Influenza viruses express two envelope proteins that are involved in virulence, neuraminidase (NA) and hemagglutinin (HA). During the virus life cycle these proteins have distinct functions in entry and release of the virus. HA plays a primary role in the binding and uptake of the virus into target cells and is the main target of neutralizing antibodies. Structurally, the HA is a 200-kDa homotrimer with an ectodomain composed of a globular head and a stalk region (
49). Both of these regions undergo posttranslational modifications in the Golgi, where glycoconjugates are added to certain sites of N-linked glycosylation. Some of these glycosylation sites, primarily in the stalk region, are indispensable to the proper folding and conformation of the HA molecule (
9,
33). During the last 4 decades of circulation in humans, N-linked glycosylation in and around the globular head has gradually increased in H3N2 subtype viruses (
36,
48). Carbohydrate attached to glycosylation sites has been previously characterized in tissue culture to be cell type specific and site specific and composed primarily of complex-type and oligomannose oligosaccharide (
27,
35). The role of these glycoconjugates in the life cycle of the virus and the evolutionary reasons behind the increasing glycosylation seen since H3N2 viruses began circulating in humans are poorly understood at present.
The presence of carbohydrate on the HA can have either positive or detrimental effects on the virus. For example, glycoconjugates that are positioned close to the cleavage site can interfere with proteolytic activation of the nascent HA0 (
22). Alternatively, replication and release of virus may be facilitated by carbohydrate that is located near the receptor binding site through a mechanism of reduced receptor affinity (
46,
47). Carbohydrate around the globular head can also potentially shield antigenic sites from immune recognition. This may contribute to antigenic drift of influenza viruses where successive glycosylation events prevent accessibility and recognition by antibodies in an immune population (
1). Additional work from Klenk at al. illustrates that carbohydrate is especially important for the interaction of HA and NA, where a balance is needed between receptor binding activity and virus release (
23). A virus containing HA with little carbohydrate modification can tightly bind the receptor, requiring greater NA activity to promote particle release. Conversely, an HA with more extensive glycosylation interacts weakly with receptors and requires a less active NA to facilitate release. Overall, the HA depends on a balance of glycosylation to mediate the proper folding of the HA, interaction of virus with receptor, and efficient particle release.
Collectins are a family of collagenous lectin molecules that are calcium-dependent carbohydrate binding proteins previously shown to bind enveloped viruses (
6,
26,
32,
41). The function of these proteins is believed to be as a first line of defense against both bacterial and viral pathogens by binding to carbohydrate moieties on the pathogen surface. In support of this concept, children with a deficiency in mannose binding lectin are more prone to a variety of serious infections (
21,
40). Multiple studies have shown that lung-resident surfactant proteins A and D (SP-A and SP-D, respectively) neutralize and aid in clearance of influenza A viruses (
7,
8,
15,
18,
19). SP-A is more effective at neutralizing influenza viruses that contain low carbohydrate content and does so via sialic acid residues present on the carbohydrate recognition domain that compete virus away from cellular sialic acids (
24). SP-D directly interacts with carbohydrate on the HA globular head. It preferentially binds to high-mannose oligosaccharides on the HA, most notably the oligosaccharide attached via amino acid 165, which is conserved in all H3N2 viruses isolated to date (
8). Data from several labs suggest that the high avidity of SP-D binding to influenza viruses is a key contributor to the virus-neutralizing capacity of the bronchoalveolar fluid (
8,
19,
43,
44).
We sought to investigate the potential cost of accumulating additional glycosylation on the globular head of the HA of influenza viruses. We constructed seven viruses expressing mutant HAs containing between 6 and 12 potential sites for N-linked glycosylation. We hypothesize that the level of glycosylation is inversely related to virulence in the naïve host; as glycosylation increases, the severity and sequelae of disease decrease due in part to improved recognition and neutralization by collectins.
MATERIALS AND METHODS
Generation of mutant viruses.
