sample buffer was added to the beads which were boiled to release all bound proteins. Western blot analysis was subsequently performed to detect for p85β and NS1(ED) proteins.

Densitometric analysis of band intensities for Western blots were performed using ImageJ. Two-tailed Student's t-test was applied to evaluate the statistical significance of differences measured from the data sets obtained in three independent experiments. P < 0.05 was considered statistically significant.

The generation of mAb 19H9 was carried out using the recombinant GST-tagged NS1(ED) protein of A/Puerto Rico/8/1934(H1N1) virus, which was expressed in and purified from E. coli. MAb were incubated with 100 μg 293T cell lysates transiently transfected to overexpress p85β protein together with indicated amount of control mAb 1A9 (lane 3) or mAb 19H9 (lanes 4-7) at 4 • C for 2 hr. After washing, bound p85β, GST and GST-NS1(ED) were probed by p85β and GST antibodies, respectively. (B), The relative p85β-NS1 interaction in the absence of 19H9 was arbitrarily set at 100%, which was used to normalize the relative binding in the presence of indicated concentrations of mAb 19H9 or control mAb 1A9.

19H9 was able to recognize the NS1(ED) upon initial screening using ELISA (data not shown). The isotype of mAb 19H9 was determined to be IgG2a, using IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche) according to the manufacturer's protocol. The Myc-tagged full-length NS1, NS1(RBD) and NS1(ED) proteins were expressed in 293T cells via transient transfection. As shown by Western blot analysis (Fig. 1A, bottom panel) , mAb 19H9 only detected full-length NS1 and NS1(ED), suggesting its specificity to ED. The expression level of NS1 proteins was verified using an anti-Myc antibody (Fig. 1A, top panel) . Since Western blot analysis only detected denatured form of proteins, immunoprecipitation assay was also performed to determine whether mAb 19H9 could recognize NS1 under native condition. As shown in Fig. 1B , mAb 19H9 immunoprecipitated full-length NS1 and NS1(ED) proteins in native state, while no binding was detected with NS1(RBD). Thus, mAb 19H9 is able to bind to both denatured and native forms of NS1 with specificity to the ED.

Furthermore, for spatial analysis of NS1 expressed in H1N1infected cells using mAb 19H9, MDCK cells were mock-or infected with A/Puerto Rico/8/1934/(H1N1) (rgPR8) generated by reverse genetics at a multiplicity of infection (MOI) of 1 for 24 hr, fixed and processed for imaging analysis. Infection was determined by immunolabeling for another viral protein, NP and the expression of NP was observed in all the cells exhibiting nuclear and cytoplasmic localization. Figure 1C demonstrates that mAb 19H9 binds to NS1 expressed in H1N1-infected cells. In addition, NS1 displayed cytoplasmic as well as nucleolar localization. This results are consistent with previous data (Greenspan, Palese and Krystal 1988; Garaigorta, Falcon and Ortin 2005; Melen et al. 2007; Tsai et al. 2017 ) on the subcellular localization of NS1 as virus strains (Newby, Sabin and Pekosz 2007) and duration of infection (Melen et al. 2007) can determine NS1 localization pattern. Thus, mAb 19H9 could serve as a useful tool for studying NS1 cellular localization, especially in relation to its functions in the cytoplasm and nucleolus.

To delineate the epitope of NS1 recognized by mAb 19H9, a series of NS1 truncation mutants consisting of residues 1-84, 1-100, 91-230 and 85-230 were generated and their expression levels in transiently transfected 293T cells were verified using an anti-Myc antibody ( Fig. 2A, upper panel) . Western blot results showed that the minimal epitope region lies in the amino acids 85-90 ( 85 PASRYL 90 ) as mAb 19H9 could only bind NS1 fragments containing this motif ( Fig. 2A, lower panel) .

Next, alanine scanning was used to determine the relative contribution of each of these residues to the interaction with mAb 19H9. NS1(ED) mutants containing a single alanine substitution (at residue 85, 87, 88, 89, 90, in the case of residues 86 and 89, A86V mutant and a more conservative Y89F mutant were also constructed) were constructed and tested for the binding of mAb 19H9. The expression levels of these mutants in transiently transfected 293T cells were verified using an anti-Myc antibody (Fig. 2B, upper panel) . As illustrated in Fig. 2B (lower panel) , the binding of mAb 19H9 to alanine mutants at position 85 or 89 was totally abolished, while the binding to the more conservative Y89F mutant was significantly reduced. On the other hand, single substitution at residues 86, 87, 88 and 90 did not affect binding of mAb 19H9. Fig. 2 shows that residues P85 and Y89 of NS1 are essential for the binding of mAb 19H9. Amino acid sequence alignment revealed that these two residues are highly conserved among the seasonal H1N1 and H3N2, as well as highly pathogenic H5N1 and H5N6 avian IAV strains (Fig. 3A) . In order to investigate whether mAb 19H9 interacts with the two residues conserved across different subtypes, NS1 of H1N1 and H3N2, as well as H5N1 and H5N6 was expressed in 293T cells via transient transfection. Indeed, mAb 19H9 was able to detect NS1 of different subtypes (Fig. 3B) . Thus, mAb 19H9 not only binds to NS1 of the homologous PR8 virus but also cross-reacts with NS1 of different IAV subtypes.

Besides testing the binding of mAb 19H9 to NS1 protein overexpressed in 293T cells, recombinant viruses with various NS1 mutations (rgPR8-NS1-P85A, rgPR8-NS1-Y89A and rgPR8-NS1-Y89F) were generated so that binding of mAb 19H9 to NS1 expressed in infected cells can be assessed. The replication potential of these recombinant viruses was first assessed by comparing their multi-step growth kinetics at 12, 24, 36 and 48 hpi in A549 cells infected with the viruses at an MOI of 0.01. As shown in Fig. 4A , all mutant viruses displayed marginally slower growth kinetics as compared to the wild-type virus, however, the difference was not statistically different.

