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Arch Virol (2007) 152: 17311742 DOI 10.

1007/s00705-007-0983-4 Printed in The Netherlands

Inhibition of inuenza viral neuraminidase activity by collectins


T. Tecle1 , M. R. White1 , E. C. Crouch2 , K. L. Hartshorn1
1 2

Department of Medicine, Boston University School of Medicine, Boston, MA, USA Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

Received 2 January 2007; Accepted 4 April 2007; Published online 22 May 2007 # Springer-Verlag 2007

Summary

The collectins, lung surfactant proteins A and D (SP-A and SP-D), contribute to innate host defense against inuenza A virus (IAV) in vivo. Although collectins bind to the viral hemagglutinin (HA) and inhibit early stages of viral infection in vitro, they also bind to the neuraminidase (NA) and inhibit NA activity. We used a variety of NA functional assays, viral strains and recombinant (mutant or wild type) collectins to characterize the mechanism of NA inhibition. NA inhibition by SP-D correlates with binding of its carbohydrate recognition domain (CRD) to oligomannose oligosaccharides on the viral hemagglutinin (HA). The effects of SP-D are additive with oseltamivir, consistent with differences in mechanism of action. NA inhibition was observed using fetuin or MDCK cells as a substrate, but not in assays using a soluble sialic acid analogue. Collectin multimerization and CRD binding properties are key determinants for NA inhibition. SP-D had greater NA inhibitory activity than mannose-binding lectin, which in turn had greater activity than SP-A. The markedly greater NA inhibitory activity of SP-D compared to SP-A may partly account for the nding
Correspondence: Kevan L. Hartshorn, Department of Medicine, Boston University School of Medicine, EBRC 414, 650 Albany Street, Boston, MA 02118, USA e-mail: khartsho@bu.edu

that deletion of the SP-D gene in mice has a greater effect on viral replication in vivo. Introduction Innate defense plays important roles in host response and viral containment in the rst several days after IAV infection [17, 18, 20, 2325, 34, 35]. Although adaptive immune mechanisms are needed for ultimate viral clearance, during the vulnerable period between initial infection and elaboration of the adaptive response (antibodies and cytotoxic T cells), the virus must be contained without excessive inammation. There are several innate immune mechanisms that participate in the early containment of IAV, including cellular elements (e.g., NK cells, neutrophils, and macrophages) and soluble inhibitors (e.g., interferons, collectins, defensins, mucins, lung and salivary scavenger receptor cysteine-rich glycoprotein 340) [8, 9, 14, 15, 17, 19, 41]. Both surfactant proteins A and D (SP-A and SP-D) inhibit IAV, and mice lacking either protein suffer from increased illness, lung inammation and viral replication after infection with wild-type human IAV strains [20, 24, 25, 42]. The effect of deletion of SP-D is more profound that deletion of SP-A, indicating that SP-D may play a more important role. Of interest, mouse-adapted strains, which have been most frequently used in animal studies of immune

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T. Tecle et al. RfSP-DnCRD is a trimeric preparation containing the neck and carbohydrate-recognition domains (NCRD) of human SP-D, which was prepared in E. coli as recently described [3]. A neck CRD trimeric preparation of bovine CL43 was produced in the same way. Human alveolar proteinosis SP-A was graciously provided by Jeffrey Whitsett, Childrens Hospital Medical Center, Cincinnati, OH. RhMBL was expressed in CHO-K1 cells, puried as described [33], and was graciously provided by Drs. Kazue Takahashi and R. Alan B. Ezekowitz (Department of Pediatrics, Harvard Med. School). The nal concentrations of endotoxin in protein samples containing the highest concentrations of collectins were $20100 pg=ml (or 612 Endotoxin units=ml using internal assay standard). Oseltamivir was a gracious gift of Roche Pharmaceuticals.

