West Nile virus nonstructural protein NS1 inhibitscomplement activation by binding the regulatoryprotein factor HKyung Min Chung*, M. Kathryn Liszewski*, Grant Nybakken†, Alan E. Davis*‡, R. Reid Townsend*‡,Daved H. Fremont†§, John P. Atkinson*†¶, and Michael S. Diamond*†¶�

Departments of *Medicine, †Pathology and Immunology, §Biochemistry and Molecular Biophysics, ‡Cell Biology and Physiology,and ¶Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Charles M. Rice, The Rockefeller University, New York, NY, and approved October 14, 2006 (received for review July 6, 2006)

The complement system, by virtue of its dual effector and primingfunctions, is a major host defense against pathogens. Flavivirusnonstructural protein (NS)-1 has been speculated to have immuneevasion activity, because it is a secreted glycoprotein, binds back tocell surfaces, and accumulates to high levels in the serum ofinfected patients. Herein, we demonstrate an immunomodulatoryfunction of West Nile virus NS1. Soluble and cell-surface-associatedNS1 binds to and recruits the complement regulatory protein factorH, resulting in decreased complement activation in solution andattenuated deposition of C3 fragments and C5b–9 membraneattack complexes on cell surfaces. Accordingly, extracellular NS1may function to minimize immune system targeting of West Nilevirus by decreasing complement recognition of infected cells.

flavivirus � immune evasion � innate immunity � pathogenesis

West Nile virus (WNV) is a single-stranded positive sense-enveloped RNA flavivirus that is related closely to other

major human pathogens, such as yellow fever virus, dengue virus(DENV), and Japanese encephalitis virus. The �11-kb genomeis translated in the cytoplasm as a polyprotein and then cleavedinto structural (capsid, membrane, and envelope) and nonstruc-tural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteinsby virus- and host-encoded proteases. The nonstructural pro-teins regulate viral transcription, translation, and replication andattenuate host antiviral responses. Outbreaks of WNV infectionnow occur annually in North America, and the elderly andimmunocompromised progress more frequently to the moresevere neurological forms of the disease, including paralysis,meningitis, and encephalitis.

The ability of viruses to cause disease depends on theircapacity to avoid detection and targeting by the host immuneresponse. RNA and DNA viruses use an array of strategies toevade the host immune system. Whereas large DNA virusesoften acquire and modify host immunodulatory genes to avoidrecognition or targeting, smaller RNA viruses have genomic sizeconstraints that prohibit the acquisition of exogenous geneswithout compromising viral replication or assembly. As such,smaller viruses tend to evolve multifunctional genes that mediateessential steps of the viral life cycle and, in some cases, modulatethe host response.

Flavivirus nonstructural protein (NS1) is an essential genethat generates a conserved 48-kDa glycoprotein. Within infectedcells, NS1 is believed to function as a cofactor for viral RNAreplication as it colocalizes with the double-stranded RNAreplicative form (1), and mutations attenuate viral RNA accu-mulation (2–4). Unlike the other nonstructural proteins, NS1 issecreted (5–7), and high levels (up to 50 �g/ml) accumulate inthe serum of WNV and DENV-infected patients and correlatewith the development of severe disease (8–12). Additionally,soluble NS1 becomes associated with the cell surface through anas-yet-undetermined process (6, 7). The mechanism(s) by whichsoluble NS1 contribute to flavivirus pathogenesis remains un-

certain, although it has been proposed to facilitate immunecomplex formation (9), elicit autoantibodies that react withplatelet and extracellular matrix proteins (13, 14), cause endo-thelial cell damage (14–17), and directly enhance infection (18).

The complement system is a family of soluble and cell-surfacemolecules that recognize pathogen-associated molecular pat-terns, altered-self ligands, and immune complexes. Complementactivation controls viral infections (19–26), including WNV (27,28), through multiple mechanisms including enhanced B and Tcell priming, generation of C5b–9 membrane attack complexesthat lyse infected cells, and production of fragments of C3 thatopsonize viral particles and chemoattract leukocytes. In re-sponse to these antiviral mechanisms, some DNA viruses haveevolved specific strategies to sabotage complement activationand neutralization by producing or incorporating complementmodulating or complement-blocking molecules (29–32).