Plasmids expressing the internal genes from influenza virus A Puerto Rico/8/34 (H1N1, referred to hereafter as PR8) and the HA and/or NA from A/Hong Kong/1/68 (H3N2) (HK68), A/Leningrad/360/86 (H3N2) (Len86), A/Sydney/5/97 (H3N2) (Syd97), and A/Panama/2007/99 (H3N2) (Pan99) were obtained from Robert Webster (St. Jude Children's Research Hospital [SJCRH]) or were cloned from viruses obtained from Webster as described previously (
20). The plasmid expressing the HA of HK68 was sequentially modified by site-directed mutagenesis (QuickChange; Stratagene, La Jolla, CA) to encode additional sites of glycosylation. Oligonucleotide primers were designed using web-based Primer X (www.Bioinformatics.org ) from the consensus sequence for HK68 to create the N-X-S/T sequon at positions 63, 126, 248, 135, and 144 using historic sequences found in natural isolates from the past 38 years. Reassortant viruses expressing an H3 HA, an N2 NA (from Syd97), and the internal genes of PR8 were rescued into a coculture of MDCK and 293T cells using the 8-plasmid reverse genetics system, as described previously (
20). Viruses were propagated for an additional passage in MDCK cells and then grown in eggs to produce stocks for use in the experiments described here. Glycosylation mutants were generated with the previous virus as a template to generate viruses containing 1, 2, 3, 4, and 5 additional glycosylation sites (Table
1). An additional mutant virus was created by disrupting the glycosylation site at amino acid 165 of the HA. The nucleotide sequences of all plasmids and of the HA and NA of all stock viruses were confirmed by Big Dye Terminator Cycle sequencing (Hartwell Center), and analysis was performed by alignment against published sequences.
Immunoprecipitation and Western blotting.
Subconfluent MDCK cells were infected at a multiplicity of infection of 0.1 with reassortant virus. At 48 h postinfection the monolayers were disrupted in phosphate-buffered saline (PBS) containing 0.5% NP-40 and immunoprecipitated with EZview Red Anti-HA Affinity Gel (Sigma Aldrich, St. Louis, MO) targeting amino acid residues 98 to 106 (YPYDVPDYA) of human influenza virus HA. Immunoprecipitates were resolved on a 10 to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Bio-Rad, Hercules, CA). Western blotting was performed with mouse monoclonal anti-HA antibody (Research Diagnostics, Concord, MA) and TrueBlot horseradish peroxidase-conjugated anti-mouse secondary antibody (eBioscience, San Diego, CA) and visualized by enhanced chemiluminescence on radiographic film.
Mice.
Female BALB/cByJ and C57BL/6J mice were obtained from Jackson Laboratories and utilized at 6 to 8 weeks of age (Bar Harbor, ME). C57BL/6J mice deficient in SP-D were generated by author S. Hawgood at the University of California at San Francisco (
4) and then bred at SJCRH. Mice were housed in groups of 4 to 6 in high-temperature, 31.2-cm by 23.5-cm by 15.2-cm polycarbonate cages with isolator lids. Rooms used for housing mice were maintained on a 12:12-h light:dark cycle at 22 ± 2°C with a humidity of 50% in the biosafety level 2 facility at SJCRH. Prior to inclusion in experiments, mice were allowed at least 7 days to acclimate to the animal facility. Laboratory Autoclavable Rodent Diet (PMI Nutrition International, St. Louis, MO) and autoclaved water were available ad libitum. All experiments were performed in accordance with the guidelines set forth by the Animal Care and Use Committee at SJCRH.
Infection model.
The dose infectious for 50% of embryonated chicken eggs (EID50) was determined by interpolation using the method of Reed and Muench and used as the basis to calculate the dose lethal for 50% of mice (MLD50) using serial dilutions of virus delivered to groups of 4 mice. For infection experiments, virus was diluted in sterile PBS and administered at a dose of 1 × 106 EID50 intranasally to mice lightly anesthetized with 2.5% inhaled isoflurane (Baxter, Deerfield, IL) in a total volume of 100 μl (50 μl per nostril). Mice were weighed at the onset of infection and each subsequent day for illness and mortality. Mice that were found to be moribund were euthanized and considered to have died that day.
Lung titers.
Mice were euthanized by CO2 asphyxiation. Lungs were aseptically harvested, washed three times in PBS, and placed in 750 μl of sterile PBS. Lungs were mechanically homogenized using an Ultra-Turrax T8 homogenizer (IKA-werke, Staufen, Germany). Lung homogenates were pelleted at 10,000 rpm for 5 min, and the supernatants were used to determine the viral titer for each set of lungs using serial dilutions on MDCK monolayers.
Pathology.
