of February 15, 2018.This information is current as Virulence in Mice

Inhibitors of the Innate Immune System and Influenza A Virus Determine Sensitivity tothe Hemagglutinin of H1N1 Subtype

-Linked Glycosylation onNSpecific Sites of

Michelle D. Tate, Andrew G. Brooks and Patrick C. Reading

ol.1100295http://www.jimmunol.org/content/early/2011/07/15/jimmun

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The Journal of Immunology

Specific Sites of N-Linked Glycosylation on the Hemagglutininof H1N1 Subtype Influenza AVirus Determine Sensitivity toInhibitors of the Innate Immune System and Virulence inMice

Michelle D. Tate,* Andrew G. Brooks,* and Patrick C. Reading*,†

Oligosaccharides on the hemagglutinin (HA) and neuraminidase of influenza Avirus (IAV) are a target for recognition by lectins of

the innate immune system, including soluble surfactant protein-D and the macrophage mannose receptor on airway macrophages.

Glycans attached to the head of H1 subtype of IAV differ markedly in number and location. A reverse genetic approach was used to

define the importance of particular N-glycosylation sites on H1 in determining sensitivity to innate immune defenses and virulence

in mice. The HA of A/PR/8/34 (PR8, H1N1) and A/Brazil/11/78 (Brazil, H1N1) express zero and four glycosylation sites on the

head of HA, respectively. Site-directed mutagenesis was used to add (PR8) or delete (Brazil) glycosylation sites, and IAVexpressing

wild-type or mutant HA were generated on a PR8 backbone. Addition or removal of particular glycans modulated sensitivity to

mouse lung fluids but was not a major factor determining susceptibility of airway macrophages to infection. PR8 is a mouse-

adapted virus, and mutations in multiple IAV genes have been shown to contribute to virulence, yet addition of glycosylation to

PR8 HA was sufficient to attenuate disease. In contrast, removal of glycans from Brazil HA resulted in severe disease and death.

These studies provide insight regarding the mechanisms by which IAV can induce disease in mice. Moreover, reduced glycosyl-

ation of HA is likely to be an important factor associated with adaptation of human IAV to growth in mouse lung. The Journal of

Immunology, 2011, 187: 000–000.

Glycans on the hemagglutinin (HA) of influenza A virus(IAV) attach through N-glycosidic linkages to asparagine(Asn) residues of the conserved glycosylation site motif

Asn-X-Ser/Thr, in which X may represent any amino acid exceptproline (1). Oligosaccharides attached to the stalk region are wellconserved between different IAV strains and are important indetermining appropriate folding and conformation of HA (2–5). Incontrast, the location and number of glycans on the globular headof HA differ markedly between IAV strains (6, 7).HA serves as the major target for neutralizing Abs, and glycans

expressed on the head of HA are likely to shield or modify antigenicsites (8), thereby preventing recognition by Abs elicited to pre-

viously circulating strains. Since its re-emergence in 1977, gly-cosylation sites have been added and lost from the HA of seasonal

H1N1 IAV, although the majority of strains have maintained three

to five sites (9, 10). Loss or gain of glycans can also affect bi-

ological properties of IAV by altering the affinity of HA for host

cell receptors (11, 12), modulating proteolytic cleavage of the

HA0 precursor (13) or disrupting the balance between receptor

binding activity and efficient particle release (14). In addition,

glycosylation on HA is important in determining sensitivity of

IAV to recognition by lectins of the innate immune system.Surfactant protein (SP)-D, a calcium-dependent (C-type) lectin

of the collectin family, binds to mannose-rich glycans on influenza

virus HA/neuraminidase (NA) glycoproteins to mediate a range

of antiviral activities in vitro (15–17). In addition, the macrophage

mannose receptor (MMR), a membrane-associated C-type lectin,

also binds oligosaccharides on HA/NA to facilitate virus uptake

and destruction by macrophages (Mf) (18, 19). Previous studies

have reported a correlation between the degree of glycosylation

on the head of HA and: 1) susceptibility to neutralization by sur-

factant protein D (SP-D) (17, 20, 21); 2) ability to bind MMR and

infect murine Mf (18, 19); and 3) virulence of different IAV in

mice (17, 20, 22). The mouse-adapted A/PR/8/34 strain (PR8,

H1N1) lacks glycosylation on the head of HA (23), is resistant to

SP-D (17, 24), poor in its ability to infect Mf (18, 19, 25), and

highly virulent in mice (22, 26). A mutant of A/Brazil/11/78

(Brazil, H1N1), resistant to the bovine collectin conglutinin, was

found to lack N-glycosylation site Asn104 (H1 numbering) from

the head of HA (27) and showed a modest reduction in sensitivity

to rat SP-D (28). Detailed analysis of the role of that particular

glycosylation site in determining sensitivity to innate immune

defenses and virulence has not been reported.

*Department of Microbiology and Immunology, University of Melbourne, Mel-bourne, Victoria 3010, Australia; and †Victorian Infectious Diseases Reference Lab-oratory, World Health Organization Collaborating Centre for Reference and Researchon Influenza, North Melbourne, Victoria 3051, Australia

Received for publication January 28, 2011. Accepted for publication June 13, 2011.

This work was supported by Project Grant 509230 from the National Health andMedical Research Council of Australia. P.C.R. is a National Health and MedicalResearch Council R.D. Wright Research Fellow. The Melbourne World Health Or-ganization Collaborating Centre for Reference and Research on Influenza is sup-ported by the Australian Government Department of Health and Ageing.

Address correspondence and reprint requests to Dr. Patrick C. Reading, Departmentof Microbiology and Immunology, University of Melbourne, Parkville, Melbourne,VIC 3010, Australia. E-mail address: preading@unimelb.edu.au

Abbreviations used in this article: Asn, asparagine; BAL, bronchoalveolar lavage;Brazil, A/Brazil/11/78; HA, hemagglutinin; IAV, influenza A virus; Mf, macro-phages; MDCK, Madin–Darby canine kidney; MMR, macrophage mannose receptor;NA, neuraminidase; Neu5Ac, N-acetylneuraminic acid; NP, nucleoprotein; PI, propi-dium iodide; PR8, A/PR/8/34; RG, reverse genetics; SA, sialic acid; SP, single-positive; SP-D, surfactant protein-D; TCID50, 50% tissue culture-infective dose;WT, wild-type.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

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We have investigated the role of glycosylation on the head ofH1 subtype IAV in modulating sensitivity to components of themurine innate immune system (SP-D and Mf) in vitro and viru-lence in mice. Site-directed mutagenesis allowed for sequentialremoval or addition of potential glycosylation sites to the HA, andreverse genetics (RG) was used to engineer 7:1 reassortant virusesexpressing either wild-type (WT) or glycosylation mutant HA ona PR8 backbone. We have removed each of the four potential sitesfrom the head of Brazil HA, as well as generating mutants inwhich multiple sites were deleted. We have also added individualN-glycosylation sites to PR8 HA and generated a mutant ex-pressing two sites on the head of HA. Data presented in thisstudy demonstrate that HA glycosylation was a critical factordetermining sensitivity to SP-D but was not a major factor influ-encing susceptibility of murine Mf to infection. RG virusesexpressing reduced glycosylation were resistant to neutralizationby mouse lung fluids and caused severe disease in mice. Using theHA of PR8 or Brazil, we demonstrate that Asn144 was particularlyimportant in determining sensitivity to murine mannose-specificlectins in vitro and disease in mice.