response to IAV, are resistant to SP-D based on loss of oligomannose oligosaccharides on the viral envelope proteins [14]. The mechanisms of anti-inuenza activity of collectins have been partially elucidated. SP-D and the serum collectins, mannose-binding lectin (MBL), bovine conglutinin, and bovine CL43, bind in a calcium-dependent manner on the viral envelope proteins, hemagglutinin (HA) and neuraminidase (NA). These collectins inhibit HA activity and infectivity and agglutinate viral particles. These effects suggest inhibition of viral binding and=or other steps early in the viral life cycle. One report has shown that MBL inhibits membrane fusion activity of the IAV HA through immobilization of membrane components [22]. SP-D and MBL have also been reported to inhibit NA activity [31]. In this paper, we use a variety of NA assays, diverse viral strains, and various wild-type or recombinant mutant collectin preparations to characterize in detail the mechanism of NA inhibition by collectins. Materials and methods
Virus preparations Inuenza A virus was grown in the chorioallantoic uid of ten-day-old chicken eggs and puried on a discontinuous sucrose gradient as previously described [12]. The virus was dialyzed against PBS to remove sucrose, aliquoted and stored at 80  C until needed. The Philippines 82=H3N2 (Phil82) strain and its bovine-serum-b-inhibitor-resistant variant, Phil82=BS, and the Mem71H-BelN=H3N1 strain, were kindly provided by Dr. E. Margot Anders (Univ. of Melbourne, Melbourne, Australia). The A=PR=8=34=H1N1 (PR-8) strain was a gift of Dr. Jon Abramson (Bowman Gray School of Medicine, Winston-Salem, NC). Post-thawing, the viral stocks contained $5 1010 plaque forming units=ml. Preparations of collectins and oseltamivir Recombinant human SP-D (RhSP-D) and recombinant rat SP-D (RrSP-D) were produced in stably transfected CHO-K1 cells as previously described [7]. For these studies the dodecameric fraction of RhSP-D was used unless otherwise specied. Recombinant bovine CL43 [16] and chimeric collectins containing the human SP-D N-terminal and collagen domains and human MBL neck and carbohydraterecognition domains (called SP-D=MBLnCRD) or the rat SP-D N-terminus and collagen domain and the bovine conglutinin neck and carbohydrate-recognition domains (SP-D=CongnCRD) were also produced in CHO cells [10, 39].

Neuraminidase (NA) inhibition assays Inuenza virus NA activity and its inhibition by collectins or oseltamivir were measured by several assays. The rst was an enzyme-linked micro plate assay in which Arachis hypogaea peanut lectin was used to detect b-D-galactose-Nacetylglucosamine sequences exposed after the removal of sialic acid from fetuin [31]. The wells of a microtiter plate were coated with 50 ml of fetuin (Sigma F-2379: 20 ml=ml in PBS) overnight at 4  C and washed with PBS. Dilutions of IAV strains with different concentrations of SP-D or oseltamivir were preincubated for 30 min at 37  C, and 50 ml of the mixture was added to fetuin-coated wells and incubated at 37  C for 2 h. After the wells were washed, 50 ml of peroxidase-labeled peanut lectin [Sigma L-6135: 20 ml=ml in BSA (0.5%)] was added to each well for 30 min. at room temperature, followed by washing and incubation with 50 ml of TMP-peroxidase (Bio-Rad Labs, Hercules, CA) for 20 min. Finally, 50 ml of 1N H2SO4 was added to the wells and the absorbance was measured by ELISA plate reader at 450 nm. A modication of this assay involved the use of 96-well plates coated with MDCK cells instead of fetuin. This modied assay was carried out otherwise identically to the fetuin assay. An alternative assay using 20 -(4-methylumbelliferyl)-a-DN-acetylneuraminic acid (MU-NANA) was used as well to test inhibition of neuraminidase by SP-D or oseltamivir. This assay was performed as described by Gubareva et al. [5]. The assay depends on increased uorescence of MU-NANA after removal of the NANA component by the neuraminidase. IAV was pre-incubated for 30 min at 37  C with SP-D or oseltamivir in microtiter plates, followed by addition of 30 ml of a 0.1 mM solution of MU-NANA in a buffer containing 33 mM 2-[N-morpholino]ethanesulfonic acid [MES] and 4 mM CaCl2 (calcium-MES buffer) at pH 6.0. The mixtures were incubated at 37  C for 60 min, and the reaction was stopped by the addition of 150 ml of stop solution (0.1 M NaOH in 80% ethanol; pH 10.0). Fluorescence was quantied at an excitation wavelength of 360 nm and an emission

Inhibition of inuenza virus NA by collectins wavelength of 465 nm with a Polar Star OPTIMA (MG: LabTECH) plate-reading uorometer. Plaque assay The plaque assay was performed as described previously [13]. Briey, six-well tissue culture plates were seeded with Madin-Darby Canine Kidney (MDCK) cell monolayers. At 100% conuence (24 days post seeding), the cells were washed twice with PBS. A 108 dilution of the Phil82 IAV strain was prepared in DMEM medium. Where indicated,