Lungs were removed immediately after euthanasia and were insufflated and fixed overnight with 2% neutral buffered paraformaldehyde. After 24 h, the lungs were transferred into 10% neutral buffered formalin for an additional 24 h; the lungs were then embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically for histopathology by an experienced veterinary pathologist (K. L. Boyd) blinded to the composition of the groups. The lung parenchyma and large airways were considered separately and assigned a grade of 0 to 3 based on the histologic character of the lesions. A score of 1 was given to mild findings including minimal infiltrates of lymphocytes and plasma cells around airways and vessels, minimal epithelial hyperplasia, minimal leukocyte infiltration of alveolar spaces, and <10% of the lung affected. A score of 2 was given for moderate findings including moderate infiltrates of lymphocytes and plasma cells around airways and vessels, moderate epithelial hyperplasia with focal necrosis, focally extensive infiltration of the alveolar spaces by leukocytes with some consolidation, focal pleuritis, and >10% but <30% of the lung affected. A score of 3 was given for more severe findings including extensive necrosis of airway epithelium and the interstitium, extensive leukocyte infiltration and consolidation, severe pleuritis, and lobar involvement.
Inhibition of hemagglutination by rhSP-D.
Recombinant human SP-D (rhSP-D) was expressed in Chinese hamster ovary cells and purified by maltose affinity chromatography as previously described (
16). Inhibition of hemagglutination was done using standard methods. Briefly, virus suspensions titrated to 4 HA units were incubated with serial dilutions of rhSP-D for 30 min at room temperature. A 0.5% suspension of chicken red blood cells was added, and the minimum concentration needed to inhibit hemagglutination was determined in quadruplicate assays. In a second experiment, 1.25 μg of rhSP-D (the minimum amount which would inhibit hemagglutination from all viruses) was incubated with serial dilutions of virus prior to hemagglutination. In parallel, virus was incubated with PBS, and the HA titer of the diluted suspension was determined. The maximal number of HA units which could be prevented from hemagglutinating red blood cells is reported.
Statistical analysis.
Comparison of survival between groups of mice was done with a log rank chi-squared test on the Kaplan-Meier survival data. Comparison of viral lung titers, weight loss, and inhibition of hemagglutination between groups was done using analysis of variance. A P value of <0.05 was considered significant for these comparisons. SigmaStat for Windows (version 3.11; SysStat Software, Inc.,) was utilized for all statistical analyses. Due to the small number of SP-D-deficient animals available for use, studies involving those mice were underpowered for statistical analysis.
DISCUSSION
Over the past nearly 40 years of circulation, H3N2 viruses have gradually acquired additional oligosaccharide content around the globular head of the protein. These progressive, adaptive changes likely occurred because they provided an evolutionary advantage. However, since the changes have been gradual and are not fixed features of the HA, we reasoned they likely also came at some cost. We constructed a series of mutant viruses differing only in sites of potential glycosylation on the globular head of the HA to test the hypothesis that the level of oligosaccharide content is inversely related to virulence and tested this hypothesis in a mouse model of infection. We demonstrate that the functional outcome of additional N-linked glycosylation on the globular head of H3N2 influenza viruses is to attenuate the severity of infection in naive mice, likely mediated by improved neutralization by SP-D. A breakpoint for virulence in mice was evident at eight total sites of glycosylation on the HA (3 sites on the globular head); for maximal hemagglutination inhibition capacity of recombinant SP-D the breakpoint was nine total sites (4 sites on the globular head). Our conclusions were supported by studies in mice deficient in SP-D, which evidenced an inverse pattern of disease, with viruses possessing the highest potential for glycosylation eliciting significant disease and mortality.
Glycosylation of surface proteins plays a role in the biology of many viruses. including Hendra (
5), Hantaan (
37), severe acute respiratory syndrome coronavirus (SARS-CoV) (
30), West Nile (
17), hepatitis C (
13), and influenza viruses. The function of surface glycoconjugates in the life cycle of many of these viruses is to aid in entry into target cells. For example, the hepatitis virus E2 and West Nile virus PrM and E proteins rely upon glycosylation to interact with immune molecules such as DC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin) and the related liver lectin L-SIGN for attachment and entry (
10,
11). The glycosylated SARS-CoV S protein and filovirus envelope glycoprotein can interact with the lectin LSECtin (liver and lymph node sinusoidal endothelial cell C-type lectin) to enhance viral uptake and infection (
14). Human immunodeficiency virus type 1 and influenza virus have also been described as using glycosylated gp120 and HA molecules to interact with and mediate entry via DC-SIGN and mannose receptor molecules on dendritic cells and macrophages (
29,
31,
45). In addition, glycosylation of the surface proteins of SARS-CoV, Nipah, Hendra and Hantaan viruses has been described to participate in infectivity, protein folding, tropism, and proteolytic processing (
2,
3,
12,
25,
28,
30,
34).