In the single-step growth kinetics assay, 293T cells were infected with wild-type and mutant viruses at an MOI of 1, harvested at 12 and 24 hpi and cell lysates were subjected to Western blot analysis to assess the level of viral protein synthesis. As shown in Fig. 4B , single mutation at either residue P85 or Y89 of NS1 did not affect the rate of viral protein synthesis in single-cycle replication, since the expression levels of viral NP in mutant viruses were comparable to that of the wild-type virus at both time points. Similarly, the level of NS1, as determined by using a rabbit anti-NS1 polyclonal antibody (Wu et al. 2016) , was also comparable for all four viruses. However, mAb 19H9 only bound strongly to rgPR8-NS1-WT virus and did not bind the three mutant viruses. Consistent with the Western blot results obtained using recombinantly expressed NS1(ED) fragments, these results suggested that residues P85 and Y89 are required for mAb 19H9 binding to viral NS1 expressed in infected cells during viral infection.

It has been shown that NS1 binds specifically and directly to the p85β regulatory subunit of PI3K to activate the PI3K/Akt pathway, which in turn supports efficient virus replication (Ehrhardt et al. 2007) . Structural study has revealed that residue Y89 of NS1 is positioned in the binding interface of NS1-p85β complex and the conservative substitution at this residue (Y89F) disrupts this interaction (Hale et al. 2010) . Since Y89 is also critical for the binding of mAb 19H9 to NS1, an in vitro GST pull-down competition assay was performed to investigate the effect of mAb 19H9 on the interaction between NS1 and p85β. Bacterially expressed and purified GST (negative control) and GST-NS1(ED) proteins were incubated with glutathione Sepharose 4B beads followed by incubation, in the presence of mAb 19H9 or control antibody, with total cell lysates containing p85β protein expressed by transient transfection. The amount of p85β protein bound to GST or GST-NS1(ED) was detected by Western blot using antibodies specific to the p85β subunit. As shown in Fig. 5A , p85β specifically bound to NS1(ED) instead of GST, since GST alone failed to pull down the p85β protein. Furthermore, mAb 19H9 inhibited the interaction between p85β and NS1(ED) in a dose-dependent manner (Fig. 5A) , implying that the binding of mAb 19H9 and p85β to NS1(ED) is competitive. Quantification by densitometry showed that there was a significant reduction in the interaction between p85β and GST-NS1(ED) when mAb 19H9 was used at 0.07 nM and 0.13 nM (Fig. 5B) . On the other hand, the presence of 0.13 nM of control antibody (mAb 1A9) showed no effect on the interaction between p85β and GST-NS1(ED). This result shows that mAb 19H9 binds to residue Y89 of NS1 and blocks its interaction with p85β.

As mAbs targeting NS1 can be used for research and diagnostic assay development, multiple hybridomas have been generated to produce anti-NS1 antibodies. For those mAbs with epitope mapped, all of them have been shown to bind to motifs in either NS1(RBD) or NS1(ED) (Tan et al. 2010; He et al. 2013; Rahim et al. 2013; Wen et al. 2015; Sun et al. 2016) . In this study, we have generated a novel mAb 19H9 and found that it interacts with two residues, P85 and Y89, of NS1 that are highly conserved in seasonal as well as newly emerged highly pathogenic avian IAV strains. Consistently, mAb 19H9 did not bind to rgPR8 mutant viruses carrying substitution at either of these positions. To our knowledge, mAb 19H9 is the first murine mAb binding at the juxtaposition between the RBD and ED of NS1. It could serve as a useful research tool for studying the conformational plasticity and dynamic changes in NS1. Interestingly, a fully human single chain antibody fragment (huScFv clone 10) was isolated from a human ScFv phage display library and found to bind to residues 75-90 of NS1 (A/duck/Thailand/144/2005(H5N1)) (Yodsheewan et al. 2013) . The overlap between the epitopes of mAb 19H9 and huScFv clone 10 suggests that the juxtaposition between the RBD and ED of NS1 is immunogenic.

The residue Y89 is part of the ED of NS1 and previous study has demonstrated that it is important for NS1's interaction with the p85β subunit of the PI3K complex (Hale et al. 2010 ). Indeed, this study has shown that the interaction between NS1(ED) and p85β was abrogated by the binding of mAb 19H9 to Y89 of NS1. On the other hand, P85 is found in the LR between the RBD and ED. While the exact role of the LR in viral replication has not been defined, five amino acids deletion (residues 80-84) is found to be implicated in the high virulence of the H5N1 strains like A/Vietnam/1203/2004/(H5N1) (Long et al. 2008 ). In addition, structural analysis showed that the NS1(ED) dimer in the full-length NS1 of A/Vietnam/1203/2004/(H5N1) exhibited significant difference from its structure when solved as an isolated domain (Bornholdt and Prasad 2008) . Structural analysis of the full length H6N6-NS1 and a mutant H6N6-NS1 80-84 further revealed that the LR influences the orientation of NS1(ED) with respect to NS1(RBD) and thus plays an important role in determining the quaternary structure of NS1 (Carrillo et al. 2014) .

The LR could be the basis for strain-specific functions associated with NS1 protein during IAV infection, possibly by determining the protein's correct subcellular localization. Using mAb 19H9, NS1 displayed cytoplasmic as well as nucleolar localization. As such, it could be used to study NS1 temporal distribution, and its interactions with host nucleolar or cytoplasmic proteins, during the course of virus infection. The interaction of mAb 19H9 with the LR of NS1 could potentially be useful in investigating the less well defined functions of this region.

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