1733 viral samples were pre-incubated with SP-D at 37  C in a shaking incubator for 30 min. Following infection of the wells with 1 ml of each dilution, the plates were incubated at 37  C. After 1 h adsorption, each well was overlaid with 2 DMEM medium containing 0.1% BSA, 0.4% agarose, 10 mg=ml of trypsin, and 10 mg=ml of DEAE-dextran and incubated at 37  C. After two days incubation in a CO2 incubator, the overlay was removed and the monolayer was stained with a 1:10 dilution of crystal violet in PBS. In some assays SP-D or oseltamivir were added after the one-hour infection of the monolayer with virus.

Fig. 1. Effect of delayed addition of SP-D on plaque assay The plaque assay was performed as described in Materials and methods using MDCK cells. The effect of pre-incubating Phil82 IAV with either RhSP-D multimers (left panel) or dodecamers (right panel) was tested (open squares). All concentrations of either SP-D signicantly inhibited plaque formation compared to control (p < 0.05; n 4). These results were compared to the effect of adding the same doses of SP-Ds 1 h after virus had been added to cells and allowed to incubate at 37  C (SP-D delayed). Delaying addition of SP-D reduced its ability to inhibit plaque formation compared to pre-incubation (p < 0.05 by ANOVA); however, plaque formation was still signicantly inhibited ( indicates p < 0.05 compared with control). The size of plaques was also reduced by SP-D added 1 h after viral infection (lower panel; 1 mg=ml SP-D dodecamers)

1734 Hemagglutination (HA) inhibition assay HA inhibition was measured by serially diluting collectin or other host defense protein preparations in round-bottom 96-

T. Tecle et al. well plates (Serocluster U-Vinyl plates; Costar, Cambridge, MA), using PBS as a diluent as described [19] and using type O human erythrocytes.

Fig. 2. SP-D inhibits viral neuraminidase activity SP-D dodecamers were pre-incubated with Phil82 IAV, and then NA activity was measured by assessment of cleavage of sialic acids from fetuin as described (panels AC). Different dilutions of virus were compared in panels A and B as indicated. In panel A, both dilutions of virus were signicantly inhibited (p < 0.05; n 5) at 500 and 1000 ng=ml of SP-D. The more dilute virus samples (100 HAU=ml) was also signicantly inhibited at 250 ng=ml. Pre-incubation of SP-D with mannose (100 mM) abrogated NA inhibition. Lower concentrations of SP-D were used in panel B, compared to determine the lower limit of activity. Signicant inhibition activity was observed at 50 and 100 ng=ml of SP-D at the lower virus dilution (p < 0.02 in each case; n 3). In each panel, the effect of SP-D was greater when tested against the more dilute virus samples. In panel C, SP-D (500 ng=ml) was pre-incubated with either mAb 246-07 or mAb 246-04 (1.3 mg=ml nal concentration) prior to incubation with IAV. The mAb 246-07 signicantly interfered with the NA inhibitory activity of SP-D (p < 0.05 for both concentrations tested; n 3). Panel D compares activity of SP-D against three differentially glycosylated IAV strains as indicated. SP-D did not signicantly inhibit NA activity of the Phil82=BS strain, but did inhibit activity of the Mem71H-BelN strain (p values shown). Activity against Phil82 was signicantly greater than activity against the other two strains (p < 0.05 by ANOVA; n 3). Concentrations of each virus that resulted in similar control levels of NA activity based on the fetuin assay were used

Inhibition of inuenza virus NA by collectins Statistics Statistical comparisons were made using Students paired, two-tailed t test or ANOVA with post hoc test (Tukeys).