Materials and MethodsMice and viruses

C57BL/6 (B6) mice were bred and housed in specific pathogen-free con-ditions at the Department of Microbiology and Immunology, University ofMelbourne (Melbourne, VIC, Australia). Male mice 6–8 wk of age wereused in all experiments. The influenza virus strains A/PR/8/34 Mount Sinai(PR8, H1N1) and BJ3109 (H3N2) were obtained from the World HealthOrganization Collaborating Centre for Reference and Research on In-fluenza, North Melbourne, VIC, Australia. Viruses were grown in 10-dembryonated eggs by standard procedures and titrated by plaque assayunder Madin–Darby canine kidney (MDCK) cells as described (29).

Construction of glycosylation mutant viruses using RG

Reassortant IAV used in this study were generated by eight-plasmid RGas previously described (30). Site-directed mutagenesis was performed tosequentially remove N-linked glycosylation sites from the Brazil HA (H1)or to add sites to the globular head of PR8 HA (H1). Removal of N-gly-cosylation sites was achieved by substituting Asn with Ala to abrogateglycosylation sequence motif Asn-X-Ser/Thr (i.e., Asn104 [NGT → AGT],Asn144 [NIT → AIT], Asn172 [NGS → AGS], and Asn177 [NLS → ALS]).To add N-glycosylation sites to PR8 HA, nucleic acid mutations wereperformed to facilitate amino acid substitutions that created glycosylationmotifs (i.e., Asn-X-Ser/Thr) at sites identical to those in the sequence ofBrazil HA (i.e., Asn104 [NGI→ NGT], Asn144 [NTNG→ NTTRG], Asn172[EGS→ NGS], and Asn177 [KLK→ NLS]). Site-directed mutagenesis wasperformed using cloned Pfu DNA polymerase (Stratagene), and parentaltemplate was then digested via incubation with DpnI (New EnglandBiolabs). Viruses generated were 7:1 reassortants consisting of the PR8backbone with WT or glycosylation mutant HA from either PR8 or Brazil.Plasmid vectors containing the seven genes of PR8 (other than HA) used tocreate RG-engineered viruses were kindly provided by Dr. Robert Webster,St. Jude Children’s Research Hospital (Memphis, TN). The HA gene of allviruses generated by RG was sequenced to ensure appropriate mutationswere retained and that additional mutations were not introduced duringgeneration of RG viruses.

Titers of infectious virus were determined using standard plaque assay(12) or immunofluorescence on MDCK cells. For immunofluorescence,monolayers of MDCK cells cultured in 96-well plates (Nunc) were in-oculated with dilutions of virus, incubated 1 h at 37˚C in 5% CO2, washed,and incubated a further 6–8 h. After incubation, cells were washed, fixedwith 80% (v/v) acetone in water, and stained for expression of newlysynthesized viral nucleoprotein (NP) using mAb MP3.10G2.IC7 (WorldHealth Organization Collaborating Centre for Reference and Researchon Influenza, Melbourne, VIC, Australia), followed by FITC-conjugatedsheep anti-mouse Ig (Silenus). Plates were viewed under 3100 originalmagnification, and the total number of fluorescent foci in duplicate wellswas used to calculate the titer of infectious virus.

In some experiments, the 50% tissue culture-infective dose (TCID50) wasdetermined using monolayers of MDCK cells in flat-bottom 96-well tissue-culture plates (Nunc). Samples were titrated and incubated at 37˚C in 5%

CO2 for 3 d in the presence of 2 mg/ml trypsin (TPCK-treated; Wor-thington Biochemical). Monolayers were assessed using light microscopyand the dilution in which pathological change in 50% of the cell culturecould be detected was used to calculate the TCID50 titer, expressed inTCID50 per milliliters of original sample.

Desialyation and resialyation of erythrocytes

Methods for the enzymatic modification of erythrocyte oligosaccharideshave been described previously (31). In brief, suspensions (10%) of humanerythrocytes were desialylated with 600 mU/ml sialidase from Clostridiumperfringens (Sigma-Aldrich) for 1 h at 37˚C, washed twice, and resialy-lated using 1.5 mM CMP-N-acetylneuraminic acid (Neu5Ac; Sigma-Aldrich) and 4 mU either b-galactoside-a2,3-sialyltransferase (Japan To-bacco, Shizuka, Japan) or b-D-galactosyl-b1,4-N-acetyl-b-D-glucosamine-a2,6-sialyltransferase (Merck) or with buffer alone (sham-treated) at 37˚Cfor 4 h. Erythrocytes were washed in TBS and used in standard HAtitrations.

Virus neutralization assay

The ability of mouse bronchoalveolar lavage (BAL) fluids and recombinantrat SP-D to neutralize virus infectivity was measured by fluorescent-focusreduction in monolayers of MDCK cells cultured in 96-well plates asdescribed (17). Naive mice were euthanized, and BAL fluids were obtainedby flushing the lungs three times with 1 ml TBS through a blunted 23-guage needle inserted into the trachea. BAL fluids were clarified by cen-trifugation and supernatants frozen at 220˚C. Recombinant rat SP-D wasa generous gift from Prof. Erika Crouch (Department of Pathology andImmunology, Washington University School of Medicine, St. Louis, MO).BAL supernatants or SP-D were diluted in TBS supplemented with 10 mMCaCl2, incubated with virus for 30 min, and added to MDCK cell mono-layers. The number of IAV-infected cells was determined at 7 to 8 hpostinfection using immunofluorescence as described above. The totalnumber of fluorescent foci in four representative fields were counted andexpressed as a percentage of the number of foci in the corresponding areaof duplicate control wells infected with virus alone (i.e., percent of viruscontrol).

Infection of mouse Mf and epithelial cells

Mouse peritoneal exudate cell Mf or the murine LA-4 lung epithelial cellline were prepared and infected with virus in eight-well chamber slides asdescribed (17). Slides were stained for expression of IAV NP as above andthen with 10 mg/ml propidium iodide (PI; Sigma-Aldrich) to visualizenuclei of adherent cells and therefore determine total cell number. Finally,slides were washed extensively, mounted in 80% v/v glycerol, and assessedusing fluorescence microscopy. The percentage of fluorescent cells wasdetermined in a minimum of four random fields with a minimum of 200cells counted for each sample. To test for inhibition of infection bymannan, cells were incubated with mannan (final concentration 10 mg/ml)for 30 min at room temperature before addition of virus.

