The infection of chicken tracheal epithelial cells with a H6N1 avian influenza virus

Sialic acids (SAs) linked to galactose (Gal) in α2,3- and α2,6-configurations are the receptors for avian and human influenza viruses, respectively. We demonstrate that chicken tracheal ciliated cells express α2,3-linked SA, while goblet cells mainly express α2,6-linked SA. In addition, the plant lectin MAL-II, but not MAA/MAL-I, is bound to the surface of goblet cells, suggesting that SA2,3-linked oligosaccharides with Galβ1-3GalNAc subterminal residues are specifically present on the goblet cells. Moreover, both α2,3- and α2,6-linked SAs are detected on single tracheal basal cells. At a low multiplicity of infection (MOI) avian influenza virus H6N1 is exclusively detected in the ciliated cells, suggesting that the ciliated cell is the major target cell of the H6N1 virus. At a MOI of 1, ciliated, goblet and basal cells are all permissive to the AIV infection. This result clearly elucidates the receptor distribution for the avian influenza virus among chicken tracheal epithelial cells and illustrates a primary cell model for evaluating the cell tropisms of respiratory viruses in poultry.     

Effects of receptor binding specificity of avian influenza virus on the human innate immune response

Humans infected by the highly pathogenic H5N1 avian influenza viruses (HPAIV) present unusually high concentrations in serum of proinflammatory cytokines and chemokines, which are believed to contribute to the high pathogenicity of these viruses. The hemagglutinins (HAs) of avian influenza viruses preferentially bind to sialic acids attached through α2,3 linkages (SAα2,3) to the terminal galactose of carbohydrates on the host cell surface, while the HAs from human strains bind to α2,6-linked SA (SAα2,6). To evaluate the role of the viral receptor specificity in promoting innate immune responses in humans, we generated recombinant influenza viruses, one bearing the HA and neuraminidase (NA) genes from the A/Vietnam/1203/2004 H5N1 HPAIV in an influenza A/Puerto Rico/8/1934 (A/PR/8/34) backbone with specificity for SAα2,3 and the other a mutant virus (with Q226L and G228S in the HA) with preferential receptor specificity for SAα2,6. Viruses with preferential affinity for SAα2,3 induced higher levels of proinflammatory cytokines and interferon (IFN)-inducible genes in primary human dendritic cells (DCs) than viruses with SAα2,6 binding specificity, and these differences were independent of viral replication, as shown by infections with UV-inactivated viruses. Moreover, human primary macrophages and respiratory epithelial cells showed higher expression of proinflammatory genes after infection with the virus with SAα2,3 affinity than after infection with the virus with SAα2,6 affinity. These data indicate that binding to SAα2,3 by H5N1 HPAIV may be sensed by human cells differently than binding to SAα2,6, inducing an exacerbated innate proinflammatory response in infected individuals.     

Infection of human airway epithelium by human and avian strains of influenza a virus

We describe the characterization of influenza A virus infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). Sialic acid receptors for both human and avian viruses, alpha-2,6- and alpha-2,3-linked sialic acids, respectively, were detected on the HAE cell surface, and their distribution accurately reflected that in human tracheobronchial tissue. Nonciliated cells present a higher proportion of alpha-2,6-linked sialic acid, while ciliated cells possess both sialic acid linkages. Although we found that human influenza viruses infected both ciliated and nonciliated cell types in the first round of infection, recent human H3N2 viruses infected a higher proportion of nonciliated cells in HAE than a 1968 pandemic-era human virus, which infected proportionally more ciliated cells. In contrast, avian influenza viruses exclusively infected ciliated cells. Although a broad-range neuraminidase abolished infection of HAE by human parainfluenza virus type 3, this treatment did not significantly affect infection by influenza viruses. All human viruses replicated efficiently in HAE, leading to accumulation of nascent virus released from the apical surface between 6 and 24 h postinfection with a low multiplicity of infection. Avian influenza A viruses also infected HAE, but spread was limited compared to that of human viruses. The nonciliated cell tropism of recent human H3N2 viruses reflects a preference for the sialic acid linkages displayed on these cell types and suggests a drift in the receptor binding phenotype of the H3 hemagglutinin protein as it evolves in humans away from its avian virus precursor.     

Receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential

The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed.     

Enhanced susceptibility of nasal polyp tissues to avian and human influenza viruses

Background

Influenza viruses bind and infect respiratory epithelial cells through sialic acid on cell surface. Differential preference to sialic acid types contributes to host- and tissue-tropism of avian and seasonal influenza viruses. Although the highly pathogenic avian influenza virus H5N1 can infect and cause severe diseases in humans, it is not efficient in infecting human upper respiratory tract. This is because of the scarcity of its receptor, α2,3-linked sialic acid, in human upper airway. Expression of sialic acid can be influenced by various factors including inflammatory process. Allergic rhinitis and nasal polyp are common inflammatory conditions of nasal mucosa and may affect expression of the sialic acid and susceptibility to influenza infection.

Methodology/Principal Finding

To test this hypothesis, we detected α2,3- and α2,6-linked sialic acid in human nasal polyp and normal nasal mucosal tissues by lectin staining and infected explants of those tissues with avian influenza viruses H5N1 and seasonal influenza viruses. We show here that mucosal surface of nasal polyp expressed higher level of α2,3- and α2,6-linked sialic acid than normal nasal mucosa. Accordingly, both H5N1 avian influenza viruses and seasonal influenza viruses replicated more efficiently in nasal polyp tissues explants.

Conclusions/Significance

Our data suggest a role of nasal inflammatory conditions in susceptibility to influenza infection, especially by avian influenza viruses, which is generally inefficient in infecting human upper airway. The increased receptor expression may contribute to increased susceptibility in some individuals. This may contribute to the gradual adaptation of the virus to human population.     

Host-mediated selection of influenza virus receptor variants. Sialic acid-alpha 2,6Gal-specific clones of A/duck/Ukraine/1/63 revert to sialic acid-alpha 2,3Gal-specific wild type in ovo

Human and animal influenza A isolates of the H3 serotype preferentially bind SA alpha 2,6Gal or SA alpha 2,3Gal linkages (where SA represents sialic acid), respectively, on cell-surface sialyloligosaccharides. Previously, we have demonstrated selection of SA alpha 2,3Gal-specific receptor variants of several human viruses which differed from the parent viruses by a single amino acid at residue 226 of the hemagglutinin which is located in the receptor binding pocket (Rogers, G. N., Paulson, J.C., Daniels, R.S., Skehel, J.J., Wilson, I.A., and Wiley, D.C. (1983) Nature 304, 76-78). In this report, the selection in the reverse direction was accomplished starting with a SA alpha 2,3Gal-specific avian virus, A/duck/Ukraine/1/63 (H3N7), yielding SA alpha 2,6Gal-specific variants that exhibit the receptor binding properties characteristic of the human isolates. Selection was again mediated at residue 226 of the hemagglutinin, in this case changing from Gln in the parent virus to Leu in the variants. Although the SA alpha 2,6Gal-specific avian virus variants were stable to passage in MDCK cells, they exhibited dramatic reversion to the SA alpha 2,3Gal-specific phenotype of the parent virus during a single passage in chicken embryos. This was in contrast to the SA alpha 2,6Gal-specific human virus isolates which were stable to passage in both hosts. The reversion of the avian virus variants in eggs provides compelling evidence for host-mediated selection of influenza virus receptor variants.     

Sialylation of human IgG-Fc carbohydrate by transfected rat alpha2,6-sialyltransferase

A recombinant IgG3 antibody with Phe-243 replaced by Ala (FA243) was expressed in a CHO-K1 parental cell line. The resulting IgG-Fc-linked carbohydrate was significantly alpha2,3-sialylated (53% of glycans), as indicated by normal- and reverse-phase HPLC analyses. Following transfection of a rat alpha2,6-sialyltransferase gene into this parental cell line, IgG-Fc-linked glycans were sialylated (60% of glycans) such that the ratio of alpha2,6- to alpha2,3-linked sialic acid was 0.9:1.0. By comparison, the wild-type IgG3 (F243) is minimally sialylated (2-3% alpha2,3-linked), thus suggesting that sialylation is controlled primarily by the protein structure local to the carbohydrate and that the two sialyltransferases compete to sialylate the nascent oligosaccharide. The additional alpha2,6-sialylation affected the function of the recombinant antibody. FA243 IgG3 having both alpha2,6 and alpha2,3-sialylation restored recognition to wild-type IgG3 levels for human FcgammaRI, FcgammaRII, and target cell lysis by complement. We discuss how sialylation linkage could modulate IgG function.     