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Results SP-D inhibits IAV plaque formation when added after establishment of viral infection Pre-incubation of IAV with SP-D dodecamers (right panel, Fig. 1) or high-molecular-weight multimers of SP-D (left panel, Fig. 1) of SP-D had strong viral neutralizing activity as assessed by the plaque assay; however, signicant residual plaque inhibition was noted when addition of SP-D was delayed by one hour after addition of IAV. In addition to a reduction in the number of plaques seen with delayed addition of SP-D, the size of plaques was reduced compared to controls (lower panels, Fig. 1). These ndings are compatible with effects of SP-D on later phases of the viral life cycle, including NA inhibition. SP-D inhibits viral neuraminidase (NA) activity by binding to viral carbohydrates SP-D inhibited NA activity of the Phil82 strain of IAV (Fig. 2). The degree of NA inhibition was dependent on the size of the viral inoculum. NA inhibition was abrogated by addition of mannose (Fig. 2; panel A). In addition, the activity was partially reduced by addition of the mAB 246-07 (Fig. 2; panel C). This antibody inhibits binding of SP-D to mannan [41]. In contrast the mAb 246-04, which does not inhibit binding to mannan, did not alter NA inhibition by SP-D. NA inhibition by SP-D correlates with the abundance of oligosaccharides on the viral HA We have previously reported that SP-D or MBL are unable to inhibit infectivity of the PR-8 strain of IAV due to the lack of mannosylated carbohydrates on the envelope proteins of this strain [13, 14, 36]. SP-D also lacked any NA inhibiting activity against the PR-8 strain (data not shown). Phil82=BS is resistant to neutralization by SP-D and serum collectins due to loss of a specic high-mannose oligosaccharide on the distal tip of the HA molecule [6].

Fig. 3. SP-D inhibits ability of viral NA to cleave sialic acid from MDCK cell monolayers In panel A, monolayers of MDCK cells in 96-well plates were exposed to Phil82 IAV, and cleavage of sialic acids from the cells was measured by binding of peanut agglutinin as in Fig. 2. Preincubation of the virus with SP-D, or pre-incubation of the MDCK cells with SP-D prior to adding virus, reduced NA activity signicantly (n 3; p < 0.02 for 1 and 2 mg=ml ml concentrations of SP-D). Adding SP-D after virus had been allowed to incubate with the cells for 30 min at 37  C had no effect. In panel B, binding of SP-D to IAV-infected or uninfected MDCK cells was tested. MDCK cells were treated with the Phil82 IAV strain or control buffer and then incubated for 6 h. Binding of SP-D dodecamers to the monolayers was then tested by ELISA. SP-D bound to both uninfected and IAV-infected monolayers (p < 0.05 for all SP-D concentrations tested; n 3 or 4 experiments), but binding to infected monolayers was signicantly greater than to uninfected cells (assessed by ANOVA)

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T. Tecle et al. Table 1. Interactions between SP-D and oseltamivir in NA inhibition assays Oseltamivir concentration (ng=ml) 0 Control buffer SP-D 50 ng=ml SP-D 100 ng=ml
a

SP-D did not inhibit NA activity of the Phil82=BS strain (Fig. 2; panel D). The Mem71H-BelN (H3N1) strain is derived from an early isolate of the H3 subtype that is less sensitive to neutralization or HA inhibition by collectins than recent H3 strains (e.g., Phil82) due to a decreased abundance of oligosaccharides on the distal tip of the HA [31]. SP-D caused a lower degree of NA inhibition for this strain than for the Phil82 strain (Fig. 2; panel D). SP-D does not inhibit binding of NA to a soluble NA substrate The above results suggest that NA inhibition by SP-D is dependent on glycosylation of the HA. It is possible therefore that SP-D binds to the HA and sterically inhibits access of the NA to surface substrates. As shown in Fig. 3, panel A, SP-D inhibits the ability of Phil82 IAV to cleave sialic acids from the surface of MDCK cells. Of interest, NA activity was inhibited whether the virus was pre-incubated with SP-D or SP-D was rst added to MDCK cells, followed by addition of virus. In panel B, SP-D is shown to bind to uninfected MDCK cells, perhaps providing explanation for the ability of SP-D to inhibit NA activity when added to MDCK cells before addition of virus. Of note, SP-D bound much more strongly to MDCK cells that were previously infected with virus (panel B). Figure 3, panel A, also shows that adding SP-D after IAV was allowed to incubate with MDCK cells for 30 min had no effect on sialic acid removal from the cells. This provides conrmation that SP-D inhibits NA activity with respect to target epithelial cells. If SP-D causes this inhibition by indirect effects resulting from binding to the HA, it would be unlikely to block access of soluble sialic acid derivatives the NA binding site. This was conrmed using the MU-NANA assay. Concentrations of 0.5 and 1 mg=ml of SP-D caused no inhibition of Phil82 IAV in this assay (e.g., NA activity was 102 0.3% of control using 1 mg=ml of SP-D dodecamers), whereas oseltamivir reduced NA activity to 12 2% of control (n 3; data not shown). Using the fetuin assay, SP-D had additive NA inhibiting effects when combined with oseltamivir (Table 1), suggesting possible differences in the mechanism of NA inhibition.