To determine the titer of infectious virus in supernatants from IAV-infected LA-4 epithelial cells, cell monolayers were incubated with 106

PFU each virus for 60 min, washed three times to remove free virus, andincubated at 37˚C in 5% CO2. At 2 and 24 h postinfection, supernatantswere removed, clarified by centrifugation, and titers of infectious virusdetermined by TCID50 on MDCK cell monolayers.

Virus infection of mice

Groups of five mice were anesthetized and infected with 105 PFU or 103

PFU IAV via the intranasal route in 50 ml PBS. Mice were weighed dailyand assessed for signs of clinical disease including inactivity, ruffled fur,labored breathing, and huddling behavior. Animals that had lost $25% oftheir original body weight were euthanized. All research complied with theUniversity of Melbourne’s Animal Experimentation Ethics guidelines andpolicies. At various times postinfection, mice were euthanized, and thelungs, nasal tissues, brain, and hearts were removed, homogenized in PBS,and clarified by centrifugation. Titers of infectious virus in tissue homo-genates were determined by standard plaque assay or immunofluorescenceon MDCK cells.

Recovery and characterization of leukocytes from mice

BAL cells and heparinized blood were obtained from mice as described(32). Thymi were pressed through 70-mm plastic mesh with the plunger ofa 3-ml plastic syringe to generate single-cell suspensions. Samples weretreated with Tris-NH4Cl (0.14 M NH4Cl in 17 mM Tris, adjusted to pH7.2) to lyse erythrocytes and washed in RPMI 1640 medium supplemented

2 INFLUENZA VIRUS H1 GLYCOSYLATION AND VIRULENCE IN MICE

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with 10% FCS. Cell numbers and cell viability were determined via trypanblue exclusion.

For flow cytometric analysis, single-cell suspensions prepared from theblood, BAL, and thymi were incubated on ice for 20 min with supernatantsfrom hybridoma 2.4G2 to block Fc receptors, followed by staining withappropriate combinations of FITC, PE, and allophycocyanin conjugated toGr-1 (RB6-8C5), CD45.2 (104), CD8a (53-6.7), CD4 (GK1.5), B220 (RA3-6B2), TCRb (H57-597), and NK1.1 (PK136). Addition of 10 mg/ml PI toeach sample was used to identify viable cells. Cells were analyzed using anFACSCalibur flow cytometer (BD Biosciences), and a minimum of 50,000PI2 cells were collected. Airway Mf were identified following cytospinand differential staining of BAL cells.

Pulmonary histopathology

Lungs were perfused, inflated, and fixed in a solution of 4% formaldehyde aspreviously described (33) before 4-mm sections were prepared and stainedwith H&E. Airway inflammation of H&E-stained lung sections wasevaluated on a subjective scale of 0–5 (0, no inflammation; 1, very mild; 2,mild; 3, moderate; 4, marked; and 5, severe inflammation) by three in-dependent readers, as described (33). Sections were blinded and random-ized, and samples corresponding to the least severe and most severeinflammation were assigned scores of 0 and 5, respectively. All sampleswere then graded for peribronchiolar inflammation (around three to fivesmall airways per section) and alveolitis in multiple random fields persection by three independent readers as described (34). Lung sections wereviewed on a Leica DMI3000 B microscope (Leica Microsystems) andphotographed at3100 original magnification unless otherwise stated usinga Leica DFC 490 camera running from the Leica Application software(Leica Microsystems).

Cytokine bead array for the detection of inflammatorymediators

The levels of IFN-g, TNF-a, IL-6, IL-10, IL-12.p70, and MCP-1 in BALsupernatants and serum were determined with the use of a BD CytometricBead Array mouse inflammation kit (BD Biosciences) according to man-ufacturer’s instructions. The detection limit was 5 pg/ml for all cytokinestested.

Assessment of lung edema and vascular leak

The lung wet-to-dry weight ratio was used as an index of lung fluid ac-cumulation during influenza virus infection. Lungs were surgically dis-sected, blotted dry, and weighed immediately (wet weight). Note that lungsused in these studies had not been subject to BAL prior to analysis. The lungtissue was then dried in an oven at 60˚C for 72 h and reweighed (dryweight). The ratio of weight-to-dry was calculated for each animal to as-sess tissue edema as previously described (35, 36). The concentration ofprotein in cell-free BAL supernatant was measured by adding Bradfordprotein dye. A standard curve using BSA was constructed and the ODdetermined at 595 nm.

Statistical analysis

For the comparison of two sets of values, a Student t test (two-tailed, two-sample equal variance) was used. When comparing three or more sets ofvalues, data were analyzed by one-way ANOVA (nonparametric) followedby post hoc analysis using Tukey’s multiple comparison test. For theanalysis of histopathological data, a Kruskal–Wallis test (nonparametric)was used followed by the Dunn’s posttest. Survival proportions werecompared using the Mantel–Cox log-rank test. A p value #0.05 wasconsidered statistically significant.

ResultsThe HA of influenza viruses plays a critical role in determiningsensitivity to innate host defenses in BAL fluid and virulence inmice

Brazil HA carries four potential sites of N-linked glycosylation onthe head, whereas PR8 lacks glycosylation on the head of HA(Fig. 1A). Both viruses express similar numbers of potential N-glycosylation sites on the HA stalk. To determine the role of HAin virulence of H1 IAV in mice, RG was used to insert the HA ofBrazil into the seven-gene backbone of PR8, thereby generatingthe 7:1 virus RG-Brazil-HA.

We first compared sensitivity of Brazil and PR8, as well as RG-Brazil-HA, to neutralization by mouse BAL fluid (Fig. 1B). Viruseswere incubated with dilutions of mouse BAL (in the presence orabsence of 10 mg/ml mannan) and the amount of infectious virusremaining determined by immunofluorescence (18, 22). The po-tent neutralizing activity of mouse BAL against Brazil was re-versed in the presence of mannan (Fig. 1B), indicating that SP-D(or a related mannose-specific lectin) was the major neutralizingactivity in lung fluids (22). PR8 was largely resistant to mouseBAL, whereas RG-Brazil-HA was neutralized to equivalent levelsto Brazil. Therefore, expression of Brazil HA was associated withenhanced sensitivity to neutralization by mouse BAL.We next examined the role of HA in determining the ability of

IAV to infect murine Mf (Fig. 1Ci). Consistent with previousreports (18, 22, 25, 26), PR8 was poor in its ability to infect Mf.In contrast, Brazil and RG-Brazil HA infected ∼60% of Mf andinfection was inhibited by mannan, a ligand of MMR. PR8, Brazil,or RG-Brazil HA did not differ in their ability to infect the murinelung epithelial (LA-4) cell line and infection of LA-4 cells wasunaffected by mannan (Fig. 1Cii).Next, mice were infected with 105 PFU of either Brazil, PR8, or

RG-Brazil-HA and monitored daily for weight loss and disease.Mice infected with Brazil or RG-Brazil-HA did not lose weight(Fig. 1D) or show visible signs of disease (data not shown) overthe 10-d monitoring period, whereas PR8-infected mice rapidlylost weight, and all animals succumbed to disease. Thus, expres-sion of Brazil HA led to increased sensitivity to neutralization bymouse BAL and enhanced susceptibility of Mf to infection. Inmice, expression of Brazil HA was associated with a marked at-tenuation in virulence.