Because widespread complement activation may cause patho-logic tissue injury, the host expresses regulatory molecules oncell surfaces and in solution. The plasma glycoprotein factor H(fH) is the major fluid-phase regulator of the alternative pathwayand, under certain circumstances, it can also act as a surface-bound inhibitor (33). fH is a 150-kDa protein composed solelyof 20 short consensus repeat domains. It attenuates alternativepathway activation by inhibiting the binding of factor B to C3b,accelerating the decay of preformed C3bBb convertases andacting as a cofactor for the serine protease factor I (fI) to cleaveC3b (34–36). Because fH is a key regulator of complementactivation, some bacteria have manipulated its function to evadecomplement system detection (37, 38). Immune evasion mech-anisms by viruses involving fH have not been described.

In this paper, we define an interaction between WNV NS1 andfH. We show that NS1 binds to fH in solution and promotes thefI-mediated cleavage of C3b, and cell surface-associated NS1attenuates the deposition of C3b and C5b–9 membrane attackcomplexes. Because it lacks any sequence homology to knowncomplement regulatory genes, WNV NS1 appears to have

Author contributions: K.M.C., M.K.L., R.R.T., D.H.F., J.P.A., and M.S.D. designed research;K.M.C., M.K.L., and A.E.D. performed research; G.N. contributed new reagents/analytictools; K.M.C., R.R.T., J.P.A., and M.S.D. analyzed data; and K.M.C., M.K.L., G.N., R.R.T.,D.H.F., J.P.A., and M.S.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Freely available online through the PNAS open access option.

Abbreviations: WNV, West Nile virus; DENV, dengue virus; fH, plasma glycoprotein factorH; fI, serine protease factor I; NHS, normal human serum; BHK, baby hamster kidney;C7d-HS, C7-deficient human serum.

See Commentary on page 18879.

�To whom correspondence should be addressed. E-mail: diamond@borcim.wustl.edu.

This article contains supporting information online at www.pnas.org�cgi�content�full�0605668103/DC1.

© 2006 by The National Academy of Sciences of the USA

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evolved a novel viral immune evasion activity, inhibiting theinflammatory and effector functions of complement.

ResultsIdentification of a Complex with WNV NS1. In a previous study,recombinant WNV NS1 was purified to near homogeneity frombaculovirus-infected SF9 cells that were cultivated under serum-free conditions (39). When NS1 was purified from infected SF9cell supernatants containing 10% FBS, despite several sequen-tial purification steps, NS1 was enriched along with one majorand several minor contaminant proteins (Fig. 1). We hypothe-sized that some of the contaminating bands could representserum ligands for NS1. We identified the dominant �150-kDaprotein by tandem MS (MALDI-TOF/TOF). Peptide fragmen-tation spectra from tryptic peptides identified the 150-kDa bandas bovine complement fH [Table 1 and supporting information(SI) Fig. 6].

WNV NS1 Directly Binds fH. Coimmunoprecipitation studies weredesigned to establish a direct interaction between WNV NS1 andfH. The presence of fH in immunoprecipitates was evaluated byWestern blot with a polyclonal antibody against human fH. Theexperiments were performed several ways with normal humanserum (NHS), NS1 of mammalian or insect origin, and purifiedfH (Fig. 2 A–D). Initially, we used a baby hamster kidney (BHK)cell line that stably propagates a WNV subgenomic replicon andexpresses the viral proteins NS1–NS5 (40). Using a quantitativecapture ELISA with two anti-NS1 mAbs (3NS1 and 10NS1; ref.39), we confirmed these cells efficiently secrete NS1 (data notshown). Supernatants from these cells or uninfected controlBHK cells were immunoprecipitated with an anti-NS1-mAb-Sepharose, washed, incubated with NHS, washed again, andsubjected to SDS/PAGE and Western blotting. Only anti-NS1-mAb-Sepharose that was incubated with NS1-containing super-natants coprecipitated a 150-kDa band that was recognized bythe anti-fH antibody (Fig. 2 A). When these studies were re-peated with NS1 purified from baculovirus-infected insect cells,similar results were observed (Fig. 2B). As an additional con-firmation, the immunoprecipitation studies with NS1 obtainedfrom mammalian (Fig. 2C) or insect cells (Fig. 2D) wererepeated with commercially available purified human fH. Im-portantly, the concentrations of NS1 (8 �g/ml) and fH (�50

Fig. 1. Copurification of WNV NS1 and bovine fH. Supernatants wereharvested from baculovirus-infected SF9 insect cells grown in 10% FBS, andNS1 was purified by nickel-affinity and size-exclusion chromatography. Silver-stained SDS/PAGE of column fractions is shown. WNV NS1 and bovine fH areindicated by arrows. The numbers across the top correspond to the differentcolumn fractions. Protein size markers are indicated to the left in kilodaltons.