5 79 6 55.5 8 33.5 5.6

10 59 7 39 10 25 6

100 0a 80.5 6 56 7.5

Results shown are mean SEM of 4 experiments. All values were signicantly reduced (p< 0.05) compared to control. Values for samples SP-D combined with oseltamivir were signicantly lower than samples treated with SP-D or oseltamivir alone (p < 0.03). The combination of SP-D 100 ng=ml and oseltamivir 10 ng=ml had signicantly lower NA activity than either SP-D or oseltamivir alone as assessed by ANOVA.

NA inhibition correlates with HA inhibition and neutralizing activity of different collectins We have previously shown that replacement of the neck and CRD of human SP-D with those of MBL to create the chimeric molecule (SP-D=MBLnCRD) results in increased HA inhibiting and viral neutralizing activity compared to either wild-type SP-D or MBL [39, 40]. Similar results were obtained by replacing the neck and CRD of rat SP-D with those of bovine conglutinin to create the chimera SP-D=CongnCRD [10]. In Fig. 4, panels A and B, we compared the NA inhibitory activity of dodecamers of the chimeras with wild-type recombinant rat SP-D or human SP-D against the Phil82 and Mem71H-BelN viral strains. Human SP-D had greater NA inhibitory activity against either viral strain than rat SP-D. Both chimeras, SP-D=MBLnCRD and SP-D=CongnCRD, had greater activity than RrSP-D against the Phil82 strain as well (Fig. 4; panel A), although they did not have greater activity than human SP-D against this strain. In contrast, the chimeras had stronger NA inhibitory activity, which exceeded that of human SP-D (or rat SP-D) against the Mem71H-BelN strain (Fig. 4; panel B). Comparison of NA inhibitory activity of SP-D to activity of other collectins MBL also caused signicant inhibition of NA activity of the Phil82 strain of IAV (Fig. 4; panels C and D), although SP-D and SP-D=MBLnCRD

Inhibition of inuenza virus NA by collectins

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Fig. 4. Comparison of NA inhibitory activity of human or rat SP-D with SP-A, MBL or the chimeric collectins SP-D= MBLnCRD and SP-D=CongnCRD NA inhibition was measured as in Fig. 2. Panel A shows the activity of dodecamers of SP-D=MBLnCRD, recombinant rat and human SP-D (RrSP-D and RhSP-D), and SP-D=CongnCRD against the Phil82 strain, while panel B shows activity of these collectins against the Mem71H-BelN strain. All of the collectins signicantly inhibited NA activity of Phil82 (p < 0.05 for all), although the activity of rat SP-D was less than the others. The chimeras caused signicantly greater inhibition of activity of the Mem71H-BelN strain than either wild-type form of SP-D. Recombinant rat SP-D did not signicantly inhibit the Mem71H-BelN strain in these experiments. In panel C, the activity of SP-A was compared to that of MBL using 200 HAU of Phil82 IAV. SP-A only caused signicantly NA inhibition at 15 mg=ml concentration. MBL caused signicantly greater NA inhibition than SP-A at 5, 10 and 15 mg=ml concentrations (p < 0.05 by ANOVA). In panel D, the activity of MBL was compared to that of SP-D=MBLnCRD (Phil82 strain; 200 HAU=ml). SP-D=MBLnCRD had signicantly greater activity than MBL. Results shown are mean SEM of 4 experiments. Indicates p < 0.05 compared to control

dodecamers or multimers were more than 1 log more potent than MBL. SP-A was signicantly less active at NA inhibition than MBL or SP-D and only

caused signicant inhibition at 15 mg=ml using the Phil82 (Fig. 4; panel C) strain of IAV. Similar results were obtained using the PR-8 strain and SP-A

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(e.g., approximately 20% inhibition of NA activity at 15 mg=ml of SP-A; data not shown). Role of multimerization in NA inhibition The SP-D=MBLnCRD chimera forms trimers, dodecamers and high-molecular-weight multimers similar to those formed by SP-D, and these differentially oligomerized forms can be isolated by gel ltration and, like SP-D, have increasing viral aggregating and neutralizing activity with increasing levels of oligomerization [7, 39, 40]. Trimers of SP-D=MBLnCRD were devoid of NA inhibiting activity, whereas highmolecular-weight multimers or dodecamers had strong activity (Fig. 5). We tested NA inhibiting activity of a preparation of trimeric fragments of SP-D composed only of the neck and CR domains (RfSP-DnCRD). This preparation did not have any detectable NA inhibiting activity at concentrations up to 5 mg=ml (data not shown).