Generation of RG influenza viruses with differing numbers ofN-glycosylation sites on HA

Expression of different H1 HA was critical in determining sensi-tivity to lectin-mediated innate defenses and virulence in mice (Fig.1). Next, a strategy was devised to sequentially remove (Brazil) oradd (PR8) N-glycosylation sites to the globular head of HA, andRG IAV were generated on a PR8 backbone. RG viruses ex-pressing WT Brazil or PR8 HA were generated as controls. ForBrazil HA, we generated single-step mutants (2104,2144,2172,and2177), double-step mutants (2104/144 and2172/177), and amutant lacking all four glycosylation sites (2104/144/172/177).For PR8 HA, we generated single-step (+104, +144, +172 and+177) mutants and a double-step mutant (+104/144). Despitemultiple attempts, infectious virus could not be rescued for the+104/144/172/177 mutant, suggesting that addition of glycosyla-tion interferes with function and/or stability of PR8 HA. In ad-dition, particular combinations of glycosylation sites could not beadded to PR8 HA, and rescued virus reverted to HA consensus(e.g., double-step +172/177).

Glycosylation on the head of HA modulates virulence of IAV inmice

To examine the effect of the removal (Brazil) or addition (PR8) ofN-glycosylation sites to the head of the HA, mice were infectedwith 105 PFU (Fig. 2A) or 103 PFU (Fig. 2B) RG virusesexpressing WT HA or expressing a mutant HA with altered gly-cosylation. Infection of mice with 105 PFU RG-Brazil-HA (WT)was not associated with weight loss (Fig. 2Ai), and similar resultswere obtained using single-step glycosylation mutants lackingAsn104 (2104) or Asn172 (2172). Glycosylation mutants lackingAsn144 alone (2144) or in conjunction with Asn104 (2104/144)rapidly lost weight and were euthanized 4 d postinfection. Single-step mutants lacking Asn177 (2177) induced mild, but significant,

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weight loss, and all mice recovered from infection, whereasmutants lacking both Asn172 and Asn177 (2172/177) were viru-lent, and all animals were euthanized. Mice infected with theglycosylation mutant lacking all four sites from the head of HA(2104/144/172/177) succumbed to disease 4 d postinfection.Mice infected with 105 PFU RG viruses expressing RG-PR8-HA

(WT) rapidly lost weight and succumbed to disease (Fig. 2Aii).Addition of single sites of N-glycosylation at Asn104, Asn144,Asn172, or Asn177 (+104, +144, +172, or +177 mutants, respectively)or addition of Asn104 and Asn144 (+104/144) did not reduce viru-lence, and all mice were euthanized 4 to 5 d postinfection.We next inoculated mice with a 100-fold lower dose (i.e., 103

PFU) of RG viruses to highlight more subtle differences betweenviruses in ability to induce disease. The 2144 Brazil HA mutantinduced disease and death in mice following inoculation with 105

PFU (Fig. 2Ai) but did not induce disease at the lower dose (Fig.2Bi), whereas 2104/144 and 2104/144/172/177 retained viru-lence. Addition of single sites to PR8 HA (i.e., +104, +144, +172or +177 mutants) did not alter weight loss or mortality, whereasthe +104/144 virus was attenuated, and all animals survived in-fection (Fig. 2Bii). Thus, deletion of glycosylation from Brazil HAwas associated with enhanced virulence, whereas addition ofglycosylation to PR8 HA attenuated disease.

Receptor specificity of HA glycosylation mutant viruses

We compared RG viruses in a range of assays to investigate factorsthat influence virulence in mice. Addition or loss of glycosylationsites can alter binding of HA to a(2,3)-Gal– or a(2,6)-Gal–linkedsialic acid (SA) (12, 37, 38). We first compared RG viruses forability to agglutinate erythrocytes from different species, and RGviruses bearing Brazil HA did not differ in ability to agglutinatehuman, chicken, turkey, guinea pig, or horse erythrocytes (data notshown). Furthermore, when human erythrocytes were desialylatedand enzymatically resialylated, RG viruses expressing Brazil HAdid not differ in ability to agglutinate erythrocytes bearing a(2,3)-Gal– or a(2,6)-Gal–linked Neu5Ac (Table I). Addition of glyco-sylation to PR8 HA did not alter binding to human, chicken,turkey, or guinea pig erythrocytes (data not shown). However,compared with WT, the +144 and +104/144 mutants were lessefficient at agglutinating erythrocytes bearing a(2,3)-Gal–linkedNeu5Ac (Table I), indicating differences in the receptor-bindingproperties of PR8 HA.

Ability of viruses differing in HA glycosylation to infect murineMf and epithelial cells

IAV infection of airway epithelial cells results in productive virusreplication (26, 39, 40), whereas influenza virus infection of Mf

is abortive, and infectious virions are not released (18, 26, 41).Therefore, the ability of glycosylation mutants to infect differentcell types might be an important factor influencing virulence.Monolayers of murine Mf or LA-4 epithelial cells were infectedwith increasing doses of RG viruses, and immunofluorescence wasused to determine the percentage of infected cells. Compared tovirus bearing appropriate WT HA, glycosylation mutants bearingthe HA of Brazil (Fig. 3A, upper panel) or PR8 (Fig. 3A, lowerpanel) did not differ in ability to infect epithelial cells. In themouse model, Mf play a critical role in limiting disease severity(26, 42), and the MMR has been implicated as a receptor for IAV

FIGURE 1. The HA of Brazil modulates sensitivity to innate immune

defenses and virulence in mice. A, Schematic of HA monomers of PR8 and

Brazil, which carry zero and four potential sites of N-linked glycosylation,

respectively, on the head of HA. Red and blue indicate the HA1 and HA2

domains, respectively, and green circles indicate the location of glyco-

sylation motif Asn-X-Ser/Thr. Images are derived from the crystal struc-

ture of the HA of A/Aichi/2/68. The amino acid numbering of specific N-

glycosylation sites on the head of HA is indicated (H1 numbering). Images

were generated using the MacPyMol software and the solved HA structure

(ID 3EYM; National Center for Biotechnology Information database). B,

Dilutions of mouse BAL in TBS containing 10 mM CaCl2 were incubated

with Brazil, PR8, or RG virus bearing the HA of Brazil (RG-Brazil-HA)

for 30 min at 37˚C and the amount of infectious virus remaining de-

termined by fluorescent focus assay and are expressed as a percentage of

a virus only control. Inhibition of neutralization was examined by addition

of mannan (mn; final concentration of 10 mg/ml) 30 min prior to virus

addition. Data are representative of two independent experiments. C,

Monolayers of murine Mf (i) or LA-4 cells (ii) were incubated with 106

PFU Brazil, PR8, or RG-Brazil-HA (black bars). After 1 h, monolayers

were washed, incubated 7 h, fixed, and stained for expression of viral NP.

