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
). 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
). 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
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.