Table 1. MS analysis of tryptic peptides of thehigh-molecular-weight protein that copurified with WNV NS1,which correspond to bovine complement fH (gi�1419424and gi�61834503)

Peptides [M�H](observed)� [M�H](calculated)

�

Mascotscore

gi�61834503DGWVPVPR 925.51 925.49 36YLGETVR 965.53 965.51 40TPVILNGQAVLPK 1,349.84 1,349.81 62IPNGVYRPELSK 1,372.8 1,372.76 50IENGFLSESTFTYPLNK 1,959.99 1,959.97 53

gi�1419424DGWVPVPR 925.51 925.49 36YLGETVR 965.53 965.51 40EAFTMGIPR 1,021.54 1,021.53 32IPNGVYRPELSK 1,372.8 1,372.76 50IENGFLSESTFTYPLNK 1,959.99 1,959.97 53

Fig. 2. Coimmunoprecipitation of WNV NS1 and human fH. Western blot-ting with a polyclonal anti-human fH after immunoprecipitation with anti-NS1-mAb-Seph4B, NHS (A and B), and supernatant from WNV replicon-expressing cells (A) or purified NS1 (B). Western blotting was also performedafter immunoprecipitation with anti-NS1-mAb-Seph4B (C and D), purifiedhuman fH, and supernatant from replicon-expressing cells (C) or purified NS1(D). BHK cell supernatant or BSA was used as negative control, respectively. fHwas identified with a sheep polyclonal anti-human fH antibody. Lanes 1 and2 of each image represent the starting material without immunoprecipitation.The arrow points to the band that corresponds to the 150-kDa human fHprotein.

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�g/ml) used in these studies were less than that (50 and 500�g/ml, respectively) observed in human plasma (8, 11, 41).

To verify the interaction between fH and NS1, a direct bindingassay was developed. Initially, insect cell-generated NS1 that wascultivated in serum-free medium was purified to homogeneity(Fig. 3A) and adsorbed to plastic. As negative controls, insectcell-generated soluble WNV envelope protein (Ecto-E; ref. 42)and BSA were adsorbed at similar concentrations in parallel.After blocking, purified fH was added, and bound fH wasdetected with a polyclonal antibody to human fH. Notably,significant and reproducible binding of fH was observed withNS1 but not Ecto-E or BSA proteins (Fig. 3B). The assay was alsoperformed in the reverse order: fH was adsorbed to plastic, andsoluble NS1 was added. With this assay, significant binding wasobserved between fH and NS1 (Fig. 3C) compared with BSAcontrols. Binding between fH and NS1 was saturable although atrue affinity constant could not be determined, because Scat-chard analysis failed to yield a model of single-site interaction,likely because both NS1 and fH exist as oligomers (Fig. 3D anddata not shown). Taken together, these experiments establish abiochemical interaction between WNV NS1 and fH.

WNV NS1 Recruits fH to Degrade C3b in Solution. Cofactor assayswere conducted to determine whether NS1 could associate withfH and activate fI-mediated cleavage of C3b in solution. Exper-iments were performed by using biotinylated human C3b, andcleavage products were identified by Western blot (Fig. 4). NHSwas mixed with purified WNV NS1 or BSA and immunopre-cipitated with an anti-NS1-mAb-Sepharose. After extensivewashing, purified human fI and biotinylated-C3b were added. Asa positive control, purified human fH was added directly to fI andbiotinylated-C3b to induce the cleavage and generation of iC3bfragments (Fig. 4A, lanes 4 and 8), in contrast to the negativecontrol (Fig. 4A, lanes 3 and 7). Anti-NS1-mAb-Sepharoseincubated with purified NS1 but not BSA precipitated a proteinor proteins from NHS with cofactor activity for C3b cleavage(Fig. 4A, lanes 2 and 6). Similar results were obtained in aheterologous system in which anti-NS1-mAb-Sepharose chargedwith NS1 precipitated cofactor activity from mouse serum forhuman C3b (Fig. 4C, lanes 2 and 6). NS1 itself lacked directcofactor activity; addition of NS1 to fI and C3b did not generateiC3b fragments, even after prolonged incubation (Fig. 4E). NS1,however, modestly enhanced the cofactor activity of fH (SI Fig.7). To confirm that cofactor activity associated with NS1 was dueto its ability to bind fH, purified human fH was substituted for