These results could indicate that multimerization is an important determinant of NA inhibition activity and suggest that viral aggregation may contribute to NA inhibiting activity by SP-D, since the trimeric preparations of SP-D or SP-D=MBLnCRD lack aggregating activity. CL43 is a bovine serum collectin that is trimeric in both natural and recombinant forms and lacks viral aggregating activity; however, it has strong viral neutralizing and HA inhibiting activity due to distinctive properties of its CRD [3, 16]. As shown in Fig. 5 (panel B), CL43 had signicant NA inhibitory activity that exceeded that of SP-A or MBL and was only slightly less than that of SP-D dodecamers. Hence, multimerization or aggregating activity (at least as assessed by a sensitive light scattering assay [16]) are not prerequisites for NA inhibition by collectins. Of interest, the truncated neck CRD preparation of CL43 (CL43nCRD) lacked NA inhibiting activity (Fig. 5), suggesting

Fig. 5. Role of multimerization of collectins in NA inhibition In panel A, the NA inhibitory activity of SP-D=MBLnCRD trimers, dodecamers and multimers was tested in comparison to that of SP-D dodecamers and multimers (NA assay as in Fig. 2). SP-D=MBLnCRD trimers had no detectable activity. SP-D=MBLnCRD multimers had signicantly greater activity than SPD multimers (p < 0.05 by ANOVA). Activity of dodecamers of either SP-D or SP-D=MBLnCRD was signicantly greater than that of multimers of SP-D (p < 0.05 by ANOVA). In panel B, the activities of wild-type recombinant bovine CL43 and CL43nCRD were compared. CL43 had signicant NA inhibitory activity, as noted, but CL43nCRD did not. All results are mean SEM of 4 experiments and Phil82 was used

Inhibition of inuenza virus NA by collectins

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that the N-terminal and collagen domains of CL43 contribute to NA inhibition. Discussion The importance of the IAV NA in vivo is evidenced by the clinical effectiveness of NA inhibitors (e.g., oseltamivir or zanamivir) [21]. The principal role of the IAV NA is to allow newly formed viral particles to bud from the cell and disseminate without binding to the cell of origin or to other viral particles [26]. It is possible that in vivo the NA also plays a role in the early phase of infection by allowing penetration through mucous layers [28]. Although a substantial portion of the in vitro antiviral activity of collectins results from binding to the HA and inhibition of early phases of viral infection, the observation that plaque formation is inhibited when SP-D is added after establishment of initial viral infection is compatible with effects on later stages of viral replication, including NA inhibition. In the current study, we provide substantial new information regarding the mechanism of NA inhibition by SP-D and its relative activity compared to other collectins. The role of SP-Ds lectin activity in NA inhibition was shown by abrogration of the activity by mannose or by a mAb directed against its monosaccharide binding pocket, and by the lack of activity against the PR-8 viral strain. The NA inhibitory activity of SP-D against several other viral strains strongly supports the concept that binding of SP-D to the HA is important for inhibiting NA. Overall NA inhibitory activity against the different strains correlated with HA inhibitory and neutralizing activity of SP-D (which depend on binding to the HA). Of particular note is the lack of NA inhibition of the Phil82=BS strain, which differs from the highly sensitive parental Phil82 strain by virtue of loss of a high-mannose oligosaccharide on the tip of the viral HA. The Mem71H-BelN strain has been shown to be less sensitive to neutralization and HA inhibition by SP-D due to reduced abundance of glycans on the distal tip of the HA [9, 11, 31]. During the evolution of the H3N2 subtype in humans there has been a progressive addition of glycans on the globular domain of the HA, especially localized in key