Inhibition of infection was examined by addition of mannan (mn; final

concentration of 10 mg/ml) to cell monolayers 30 min prior to virus ad-

dition (gray bars). Data represent the mean percent infection (6 1 SD)

from three independent experiments. D, Groups of five mice were infected

with 105 PFU Brazil, PR8, or RG-Brazil-HA. Mice were monitored daily

for weight loss, and animals that lost $25% of their original body weight

were euthanized. Data represent the mean percent weight change 6 1 SD.

Data are representative of two independent experiments.

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infection of murine Mf (18, 19). Despite this, we did not observemajor differences between viruses bearing WT or glycosylationmutant HA in their ability to infect Mf (Fig. 3B). It should benoted that the +104/144 PR8 HA mutant was, however, associatedwith a modest increase in ability to infect Mf compared with itscorresponding WT virus. RG virus bearing the WT Brazil HA andthe Brazil HA 2104/144/172/177 mutant were equally efficient intheir ability to infect murine Mf, and infection of Mf by eithervirus was reduced to ,15% in the presence of 10 mg/ml mannan(data not shown).

Sensitivity of glycosylation mutants to neutralization by mouseBAL or rat SP-D

Pulmonary surfactant contains proteins of the innate immunesystem and acts as a barrier to influenza virus infection of theairways. SP-D, a mannose-specific lectin of the collectin family,represents the major neutralizing activity in mouse BAL againstglycosylated influenza viruses (Fig. 1) (22). Therefore, we de-termined if addition or removal of glycosylation sites on HA im-pacted on sensitivity to mouse BAL or to recombinant rat SP-D.RG virus bearing WT Brazil HAwas sensitive to neutralization bymouse BAL (Fig. 4A, left panel) or rat SP-D (Fig. 4B, left panel).Removal of Asn104 or Asn172 did not alter sensitivity to neutral-ization by BAL or SP-D and viruses did not induce disease (Fig.2A). Glycosylation at Asn144 and Asn177 was, however, important

in determining sensitivity to both mouse BAL and SP-D, andsingle-step mutants lacking each of these sites showed enhancedvirulence in mice (Fig. 2A, left panel). RG 2104/144 and 2104/144/172/172 Brazil HA mutants were largely resistant to neu-tralization by either BAL or SP-D. Note that the neutralizing ac-tivity of mouse BAL (Fig. 4A, right panel) and rat SP-D (Fig. 4B,right panel) for RG virus bearing WT Brazil HA was reversed inthe presence of mannan, whereas an equivalent amount of BAL/SP-D did not neutralize 2104/144/172/172 Brazil HA. The neu-tralizing activity of BAL/SP-D for 2104, 2144, 2172, or 2177Brazil HA mutants was also reversed in the presence of mannan(data not shown).Addition of a single glycosylation site at Asn144 of PR8 HA

(+144) increased sensitivity to neutralization by mouse BAL (Fig.4C, left panel) and SP-D (Fig. 4D, right panel), but this virus didnot differ from WT in virulence (Fig. 2). Sequential addition ofglycosylation sites (+104/144) led to a further increase in sensi-tivity to both mouse BAL and SP-D, as well as attenuation ofvirulence. Neutralization of the +104/144 mutant by mouse BAL(Fig. 4C, right panel) and SP-D (Fig. 4D, right panel) was re-versed in the presence of mannan. Overall, these results demon-strate an inverse correlation between sensitivity to mouse BALand disease severity in mice. For Brazil and PR8, glycosylation atAsn144 and, to a lesser extent Asn177, are important determinantsof sensitivity to mouse BAL and disease severity.

Table I. Receptor specificity of RG viruses

Hemagglutination Titera

HA Expressed by RG Virus HA Glycosylation Mutant Native Asialo a(2,3)-SA a(2,6)-SA

Brazil WT 64 ,1 8 162104 64 ,1 8 162144 64 ,1 16 32

2104/144 64 ,1 8 162104/144/172/177 64 ,1 8 16

PR8 WT 64 ,1 16 ,1+104 64 ,1 16 ,1+144 64 ,1 2 ,1

+104/44 64 ,1 4 ,1

HA assays were performed using standard procedures, and data are expressed as HA units.aViruses bearing WT or glycosylation mutant HA were compared for their ability to agglutinate desialylated (Asialo) and

enzymatically resialylated erythorocytes. Human erythrocytes were desialylated with bacterial sialidase from C. perfringens for1 h at 37˚C and enzymatically resialylated with Neu5Ac in the presence of either a(2,3)-ST or a(2,6)-ST. Native humanerythrocytes were included for comparison.

FIGURE 2. HA glycosylation modulates virulence of

H1 subtype IAV in mice. Groups of five mice were

infected via the intranasal route with 105 PFU (A) or

103 PFU (B) of RG viruses bearing the WT HA of

Brazil or PR8, or with glycosylation mutants of Brazil

(i) or PR8 (ii) HA. Mice were monitored daily for

weight loss. Mice that lost $25% of their original body

weight were euthanized. Data represent the mean per-

cent weight change 6 1 SD and are representative of

two independent experiments.

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Replication of RG glycosylation mutant viruses in vitro andin vivo

Next, we investigated parameters associated with disease severityfollowing infection of micewith RG virus expressing: 1)WTBrazilHA (“RG-Brazil WT”) or RG-Brazil HA -104/144/172/177 (“RG-Brazil 24”); or 2) WT PR8 HA (“RG-PR8 WT”) or RG-PR8 HA+104/144 (“RG-PR8 +2”). Analyses were performed at day 6postinfection, the time at which mice infected with RG-Brazil 24and RG-PR8 WT viruses succumbed to disease.First, mice were infected with 103 PFU each virus, and titers of

infectious virus in the upper (nasal tissues) and lower (lung) re-spiratory tract were determined (Fig. 5A). Infectious virus was notdetected in lungs (Fig. 5Ai) and was present at low titers in nasaltissues (Fig. 5Aii) of mice inoculated with RG-Brazil WT, whereashigh titers were detected in the airways of RG-Brazil 24-infectedmice (∼2600-fold and ∼20-fold higher titers in lungs and nasaltissues, respectively). Viral titers in lungs and nasal tissues of miceinfected with RG-PR8 +2 were lower than from mice infectedwith RG-PR8 WT (∼30-fold decrease in both lungs and nasaltissues).Given the marked differences in replication of RG viruses

in vivo, we compared their ability to replicate in mouse LA-4 cellsto determine if there were intrinsic differences between virusesin their ability to replicate in airway epithelial cells. In theseexperiments, LA-4 cells were infected with 106 PFU RG-BrazilWT, RG-Brazil 24, RG-PR8 WT, or RG-PR8 +2 viruses, andsupernatants removed at 2 and 24 h postinfection were assayed forinfectious virus by TCID50. Titers of virus were similar at 2 and24 h for RG-Brazil WT versus RG-Brazil 24 and for RG-PR8WT versus RG-PR8 +2 (data not shown). These data indicatethat differences in the ability of virus to replicate in the airwaysof mice are likely due to differences in susceptibility to host de-fenses rather than defects in their ability to infect and replicate intarget cells.