NHS and mixed with purified WNV NS1 or BSA. After immu-noprecipitation, cofactor activity and production of iC3b weremarkedly enhanced in the presence of NS1 (Fig. 4B, lane 2).Analogously, when serum from fH�/� mice was used, NS1-

Fig. 3. WNV NS1 and human fH binding by ELISA. (A) SDS/PAGE of purified WNV NS1 and human fH. WNV NS1 was purified from baculovirus-infected SF9 cellsgrown under serum-free conditions (lane 1). Human fH (lane 2) was commercially purchased. The positions of marker proteins are indicated to the left of thegel. (B) fH binds to solid-phase WNV NS1. Microtiter plates were coated overnight with purified NS1, WNV envelope (Ecto-E) protein, or BSA. After blocking,purified fH was added and detected with a polyclonal antibody against human fH. Results are representative of at least three independent experimentsperformed in duplicate. The error bars indicate standard deviations. (C) NS1 binds to solid-phase human fH. The microtiter plate was coated with human fH orBSA as control, blocked, and then incubated with purified WNV NS1. Subsequently, after washing, NS1 binding was detected with mAb 3NS1 or 22NS1.Experiments were performed in duplicate, and the results are representative of at least three independent experiments. (D) Saturation binding of fH and NS1.Increasing concentrations of fH were added to NS1 on microtiter plates and evaluated for binding by ELISA. The curve is representative of three independentexperiments performed in duplicate.

Fig. 4. WNV NS1 recruits fH in solution to degrade C3b. NS1 or BSA wasincubated with 9NS1 or 22NS1 mAb Sepharose overnight at 4°C. The charged oruncharged beads were mixed with 10% NHS (A), purified human fH (B), 10%C57BL/6 mouse serum (MS, C), or 10% congenic fH�/� MS (designated by triangle,D), washed extensively, and then incubated with biotinylated C3b and fI for 7 or14 min (A and B) or 20 or 40 min (C and D). In each series of experiments, a buffernegative control (lanes 3 and 7) was included that contained only biotinylatedC3b, fI, and anti-NS1-mAb-Sepharose. In E, the NS1-charged beads were addeddirectly to biotinylated C3b and fI. Reactions were stopped with SDS-reducingsample buffer, and C3b fragments were visualized after Western blot. C3bfragments are labeled at the right of the gel, and cofactor activity is establishedby the appearance of the �1 fragment (�67 kDa) of the C3b ��-chain. The bandmarked � represents the small amount of C3 in the preparation that was notcleaved to C3b. Its thioester bond, however, is cleaved, because it is also suscep-tible to degradation. Under longer exposure, the �2 degradation fragment (�41kDa) was also observed (data not shown). Data are representative of at least twoto three independent experiments.

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charged Sepharose no longer cleaved human C3b to iC3b (Fig.4D, lane 2). Collectively, these experiments establish that NS1recruits the cofactor activity of fH to degrade C3b in solution.