antigenic areas. There is evidence that these glycans protect the HA from antibody-mediated neutralization, although they render the more recent strains (1980s onward) more susceptible to inhibition by SP-D or MBL [31, 32]. We have shown that SP-D binds strongly to NA of Mem71H-BelN on Western blots [11], although inhibition of enzymatic activity of this strain is signicantly reduced compared to inhibition of Phil82. Overall, these results suggest that NA inhibition by SP-D results at least in part from binding to the HA. Reading et al. also found that there was a substantial difference in the ability of SP-D to inhibit NA activity of earlier and more recent H3N2 strains despite apparently equivalent binding to NA of these strains on Western blots [31]. Many of our other ndings are compatible with this concept as well. In order to conrm that NA inhibition by SP-D was not an artifact of the use of fetuin, we repeated the assay using MDCK cells as the substrate for NA activity measurement. These results conrmed that SP-D inhibits cleavage of sialic acids from the surface of target cells when the virus is incubated with SP-D at or before addition of virus to the cells. Addition of SP-D after allowing time for the virus to interact with the cells showed no activity, as expected, conrming that SP-D was not interfering with the assay in a nonspecic manner. The ability of SP-D to bind to MDCK cells and inhibit NA activity of subsequently added virus is of interest, and future studies will determine if similar effects occur with respiratory epithelial cells. Lack of inhibition in the MU-NANA assay is still consistent with physiologically relevant NA inhibition by inhibitors that do not bind directly to the sialic acid binding pocket of the NA. The MU-NANA assay is most sensitive for inhibitors which directly occlude the binding site of the NA (like oseltamivir or related small-molecule sialic acid analogues). There are many examples of physiologically relevant alterations in NA activity detected using cells or fetuin as substrates but not by soluble sialic-acid-based inhibitors. For example, development of resistance to NA inhibitors can result from changes in either the NA or the HA because reduced HA binding afnity can result in loss of dependence on the NA [30]. Alterations in NA activity resulting from stalk

1740 Table 2. Comparative IC50 values for NA inhibition by collectins Collectin preparation RhSP-D RrSP-D RhSP-D=MBLnCRD RrSP-D=ConglutininnCRD RhMBL SP-A RCL43 RfSP-DnCRD CL43Ncrd Description of collectin recombinant dodecamer recombinant dodecamer recombinant dodecamer recombinant dodecamer recombinant octadecamer natural octadecamer recombinant trimer recombinant truncated trimer recombinant truncated trimer IC50 for Phil82 (in ng=ml) 150 700 150 200 3500 >16000 2400 >5000 >5000

T. Tecle et al.

IC50 for Mem71H-BelN (in ng=ml) 2000 IC50 not reached 300 500 ND ND ND ND ND

Results shown are approximate values determined using 100 HAU of the indicated viruses and are derived from Figs. 37. ND indicates not determined.

truncations of the NA or from alterations of the HA molecule are only evident on cell-based assays and not on the MU-NANA assay [1, 29]. It has also been shown that some anti-NA monoclonal antibodies can inhibit NA activity using fetuin as a substrate but not using the trisaccharide neuraminyl-lactose [38], presumably because they block access of the larger, but not the smaller, substrate. The results with recombinant wild-type and mutant collectins also suggest the importance of steric blockade as mechanism of NA inhibition by collectins. Table 2 shows the comparison of approximate IC50 values for NA inhibition by the collectins used in this paper. Overall, the activity of the various preparations in NA inhibition corresponds to their activity in neutralizing and HA inhibition assays. However, it is notable that the truncated neck CRD form CL43 lacked NA inhibitory activity since this preparation has HA inhibiting and neutralizing activity [3]. In contrast, full-length CL43 did inhibit NA activity signicantly. One plausible explanation for this nding is that the large N-terminal and collagen domains of full-length collectins provide important extra steric hindrance (comparable to the added steric hindrance of full-length antibodies as compared to FAb fragments) [4], which is important for occluding access of the NA to surfaces. The greater NA inhibitory activity of human SP-D or SP-D=MBLnCRD dodecamers compared to MBL may also result from the much more extended conguration of the SP-D collagen domain allowing for greater steric interference with access of the