Pulmonary histopathology following infection of mice with RGglycosylation mutants

We examined lung sections from mice infected with 103 PFU RG-Brazil WT, RG-Brazil 24, RG-PR8 WT, or RG-PR8 +2 viruses atday 6 postinfection. Lung sections from naive animals were in-cluded for comparison. Lung sections were blinded and scored forperibronchiolar inflammation and alveolitis by three independentreaders (Fig. 5B). Both peribronchiolar inflammation and alveo-litis were more severe in the lungs of mice infected with RG-

Brazil 24 compared with animals infected with RG-Brazil WT,and lung scores from RG-Brazil24-infected mice were equivalentto those from mice infected with RG-PR8 WT (Fig. 5C). Pul-monary pathology was not significantly different between RG-PR8 WT and RG-PR8 +2 in two independent experiments.

Cellularity and production of inflammatory mediators in theairways of mice infected with RG-Brazil 24 and RG-PR8 +2

The total number of cells in BAL was determined 6 d postinfectionwith 103 PFU RG-Brazil WT, RG-Brazil24, RG-PR8 WT, or RG-PR8 +2 viruses. Low cell numbers were recovered from the air-ways of mice infected with RG-Brazil WT (Fig. 6A). Infection ofmice with an equivalent dose of RG-Brazil -4 led to recruitmentof large numbers of leukocytes, and cell numbers were ∼3.5-foldhigher than from mice infected with RG-Brazil WT. Infection withRG-PR8 WT or with RG-Brazil 24 resulted in similar BAL cellnumbers. BAL cell numbers from mice infected with RG-PR8 +2were reduced ∼2-fold compared with mice infected with RG-PR8WT.To examine the cellular infiltrate in detail, BAL cells were

analyzed by flow cytometry at day 6 postinfection and numbersof neutrophils, NK cells, T cells, CD8+ T cells, and B cells de-termined (Fig. 6B). Numbers of airway Mf were determined bycytospin and differential staining of BAL cells. Neutrophils werethe predominant cell type following infection with RG-Brazil 24,RG-PR8 WT, or RG-PR8 +2, whereas few neutrophils were re-covered from mice infected with RG-Brazil WT. Infection withRG-Brazil 24 was associated with increased neutrophils, T cells,B cells, and airway Mf compared with RG-Brazil WT. In con-trast, RG-PR8 +2 recruited fewer neutrophils, T cells, or B cellscompared with RG-PR8 WT.Compared to mice infected with RG-Brazil WT, pulmonary in-

flammation in RG-Brazil 24-infected animals was associated withenhanced BAL IL-6 (Fig. 6C; p , 0.05, one-way ANOVA), but notMCP-1, TNF-a, or IFN-g. Levels of IFN-g showed a tendency tobe enhanced from RG-Brazil 24-infected mice; however, this wasnot significant two independent experiments. Infection of mice withRG-PR8 WT resulted in BAL levels of TNF-a, IFN-g, MCP-1, andIL-6 that were significantly higher than those from mice infectedwith RG-PR8 +2 (p , 0.05, one-way ANOVA).

Vascular leak, pulmonary edema, and systemic responsesfollowing infection of mice with RG viruses

Severe influenza infections are associated with vascular leak andpulmonary edema (26, 33) as well as systemic manifestations,

FIGURE 3. Influence of HA glycosylation on

ability of RG IAV to infect murine cells. Mono-

layers of LA-4 epithelial cells (A) or mouse Mf

(B) were incubated with increasing doses of RG

viruses bearing WT HA of Brazil (upper panels) or

PR8 (lower panels) or with HA glycosylation mu-

tant viruses. After 1 h, monolayers were washed,

incubated a further 7 h, and then fixed and stained

for expression of viral NP. Data represent the mean

percent infection (6 1 SD) from three independent

experiments. *Significant increase compared with

virus expressing corresponding WT HA (p , 0.05,

one-way ANOVA).

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including leukopenia and thymic atrophy (43–46). Compared to

mice infected with RG-Brazil WT, high levels of total BAL pro-

tein were recovered from RG-Brazil 24-infected mice (Fig. 7A)

and enhanced wet/dry lung ratios observed (Fig. 7B). RG-PR8 +2-

infected mice showed reduced vascular leak and lung edema

compared with mice infected with RG-PR8 WT. We also recorded

reductions in total blood leukocyte numbers from mice infected

with RG-Brazil 24 or RG-PR8 WT compared with naive animals

(Fig. 7C) and this was associated with reduced B cells, CD8+

T cells, and CD4+ T cells. Infection with RG-PR8 +2 led to in-

creased neutrophil and B cell numbers, whereas CD8+ T cells and

CD4+ T cells were unaffected. No differences were noted in cell

numbers between mice infected with RG-Brazil WT and naive

animals. Finally, the cellularity of thymi from mice infected with

RG-Brazil 24 or RG-PR8 WT viruses were reduced compared

with mice infected with RG-Brazil WT and RG-PR8 +2 viruses,

respectively (data not shown). In each case, flow cytometric

analysis indicated a specific reduction in double-positive thymo-

cytes and more single-positive (SP) CD4+CD82 thymocytes (Fig.

7D), whereas numbers of CD42CD8+ SP and CD42CD82

double-negative thymocytes did not differ.

DiscussionAnalysis of H1 sequences (1918–2010) indicate that glycosylationon the receptor binding domain of HA is absent from 1918 and2009 pandemic virus strains but is present on nearly all seasonalIAV (47), consistent with a role for glycosylation in mediatingevasion of Ab-mediated neutralization in the human population.Accumulation of glycans in the vicinity of antigenic sites on HAmay provide an evolutionary advantage in an immune populationby shielding antigenic sites (7, 48, 49); however, these beneficialeffects are balanced by the enhanced recognition of glycosylatedIAV by collectins of the innate immune system (17, 20, 50). Gly-cosylated IAV are highly sensitive to neutralization by collectinsand show marked attenuation in mice (17, 20, 51). In this study, wehave used a mouse model of IAV infection to systematically definethe role that particular glycosylation sites on seasonal H1 IAV playin modulating sensitivity to innate immune defenses and virulence.Hemagglutination inhibition studies using different H1N1 IAV

led Hartshorn et al. (50) to propose that Asn144 and Asn172 wereimportant determinants of sensitivity to human SP-D. Using RGviruses, we confirm that Asn144 is an important determinant ofsensitivity to mouse BAL, a rich source of SP-D (22), as well asvirulence in mice. Deletion of Asn144 from Brazil HA reduced