WNV NS1 Attenuates C3b and C5b–9 Deposition on Cell Surfaces. Inaddition to accumulating in serum of WNV-infected patients(11), NS1 also binds back to the cell surface of infected cells. Wehypothesized that cell surface-associated NS1 could recruit fH toaccelerate the decay of the alternative pathway C3bBb conver-tase; this would reduce deposition of C3 ligands and inhibitformation of the C5b–9 membrane attack complex. To test this,we modified an established CHO cell assay of complement

activation and C3 deposition that requires classical pathwayinitiation and alternative pathway amplification (43–45). CHOcells were transfected with an empty retroviral vector or one thatencoded for full-length secreted WNV NS1. Several NS1-expressing (CHO-NS1) and vector alone (CHO-V) lines wereidentified and grown in serum-free media. As expected, WNVNS1 was detected in supernatants and on the cell surface ofCHO-NS1 transfectants but not in vector-transfected cells (Fig.5A). The level of NS1 on the CHO cell surface was equivalentto that observed on several cell types infected with WNV (datanot shown). To assess whether cell-associated NS1 modulated C3deposition, CHO-NS1 or CHO-V cells were preincubated withC7-deficient human serum (C7d-HS) to enhance interactionwith fH. These cells were sensitized with a polyclonal antibodyagainst CHO cell surface antigens, incubated with C7d-HS as acomplement source, and analyzed for C3b deposition by flowcytometry. Notably, cell surface-associated NS1 decreased theC3b deposited on the cell surface (Fig. 5B, 11 � 2% onCHO-NS1 vs. 22 � 3% on CHO-V, P � 0.01). This effect wasnot due to NS1-dependent blockade of CHO surface antigensites, because no change in antibody binding was observed in thepresence or absence of NS1 (data not shown). Importantly, aftera short incubation with human serum, markedly higher levels offH were bound by CHO-NS1 compared with CHO-V cells (Fig.5C). Thus, a decrease in C3b deposition on CHO-NS1 cells wasassociated with recruitment of soluble fH.

Because the lower levels of C3b on CHO-NS1 cells couldtranslate into decreased complement-dependent lysis, experi-ments were performed to determine the effect of NS1 ondeposition of the C5b–9 membrane attack complex. CHO-NS1or CHO-V cells were preincubated with NHS, sensitized withpolyclonal antibody against CHO cell antigens, incubated withNHS as a complement source, and analyzed for C5b–9 deposi-tion. After incubating cells with 10% NHS, we observed thatexpression of cell surface-associated NS1 on CHO cells resultedin a 4- to 5-fold decrease in detectable C5b–9 deposition (2.5 �0.6% vs. 11.4 � 3%, P � 0.01; Fig. 5D). Similar results wereobserved when cells were incubated with 25% NHS (8 � 1% forCHO-NS1 and 27 � 5% for CHO-V, P � 0.01). Overall, our datasuggest that cell surface-associated NS1 attenuates C3b deposi-tion and C5b–9 membrane attack complex formation.

DiscussionFor viruses to infect mammalian hosts productively, they evolveor acquire genes that attenuate or disable innate and adaptiveimmune responses. Because flavivirus NS1 is a secreted glyco-protein that binds back to cell surfaces and accumulates inserum, it has been speculated to have an immune evasivefunction. In this regard, our data suggest an immunomodulatoryfunction for WNV NS1: it inhibits complement activation insolution and on cell surfaces. We used several experimentalapproaches to demonstrate a functional interaction betweenWNV NS1 and fH: (i) NS1 efficiently bound fH in ELISA andcoimmunoprecipitation experiments; and (ii) NS1, by itself,lacked cofactor activity for fI-mediated cleavage of C3b; how-ever, fH–NS1 complexes provide cofactor activity for the deg-radation of C3b in solution and (iii) cell surface NS1 bound fHand decreased the deposition of C3 fragments and C5b–9complexes upon complement challenge.

Our finding that WNV NS1 interacts with a complementregulatory protein initially surprised us. Several decades ago,f lavivirus NS1 was originally identified as a ‘‘soluble comple-ment fixing antigen’’ (46, 47), and vascular leakage associatedwith severe DENV infection correlated with complement con-sumption and the accumulation of NS1 in serum (8–10, 12, 48).Moreover, soluble DENV NS1 appeared to activate complementin vitro and was associated with increased levels of C5b–9terminal complexes in serum (12). Subsequent experiments