NA to cell surfaces. The low activity of SP-A is particularly interesting. One possible explanation for this is that the mechanism of binding and neutralization used by SP-A is less effective for NA inhibition. SP-A binds to the viral HA by attachment of the virus to a large sialic-acid-bearing glycan on the CRD of SP-A [2]. This type of attachment may be less effective for steric hindrance of the NA since the collectin may be less closely apposed to the viral surface. It remains possible that collectins inhibit NA activity of intact viruses by binding directly to the NA, since binding to the NA has been demonstrated by Western blot [11, 27]. The N2 NA has a conserved oligomannose glycan at residue 200, which is on the surface of the tetramer and could serve as an attachment site for collectins [37]. However, there are approximately 5-fold more HA molecules than NA molecules on the viral surface, and the HA has a more extended conguration that may make it more accessible. There is an additional oligomannose glycan on the undersurface of the N2 head region which might only become accessible when the NA is solubilized, resulting in binding on Western blots which does not occur with intact virions. Further studies using recombinant viruses generated by reverse genetics should be able to denitively establish the relative roles of binding to NA and HA in NA inhibition mediated by collectins. Although we cannot exclude a role for direct binding to NA, we propose that collectins cause signicant NA inhibition principally by binding to the

Inhibition of inuenza virus NA by collectins

1741 8. Hartshorn K, Reid K, White M, Morris S, Jensenius J, Crouch E (1996) Neutrophil deactivation by inuenza A viruses: mechanisms of protection after viral opsonization with collectins and hemagglutination-inhibiting antibodies. Blood 87: 34503461 9. Hartshorn K, White M, Shepherd V, Reid K, Jensenius J, Crouch E (1997) Mechanisms of anti-inuenza activity of pulmonary surfactant proteins A and D: comparison with other collectins. Amer J Physiol 273: L1156L1166 10. Hartshorn K, Sastry K, Chang D, White M, Crouch E (2000) Enhanced antinuenza activity of a recombinant pulmonary surfactant protein D and serum conglutinin fusion protein. Amer J Physiol 278: L90L98 11. Hartshorn K, White M, Voelker D, Coburn J, Zaner K, Crouch E (2000) Mechanism of binding of surfactant surfactant protein D to inuenza A viruses: importance of binding to hemagglutinin to antiviral activity. Biochem J 351: 449458 12. Hartshorn KL, Collamer M, Auerbach M, Myers JB, Pavlotsky N, Tauber AI (1988) Effects of inuenza A virus on human neutrophil calcium metabolism. J Immunol 141: 12951301 13. Hartshorn KL, Sastry K, White MR, Anders EM, Super M, Ezekowitz RA, Tauber AI (1993) Human mannosebinding protein functions as an opsonin for inuenza A viruses. J Clin Invest 91: 14141420 14. Hartshorn KL, Crouch EC, White MR, Eggleton P, Tauber AI, Chang D, Sastry K (1994) Evidence for a protective role of pulmonary surfactant protein D (SP-D) against inuenza A viruses. J Clin Invest 94: 311319 15. Hartshorn KL, Liou LS, White MR, Kazhdan MM, Tauber JL (1995) Neutrophil deactivation by inuenza A virus: role of hemagglutinin binding to specic sialic acid-bearing cellular proteins. J Immunol 154: 39523960 16. Hartshorn KL, Holmskov U, Hansen S, Zhang P, Meschi J, Mogues T, White MR, Crouch EC (2002) Distinctive anti-inuenza properties of recombinant collectin 43. Biochem J 366: 8796 17. Hartshorn KL, White MR, Mogues T, Ligtenberg T, Crouch E, Holmskov U (2003) Lung and salivary scavenger receptor glycoprotein-340 contribute to the host defense against inuenza A viruses. Am J Physiol Lung Cell Mol Physiol 285: L1066L1076 18. Hartshorn KL, Ligtenberg A, White MR, van Eijk M, Hartshorn M, Pemberton L, Holmskov U, Crouch E (2006) Salivary agglutinin and lung scavenger receptor cysteine-rich glycoprotein 340 have broad anti-inuenza activities and interactions with surfactant protein D that vary according to donor source and sialylation. Biochem J 393: 545553 19. Hartshorn KL, White MR, Tecle T, Holmskov U, Crouch EC (2006) Innate defense against inuenza A virus: activity of human neutrophil defensins and inter-

viral HA and steric hindrance of access of the NA to surface-bound substrates (e.g., cell surfaces). This NA inhibitory activity appears to play a signicant role in the overall antiviral activity of SP-D and, to a lesser extent, MBL. SP-A, in contrast, has minimal NA inhibitory activity, which could account in part for the relatively reduced impact of SP-A (as compared to SP-D) gene deletion on IAV viral replication in mice. It will, of course, be important to demonstrate in vivo inhibition of NA activity by collectins in future studies using similar models and selected viral strains. Acknowledgements
This work was supported by NIH grants HL69031 (KLH) and HL29594 and 44015 (EC).

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