FIGURE 4. Neutralization of RG glycosylation

mutants by mouse BAL and rat SP-D. Dilutions of

mouse BAL supernatant (A, C, left panels) or

recombinant rat SP-D (B, D, left panels) in TBS

supplemented with 10 mM CaCl2 were mixed with

RG viruses bearing WT HA of Brazil (A, B) or PR8

(C, D) or with HA glycosylation mutant viruses and

incubated for 30 min at 37˚C before addition to

MDCK cell monolayers. Inhibition of neutralization

(A–D, right panels) was examined by addition of

mannan (mn; final concentration of 10 mg/ml) to

either BAL (final dilution 1/4) or SP-D (final con-

centration 150 ng/ml for Brazil HA, 2000 ng/ml for

PR8 HA) 30 min prior to virus addition. The amount

of infectious virus remaining was determined by

fluorescent focus assay at 8 h postinfection. Data are

expressed as a percentage compared to a virus only

control and are representative of two independent

experiments. Note that 24 refers to the Brazil HA

glycosylation mutant lacking all four glycosylation

sites (2104/144/172/177).

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sensitivity to mouse BAL and increased virulence in mice.Moreover, simultaneous addition or deletion of Asn104 and Asn144from PR8 or Brazil HA, respectively, led to marked changes insensitivity to mouse BAL and virulence when compared with lossof either site alone. These findings suggest that simultaneousbinding of SP-D to multiple glycans on IAV HA is likely to in-crease the overall affinity of binding and therefore antiviral ac-tivity. Single-site deletion of Asn177, but not Asn172, reduced sen-sitivity to mouse BAL/SP-D and increased virulence in mice, afinding that may reflect differences in ligand specificity betweenhuman and mouse SP-D (52). Of interest, different collectins alsodisplay a distinct specificity for oligosaccharides expressed onIAV. A mutant of Brazil (H1N1) selected for resistance to con-glutinin, a bovine collectin, lacked Asn104 from the head of HA(27) but remained somewhat sensitive to SP-D (28). In our studies,RG-Brazil 2104 was resistant to conglutinin (data not shown);however, its sensitivity to mouse BAL and purified SP-D wassimilar to that of virus expressing WT HA. Thus, Asn104 repre-

sents a critical ligand for conglutinin on H1 IAV, but does notrepresent the major glycan species recognized by rodent SP-D.A number of studies have reported an association between loss of

potential glycosylation sites fromHA and adaptation of human IAVto growth in mice. Smee et al. (53) reported elimination of gly-cosylation sites Asn71 and Asn104 following adaptation of A/NewCaledonia/20/99 (H1N1) to growth in mouse lung. Furthermore,sequential passage of A/USSR/90/77 (H1N1) in mouse lung ledto loss of Asn104 and Asn144, such that only Asn172 and Asn177remained on the head of HA, and the resultant virus replicatedto high titers in mouse lung (54, 55). However, Shilov et al. (54)reported that loss of Asn144 alone was associated with resistanceto inhibitors in mouse serum, and this mutant was as pathogenicfor mice as the mutant lacking both Asn104 and Asn144. Duringadaptation of human IAV to growth in mice, studies have shownthat mutations in additional genes, including PB2, PB1-F2, PA, M,and NS1 (56–60), can enhance virus replication and contribute tovirulence. Moreover, mutations in HA that affect receptor binding

FIGURE 5. Increased glycosylation of H1 correlates with reduced virus growth and inflammation in the respiratory tract of mice. Mice were infected

with 103 PFU RG viruses expressing WT HA of Brazil (RG-Brazil WT) (i) or Brazil HA lacking glycosylation sites Asn104, Asn144, Asn172, and Asn177(RG-Brazil -4) (ii) or the WT HA of PR8 (RG-PR8 WT) (iii) or the PR8 HAwith glycosylation sites added at Asn104 and Asn144 (RG-PR8 +2) (iv). Mice

were killed and analyzed at day 6 postinfection. A, Titers of infectious virus in lung and nasal tissue homogenates. Bars represent the mean viral titer from

a group of five mice6 1 SD. The detection limit of the assay (0.9) is indicated by the dotted line. *Virus titers different to those from mice infected with RG

virus bearing corresponding WT HA (p , 0.01, one-way ANOVA). B, Representative images of inflammation in lung sections following H&E staining.

Images are shown at original magnification 3100 (left panels) and 3400 (right panels). C, Histopathological scores for lung sections from naive (N)

animals and from mice infected with RG viruses. Lung sections were randomized and scored blinded for alveolitis (i) and peribronchiolar inflammation (ii)

on a scale of 0–5. Data shown represent scores from individual mice (as indicated by circles) and median values (as indicated by bar) obtained from one out

of three readers. Samples were compared for statistical significance using the Kruskal–Wallis test. For each reader, significant differences were observed in

immunopathology scores from mice infected with RG-Brazil WT compared with those infected with RG-Brazil 24 mutant (p , 0.01, alveolitis and

peribronchiolar inflammation). Data are pooled from two independent experiments for analysis (n = 5 per experiment).

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or cleavage have also been implicated in adaptation of human IAVto mice (53, 61, 62). A strength of our RG studies lies in the use ofgenetically defined viruses to determine whether specific changesin HA impact on virulence, removing complications associatedwith introduction of mutations in additional genes during adap-tation to growth in mice. Using this approach, we have defined theimportance of particular glycosylation sites on H1 HA in modu-lating sensitivity to mouse BAL (as well as purified SP-D) andvirulence.

IAV strain PR8 is a mouse-adapted strain that induces an in-terstitial pneumonia similar to that seen in human cases of viralpneumonia (63, 64). PR8 was adapted to mice by .300 sequentialpassages in mouse lung (65) and is likely to have acquired muta-tions associated with increased virus replication in murine tissuesand/or evasion of innate host defenses. Using RG and eight plas-mids expressing each of the genes of PR8, we demonstrate thataddition of two potential glycosylation sites (Asn104/Asn144) to PR8HA was sufficient to mitigate other mutations introduced into thePR8 genome as a result of adaptation to mouse lung. RG-PR8 +2was sufficiently attenuated such that it was no longer lethal in micedespite replicating to similar levels as RG-PR8 WT in vitro.RG-Brazil 24 and RG-PR8 WT both induced severe disease

and death in mice, although viral titers in the lungs of RG-Brazil24-infected mice were ∼100-fold lower than in mice infected

FIGURE 7. Vascular leak, pulmonary edema, lymphopenia, and thymic

atrophy are associated with severe disease. Groups of five mice infected

with 103 PFU RG-Brazil WT, RG-Brazil 24, RG-PR8 WT, or RG-PR8 +2

were killed and analyzed 6 d postinfection. Naive animals (N) were in-

cluded for comparison. Total protein levels in cell-free BAL (A) and lung

wet-to-dry ratios (B) were determined. Bars represent the mean6 1 SD. C,

Cell types in blood were determined by flow cytometry. Numbers of total

leukocytes (CD45+), neutrophils (Gr-1high), T cells (TCRb+), CD4+ T cells

(CD4+ TCRb+), CD8+ T cells (CD8+ TCRb+), and B220+ cells were de-

termined. Viable counts of blood were performed and cell numbers

expressed per milliliter of blood. Bars represent the mean6 1 SD. D, Flow

cytometric analysis of thymi cell suspensions to determine double-negative

(CD42CD82), double-positive (CD4+CD8+), and SP (CD4+ or CD8+)

thymocytes. Bars represent the average 6 1 SD. Data are representative of

two independent experiments. *Cell numbers significantly different to

naive controls (p , 0.05, one-way ANOVA).