Fig. 5. Inhibition of complement deposition by cell surface-associated NS1.(A) Flow cytometry histograms showing cell surface-associated expression ofWNV NS1 on CHO-NS1 stable cell transfectants (green line). As a negativecontrol, CHO-V cells transfected with the parent retroviral vector were stained(purple line). (B) WNV NS1 inhibits C3b deposition on CHO cells. CHO-V andCHO-NS1 cells were preincubated with C7d-HS, sensitized with an anti-CHOantibody, and incubated with C7d-HS in GVB-Mg2�-EGTA buffer. After wash-ing, cells were stained with mAb against human C3d and analyzed by flowcytometry. The results are the average of three independent experimentsperformed in duplicate, and the difference in C3b deposition between CHO-Vand CHO-NS1 cells was statistically significant (P � 0.01). (C) fH binding toCHO-V and CHO-NS1 cells. Cells were incubated with medium or 10% NHS forthe indicated times (in minutes), washed extensively, and analyzed by Westernblot with a sheep anti-human fH antibody. The �150 kDa fH is denoted by thearrow. (D) WNV NS1 inhibits C5b–9 deposition on CHO cells expressing WNVNS1. CHO-V and CHO-NS1 cells were preincubated with NHS, sensitized withan anti-CHO antibody, incubated with 10% (Upper) or 25% (Lower) NHS, andthen immunostained with murine mAb against human C5b–9 (Right). Theresults are the average of three independent experiments performed induplicate, and the difference in C5b–9 deposition between CHO-V and CHO-NS1 cells was statistically significant (P � 0.01). For negative control staining(Left), complement-activated cell lines were stained with only anti-mouse IgGconjugated with FITC. Purple and green lines refer to CHO-V and CHO-NS1cells, respectively.

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indicate that DENV NS1 does not activate complement directlybut instead forms immune complexes with natural or DENVimmune antibody, which then lead to complement consumptionand C5b–9 complex formation (P. Avirutnan, J.P.A., andM.S.D., unpublished results). It is possible that WNV and DENVNS1 have evolved distinct abilities to regulate complementactivation. Consistent with this, complement activation and C3consumption after WNV infection in mice peaks in serum within2 days and gradually normalizes with kinetics that parallel thedetection and accumulation of NS1 in serum (27). In compar-ison, C3 and C4 consumption after DENV infection in humansincreases over time and correlates with the most severe clinicalphenotype, the capillary leak syndrome of dengue hemorrhagicfever (48). Future studies are required to resolve whether NS1from other flaviviruses interacts similarly with fH.

The mechanism by which WNV NS1 modulates complementactivation is novel among viruses. Herpesviruses and poxvirusessabotage complement activation by incorporating and modifyingcomplement modulating or complement-blocking molecules(29–32). In contrast, NS1 itself lacks any sequence homology toknown complement regulatory genes and indeed lacked directcofactor activity. Instead, NS1 binds to and recruits fH toattenuate complement activation in solution and on cell surfaces.This latter function of converting fH into a membrane-associatedcomplement regulatory protein is reminiscent of the immuno-modulatory activities of surface proteins and carbohydrates ofStreptococcus, Neisseria, Yersinia, and Borrelia, which bind fH toregulate complement activation and reduce opsonization andphagocytosis (49–52).

The present study demonstrates that WNV NS1 on cellsurfaces inhibits deposition of C3b and the C5b–9 membraneattack complex. A mechanistic appreciation of how and whysoluble flavivirus NS1 binds to cell surfaces has been lacking,although a recent study suggested that it might directly enhanceDENV infection in hepatocytes (18). Our study suggests that cellsurface-associated NS1 could minimize immune system activa-tion and priming and the generation of inflammatory ligands(e.g., C3a or C5a) and opsonins (e.g., C3b or iC3b). By decreas-ing complement activation and therefore the number of C5b–9membrane attack complexes on infected cells, NS1 also coulddelay or prevent complement-dependent lysis of infected cells,thereby allowing for continued production and secretion ofvirus. Although structural studies are lacking, prior mappingstudies have suggested that NS1 may have distinct domains (14,39, 53); studies are under way to identify the region of WNV NS1that interacts with fH and analogously the region on fH thatbinds WNV NS1. A detailed molecular understanding of theinteraction between WNV NS1 and fH may, in part, explaindifferences in pathogenesis and disease among flaviviruses andfacilitate the development of inhibitors that block immuneevasion.