It has become clear that the addition of glycosylation in many viruses is also a mechanism for viral evasion and persistence. Evidence for this view derives from studies where successively adding additional sites for linkage of oligosaccharide by site-directed mutagenesis provided influenza A viruses with the ability to evade the host response without negatively impacting survival and biological activity (
1). The additional sugar on the globular head resulted in a decrease in receptor binding and did not affect fusion activity, but, importantly, the viruses were now more resistant to antibody recognition. Skehel et al. showed that the introduction of a site for glycosylation at amino acid position 63 in the X-31 (H3N2) virus resulted in a lack of recognition by monoclonal antibody directed against X-31 (
38). Thus, acquisition of carbohydrates on the globular head of the HA of influenza viruses may be an evolutionary adaptation allowing further circulation in an immune population. The trend toward accumulation of sites for potential glycosylation can be seen in both the H3N2 and H1N1 lineages. From its introduction into the human population in 1918, the H1N1 viruses have progressed from 4 sites of potential glycosylation, all within the stalk region, to 8 sites for potential glycosylation, 4 of which are now in the globular head. In the H3N2 strains, the pandemic strain at its introduction contained 6 sites within the HA1 subunit, 2 of which were on the globular head. Currently circulating H3N2 viruses now have 13 potential sites for glycosylation, the original 4 sites in the stalk region and 9 sites on the globular head.
In this study we have engineered five additional sites of glycosylation into the globular head of HK68 to recapitulate the acquisition of glycosylation that has occurred in circulating H3N2 strains over the last 38 years. Additionally, we created a reverse mutant by removing the site for glycosylation in antigenic site B [Δ167 (−1)] which has been implicated as a potential site of recognition by the lung collectin SP-D (
19). Our data demonstrate that there is generally an attenuation of disease severity in naïve mice as the level of glycosylation increases. However, in our model the Δ167 (−1) virus did not appear to be any more virulent than wild type. We found a breakpoint between 8 and 9 sites of glycosylation (3 to 4 additional sites on the globular head) for virulence and neutralization, suggesting that after this point the effects mediated by SP-D are maximized. From these experiments we cannot distinguish whether this breakpoint derives generally from reaching a plateau in carbohydrate content or is specific to the particular sites we engineered into the virus. Data from SP-D-deficient animals support our proposed mechanism since mice infected with viruses of higher potential glycosylation were not attenuated in these animals as they were in fully competent hosts. In fact, our data suggest that clearance via other mechanisms, such as SP-A, may be more important for the less glycosylated viruses, perhaps because the lack of carbohydrates on the surface improves access of SP-A to its site of binding. The differences between the breakpoint for virulence (8 sites) and inhibition of hemagglutination (9 sites) may be due to the contribution of SP-A. Further study using recombinant SP-A and animals deficient in SP-A or both SP-A and SP-D is warranted to dissect the relative contribution of each.
These findings have important implications for our understanding of influenza biology and host interaction. Pandemic strains from this century have contained few sites for glycosylation on the globular head where the carbohydrates attached there might be accessible to collectins. The HA of the H1N1 strain of 1918 has been shown to play a major role in the virulence of that virus (
42) and had glycosylation sites only in the stalk. The H2N2 pandemic strain of 1957 contained only 1 site on the globular head, and the H3N2 strain of 1968 had just 2 sites. Highly pathogenic avian influenza viruses of the H5N1 subtype which have recently crossed over into humans have a total of only six potential sites for glycosylation, excluding 1 site in the cytoplasmic tail which is unlikely to be glycosylated (
39). During human infections with H5N1 strains, the lack of neutralization by collectins could potentially contribute to the high virulence.
The presence of glycosylation may have important implications for vaccine design as well. A strong neutralizing antibody response may be dependent on access to the surface of the HA protein, which may be blocked by carbohydrates. Thus, standard vaccines made from recently circulating, highly glycosylated viruses may elicit poor responses; using genetic engineering to remove potential glycosylation sites may alleviate this problem and improve the vaccines (
4). However, our data suggest that this approach may affect virulence if applied to live attenuated influenza vaccines. The balance between attenuation and immunogenicity would have to be carefully considered.
In summary, our study demonstrates that the level of potential glycosylation impacts the disease severity and outcome of infection in naïve animals. The likely mechanism explaining this observation is neutralization and clearance of the virus, mediated by the collectin SP-D. This may provide a balance for the benefits, such as evasion of the immune response, garnered as the virus accrues more surface carbohydrates. Further analysis of the impact of both SP-D and SP-A, particularly in more complex systems where preexisting immunity is present, would be of interest. An exploration of the role of collectins in a model such as ferret that better approximates the disease in humans would also be important to better understand the biology of influenza in the lung.