FIGURE 6. Increased cellularity and production of inflammatory me-

diators in the airways of mice infected with RG viruses. Groups of five

mice were infected with 103 PFU RG-Brazil WT, RG-Brazil 24, RG-PR8

WT, or RG-PR8 +2. At day 6 postinfection, mice were killed and analyzed.

Naive animals (N) were included for comparison. Total BAL cells (A) and

BAL leukocyte (B) subsets were determined by flow cytometry and

numbers of neutrophils (Neut; Gr-1high), NK cells (NK1.1+TCRb2),

BB220+ cells, and CD8+ cells (CD8+) are shown. Airway macrophages

were identified following cytospin and differential staining of BAL cells.

Bars represent the mean cell number 6 1 SD. C, Levels of IL-6, MCP-1,

IFN-g, and TNF-a in cell-free BAL were determined by cytokine bead

array. virulence in Bars represent the mean concentration (pg/ml) and in-

dividual mice are shown as circles. Pooled data from two independent

experiments are shown and were used for statistical analysis (n = 5 mice

per experiment). The detection limit of each mediator (5 pg/ml) is in-

dicated as a dotted line. A–C, *Significantly different compared with

samples from mice infected with RG virus bearing the corresponding WT

HA (p , 0.05, one-way ANOVA).

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with RG-PR8 WT. Although resistance to neutralizing proteins inBAL is likely to be a major factor contributing to virulence ofH1 IAV, other intrinsic features of HA contribute to enhanced PR8replication in the airways. Receptor specificity differences be-tween Brazil [specificity for a(2,3)-Gal and a(2,6)-Gal-linked SA]and PR8 [specificity for a(2,3)-Gal-linked SA] are likely to beimportant, as a(2,3)-Gal-SA is the predominant linkage expressedin mouse lung (66, 67). Moreover, studies have demonstrated thatadaptation of human influenza viruses to growth in mouse lung isassociated with a switch in receptor specificity to favor binding tomouse respiratory cells (61, 62).Results presented in this study implicate potential glycosyla-

tion sites at Asn144 and Asn177 on H1 as important determinants inmodulating sensitivity to SP-D and virulence in mice. Thesefindings suggest that Asn144 and Asn177 carry glycans readilybound by SP-D (e.g., high mannose-type glycans), whereas othersites on the head of HA (Asn104 and Asn172) carry glycans that arenot readily accessible and/or bear terminal glycans bound lessefficiently by SP-D (such as complex glycans terminating in ga-lactose). We could not definitively confirm a change in molecularmass of HA for any of the single-step mutants relative to WTHA using Western blot, and both HA (nonreducing) and HA1

(reducing) appeared as diffuse bands, likely due to microhetero-geniety of glycans on the HA of egg-grown virus (data not shown).Although our data suggest that the mutations introduced alterglycosylation, it is possible that changing the underlying sequenceof the protein alone (i.e., in the absence of a glycan attachment)might influence sensitivity to mouse BAL and virulence. However,neutralization by mouse BAL and purified rodent SP-D were re-versed in the presence of mannan, demonstrating that mannose-specific lectins are the major neutralizing activity in mouse BAL.Biochemical analyses of the HA of A/USSR/90/77, an H1N1 viruswith an identical pattern of HA glycosylation to that of Brazil(68), demonstrated a mixture of complex, high-mannose, and hy-brid-type glycans (69), although the nature of glycans expressedat each glycosylation site was not determined. Further experimentsare required to confirm the presence of glycan as well as the natureof specific oligosaccharides expressed at Asn104, Asn144, Asn172,and Asn177 of the Brazil and PR8 HA.It is well established that glycosylation of HA can inhibit rec-

ognition by Abs (48, 70). In recent studies, addition of Asn142 andAsn177 to pandemic H1N1 HA trimers was associated with re-duced sensitivity to neutralizing Abs raised to WT pandemicHA (47), suggesting that glycan shielding may be an importantmechanism by which pandemic viruses entering the human pop-ulation evolve into seasonal influenza virus strains. However,analysis of pandemic H1N1 HA sequences submitted to the Na-tional Centre for Biotechnology Information indicate the addi-tion of potential glycosylation sites at Asn179 (e.g., A/Quito/WR1589N/2009 and A/Tallinn/INS374/2010, accession numbersADM14775 and ADM31858, respectively) and Asn136 (e.g., A/Netherlands/1493b/2009 and A/Athens/INS257/2009, accessionnumbers ADJ40554 and ADK33831, respectively) in some H1N1pandemic strains, and these are distinct to sites recorded on theglobular head of seasonal H1N1 IAV. It will be of major interestto define the role of novel glycans added to the pandemic H1N1HA in regard to masking antigenic epitopes on HA as well asmodulating sensitivity to collectins of the innate immune system.Influenza virus infection of murine Mf involves binding of

HA to cell-surface SA as well as interactions between MMR andglycans expressed on HA/NA glycoproteins (18, 19). We havepreviously demonstrated that receptor specificity of HA is im-portant in determining ability to infect murine Mf as these cellsexpress high levels of a(2,6)-Gal–linked SA (71). Moreover, re-

sialylated Mf expressing a(2,3)-Gal–linked SA were permissiveto MMR-mediated infection by PR8, a virus lacking glycans onthe head of HA but with a receptor preference for a(2,3)-Gal–linked SA. In this study, RG-Brazil WT and RG-Brazil 24-infected native Mf to similar levels (Fig. 3B), and infection wasinhibited in the presence of mannan (data not shown). Bothviruses show a preference for a(2,6)-Gal–linked SA (Table I), thepredominant SA linkage expressed by Mf (71), further high-lighting the importance of the match between HA specificity andthe particular SA linkages expressed at the Mf surface. The en-hanced virulence of the RG-Brazil 24 was not associated withreduced ability to infect Mf, but rather with reduced sensitivityto soluble mannose-specific lectins present in murine lung fluids.Together, our data highlight the importance of specific sites ofN-linked glycosylation on the H1 HA in determining sensitivityto SP-D and therefore virulence in mice.

AcknowledgmentsWe thank Dr. Robert Webster, St. Jude Children’s Research Hospital,

Memphis, TN, for provision of the plasmid vector used to create the

reverse-engineered viruses for this study.

DisclosuresThe authors have no financial conflicts of interest.

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