Materials and MethodsPurified Proteins. Expression and purification of recombinantWNV NS1 from insect cells were described previously (39). Insome experiments, SF9 insect cells were cultivated in Grace’smedia (JHR Bioscience, Lenexa, KS) with 10% FBS. Purifiedhuman fH, C3b, and fI were obtained commercially (AdvancedResearch Technologies, San Diego, CA), aliquoted, and storedat �80°C.

MALDI-TOF MS. Peptide pools were prepared from SDS/PAGEgels by using a previously described method (54). Gel bands from1D gels were transferred to a 96-well plate (Axygen Scientific,Union City, CA). After trypsin digestion, an aliquot was re-moved from each well and placed in a microfuge tube containingMALDI Matrix (Agilent Technologies, Palo Alto, CA). The

tubes were vortexed and microfuged, and 1 �l was spotted ontoa stainless-steel target (192 spot plate) for MS, as described (55).

NS1-Binding Assays. Coimmunoprecipitation. Coimmunoprecipita-tions were performed with NHS, NS1 generated from baculo-virus-infected SF9 cells or a BHK cell line that stably propagatesa subgenomic replicon of WNV (40), and purified human fH.

NS1 from WNV replicon and NHS or purified fH. BHK cellsexpressing the WNV replicon or uninfected control BHK cellswere cultured in serum-free DMEM. Supernatants were immu-noprecipitated with anti-NS1-Sepharose 4B beads [9NS1-Seph4B (39)]. After washing, the beads were incubated withNHS (70 �l) or purified fH (35 �g) in serum-free DMEM orGVB-Mg2�-EGTA overnight at 4°C. The beads were washedfour times with GVB-Mg2�-EGTA or serum-free DMEM mediaat 4°C.

NS1 from baculovirus-infected SF9 insect cells and NHS. NHS (70�l) and purified NS1 (70 �l, 100 �g/ml) from baculovirus-infected SF9 insect cells, cultivated without FBS, were mixed,incubated overnight at 4°C, and immunoprecipitated for 5 h at4°C with 9NS1-Seph4B. The beads were washed four times withserum-free DMEM media at 4°C.

Purified NS1 and fH. Purified NS1 (7 �g) or BSA (7 �g) wasimmunoprecipitated with 22NS1-Seph4B (39). Beads werewashed, incubated with purified fH (55 �g) in GVB-Mg2�-EGTA overnight at 4°C, and washed four times. All immuno-precipitates were boiled in SDS sample buffer and separated byreducing 8% SDS/PAGE. Western blot analysis was performedas described (39) by using a 1/2,000 dilution of a sheep anti-human fH antibody (Biodesign, Saco, ME) and a 1/3,000 dilutionof secondary antibody conjugated with HRP.ELISA. fH binding to NS1. Insect cell-derived WNV NS1 (�0.5 �g)was adsorbed overnight at 4°C to microtiter plates. Nonspecificsites were blocked [150 mM NaCl/25 mM Tris�HCl (pH 7.5)/2%BSA/0.025% Nonidet P-40/0.025% NaN3]. Human fH (50 �l of10 �g/ml stock) was added to the wells and incubated for 1.5 hat room temperature. After washing four times [150 mM NaCl/25mM Tris�Cl (pH 7.5)/0.5% BSA/0.025% Nonidet P-40/0.025NaN3], plates were incubated with a 1/200 diluted sheep anti-human fH antibody for 1 h at room temperature. After anadditional wash, plates were incubated with HRP-conjugatedrabbit anti-sheep IgG and developed after addition of 3,3�,5,5�-tetramethyl-benzidine (TMB) substrate.

NS1 binding to fH. Microtiter plates were coated with 2.5 �g ofhuman fH in PBS overnight at 4°C. After blocking, WNV NS1 (0.5�g) was added and incubated for 1.5 h at room temperature. Afterwashing, the plates were incubated serially with 10 �g/ml anti-WNVmAbs 3NS1 or 22NS1 (39) and donkey anti-mouse IgG-conjugatedHRP (1/600 dilution) and developed with tetramethyl-benzidinesubstrate.

C3b Cofactor Assay. C3b cofactor activity was assessed as de-scribed (43, 44). This assay analyzes the breakdown products ofbiotinylated human C3b in solution. The fragments of thecleavage reaction, after addition of human fI and NS1 coimmu-noprecipitates, were assessed by Western blot. Human C3b wasbiotinylated by using a 50-molar excess of EZ-Link Sulfo NHS-LC-biotin (Pierce, Rockford, IL). For the cofactor assay, alldilutions were prepared in isotonic buffer [10 mM Tris�Cl (pH7.5)/150 mM NaCl] for cofactor assays with human serum or fHor low salt buffer [10 mM Tris�Cl (pH 7.5)/25 mM NaCl] forcofactor assays with mouse serum. Biotinylated C3b (�200 ng)and human fI (400 ng) were mixed, and immunoprecipitatesfrom 9NS1- or 22NS1-Seph4B were added in a total of 60 �l.After an incubation at 37°C, an equal volume of SDS-reducingsample buffer was added, the samples were heated to 95°C for3 min, and Western blotting was performed. Membranes wereprobed with 1/1,000 dilution of ExtrAvidin-HRP (Sigma, St.

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Louis, MO), and signal was detected with ECL Western blotreagent (Amersham Biosciences, Piscataway, NJ).

CHO Cell Lines. CHO-NS1 stable transfectants were generated asfollows: an initiator methionine, the last 72 nucleotides of WNVE (endogenous signal sequence), and entire NS1 protein fromthe New York 1999 strain were amplified from the infectiouscDNA clone by high-fidelity PCR. The PCR product was di-gested and inserted in the BamHI and SalI site of pBabe-puromycin retroviral expression vector. After sequencing, pB-abe-WNV NS1 or the parent vector was transfected intoCHO-K1 cells (45) by using the LT1 transfection reagent (Mirus,Madison, WI). Stable transfectants were selected with Ham’sF-12 medium supplemented with puromycin (10 �g/ml). Clonesof NS1 (CHO-NS1) or vector-containing (CHO-V) cells wereisolated and adapted into serum-free CHO-S-SFM II (Invitro-gen, Carlsbad, CA) supplemented with puromycin (8 �g/ml).

Complement Activation on the Surface of CHO Cells. Complementactivation and detection of split products on CHO cells were amodification of published protocols (43–45). To measure cellsurface C3b or C5b–9 deposition, CHO cells were resuspendedin Ham’s F-12 medium and preincubated with 10% C7d-HS (giftof P. Densen, University of Iowa, Iowa City, IA). After a 1-hourincubation in ice, the cells were washed and incubated with goatanti-CHO surface antigen IgG (Cygnus Technologies, South-port, NC: 10 �g/ml) for 20 min on ice. Subsequently, the cellswere washed and resuspended in 75 �l of GVB-Mg2�-EGTA,and 75 �l of 18% C7d-HS was added. Cells were incubated at

37°C for 30 min and washed, and C3b deposition was detectedwith a murine mAb to human C3d (5 �g/ml; Quidel, San Diego,CA). To measure C5b–9 deposition, cells were pretreated with10% NHS, incubated with 0.5 mg/ml goat anti-CHO IgG, andthen incubated with 10% or 25% of NHS as the complementsource. CHO cells were immunostained with 10 �g/ml murinemAb against human C5b–9 and analyzed by flow cytometry.

fH Binding to CHO Cell Lines. CHO-NS1 or CHO-V cells wereharvested, resuspended in 10% NHS in Ham’s F12 medium, andincubated on ice. Cells were washed four times and lysed withSDS sample buffer, and lysates were separated by SDS/PAGE.The level of bound fH was analyzed by Western blot with a sheepanti-human fH antibody.

Statistical Analysis. All data were analyzed with Prism software(GraphPad, San Diego, CA). Statistical significance was deter-mined by using a two-tailed nonpaired Student’s t test.

We thank J. Malone, S. Schaecher, and B. Thompson for help withexperiments and M. Pickering (Imperial College, London, U.K.) and M.Botto (Imperial College) for the gift of fH�/� mouse serum. This workwas supported in part by the National Institutes of Health–NationalInstitute of Diabetes and Digestive and Kidney Diseases (Grant P30DK52574), the National Institute of Allergy and Infectious Diseases[Grant U54 AI057160 to the Midwest Regional Center of Excellence forBiodefense and Emerging Infectious Diseases Research (MRCE)], thePediatric Dengue Vaccine Initiative, and institutional funds to theWashington University Proteomics Center.

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