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.     

Molecular dynamics simulation of the effects of single (S221P) and double (S221P and K216E) mutations in the hemagglutinin protein of influenza A H5N1 virus: a study on host receptor specificity

Avian influenza viruses of subtype H5N1 circulating in animals continue to pose threats to human health. The binding preference of the viral surface protein hemagglutinin (HA) to sialosaccharides of receptors is an important area for understanding mutations in the receptor binding site that could be the cause for avian-to-human transmission. In the present work, we studied the effect of two receptor binding site mutations, S221P singly and in combination with another mutation K216E in the HA protein of influenza A H5N1 viruses. Docking of sialic acid ligands corresponding to both avian and human receptors and molecular dynamics simulations of the complexes for wild and mutant strains of H5N1 viruses were carried out. The H5N1 strain possessing the S221P mutation indicated decreased binding to α2,3-linked sialic acids (avian receptor, SAα2,3Gal) when compared to the binding of the wild-type strain that did not possess the HA-221 mutation. The binding to α2,6-linked sialic acids (human receptor, SAα2,6Gal) was found to be comparable, indicating that the mutant strain shows limited dual receptor specificity. On the other hand, the S221P mutation in synergism with the K216E mutation in the binding site, resulted in increased binding affinity for SAα2,6Gal when compared to SAα2,3Gal, indicative of enhanced binding to human receptors. The in-depth study of the molecular interactions in the docked complexes could explain how co-occurring mutations in the HA viral protein can aid in providing fitness advantage to the virus, in the context of host receptor specificity in emerging variants of H5N1 influenza viruses.     

人呼吸道禽流感病毒受体的分布趋势

禽类流感病毒和人类流感病毒具有很强的受体识别特异性,分别与唾液酸α-2,3Gal和α-2,6Gal受体分子结合而感染各自的宿主细胞．这种受体结合特异性是流感病毒在禽类和人类之间跨种属传递的主要障碍．应用凝集素组织化学染色技术,探讨人呼吸道各解剖学部位流感病毒唾液酸受体的分布特征．结果显示,唾液酸α-2,3Gal受体, 即禽类流感受体,主要分布在下呼吸道的呼吸部即呼吸细支气管和肺泡, 而在主气管、支气管和细支气管仅少量分布．相反,人类流感病毒受体,唾液酸α-2,6Gal受体在气管、支气管呈高密度分布,随着支气管分级逐渐降低分布减少,至肺泡分布最少．但比较人呼吸道发育成熟过程中,唾液酸α-2,3Gal和α-2,6Gal受体的表达,未发现明显差别．禽流感H5N1病毒体外感染人呼吸道组织试验结果表明,肺泡上皮较支气管和气管上皮易感染,与唾液酸α-2,3Gal受体分布特点相符合．结果提示,人呼吸道可被禽流感病毒感染,目前H5N1病毒极少发生人传人的特点,可能与个体间上呼吸道唾液酸α-2,3Gal受体表达差异有关．     

Correlation between sialic acid expression and infection of murine macrophages by different strains of influenza virus

The mouse-adapted A/PR/8/34 (PR8; H1N1) virus infects airway macrophages poorly and is virulent in mice. Herein, we have investigated factors contributing to the ability of PR8 to evade murine macrophages. We demonstrate that the hemagglutinin of PR8 binds preferentially to α(2,3)-linked sialic acid (SA) and that murine macrophages express α(2,6)-linked SA. Moreover, resialylation of macrophages to express α(2,3)-linked SA restored susceptibility to PR8. Thus, during adaptation of human influenza viruses to growth in mice, a switch in receptor specificity from α(2,6)-linked SA to α(2,3)-linked SA is likely to favour evasion of attachment, entry and destruction by airway macrophages.     

Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities

Influenza A virus specificity for the host is mediated by the viral surface glycoprotein hemagglutinin (HA), which binds to receptors containing glycans with terminal sialic acids. Avian viruses preferentially bind to alpha2-3-linked sialic acids on receptors of intestinal epithelial cells, whereas human viruses are specific for the alpha2-6 linkage on epithelial cells of the lungs and upper respiratory tract. To define the receptor preferences of a number of human and avian H1 and H3 viruses, including the 1918 H1N1 pandemic strains, their hemagglutinins were analyzed using a recently described glycan array. The array, which contains 200 carbohydrates and glycoproteins, not only revealed clear differentiation of receptor preferences for alpha2-3 and/or alpha2-6 sialic acid linkage, but could also detect fine differences in HA specificity, such as preferences for fucosylation, sulfation and sialylation at positions 2 (Gal) and 3 (GlcNAc, GalNAc) of the terminal trisaccharide. For the two 1918 HA variants, the South Carolina (SC) HA (with Asp190, Asp225) bound exclusively alpha2-6 receptors, while the New York (NY) variant, which differed only by one residue (Gly225), had mixed alpha2-6/alpha2-3 specificity, especially for sulfated oligosaccharides. Only one mutation of the NY variant (Asp190Glu) was sufficient to revert the HA receptor preference to that of classical avian strains. Thus, the species barrier, as defined by the receptor specificity preferences of 1918 human viruses compared to likely avian virus progenitors, can be circumvented by changes at only two positions in the HA receptor binding site. The glycan array thus provides highly detailed profiles of influenza receptor specificity that can be used to map the evolution of new human pathogenic strains, such as the H5N1 avian influenza.     

Impact of Fc N-glycan sialylation on IgG structure

ABSTRACT

Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.     

Human-specific regulation of alpha 2-6-linked sialic acids

Many microbial pathogens and toxins recognize animal cells via cell surface sialic acids (Sias) that are alpha 2-3- or alpha 2-8-linked to the underlying glycan chain. Human influenza A/B viruses are unusual in preferring alpha 2-6-linked Sias, undergoing a switch from alpha 2-3 linkage preference during adaptation from animals to humans. This correlates with the expression of alpha 2-6-linked Sias on ciliated human airway epithelial target cells and of alpha 2-3-linked Sias on secreted soluble airway mucins, which are unable to inhibit virus binding. Given several known differences in Sia biology between humans and apes, we asked whether this pattern of airway epithelial Sia linkages is also human-specific. Indeed, we show that since the last common ancestor with apes, humans underwent a concerted bidirectional switch in alpha 2-6-linked Sia expression between airway epithelial cell surfaces and secreted mucins. This can explain why the chimpanzee appears relatively resistant to experimental infection with human Influenza viruses. Other tissues showed additional examples of human-specific increases or decreases in alpha 2-6-linked Sia expression and only one example of a change specific to certain great apes. Furthermore, while human and great ape leukocytes both express alpha 2-6-linked Sias, only human erythrocytes have markedly up-regulated expression. These cell type-specific changes in alpha 2-6-Sia expression during human evolution represent another example of a human-specific change in Sia biology. Because the data set involves multiple great apes, we can also conclude that Sia linkage expression patterns can be conserved during millions of years of evolution within some vertebrate taxa while undergoing sudden major changes in other closely related ones.     

Entry of Influenza A Virus with a α2,6-Linked Sialic Acid Binding Preference Requires Host Fibronectin

The receptor binding specificity of influenza A virus is one of the major determinants of viral tropism and host specificity. In general, avian viral hemagglutinin prefers to bind to α2,3-linked sialic acid, whereas the human viral hemagglutinin prefers to bind to α2,6-linked sialic acid. Here, we demonstrate that host fibronectin protein plays an important role in the life cycle of some influenza A viruses. Treating cells with anti-fibronectin antibodies or fibronectin-specific small interfering RNA can inhibit the virus replication of human H1N1 influenza A viruses. Strikingly, these inhibitory effects cannot be observed in cells infected with H5N1 viruses. By using reverse genetics techniques, we observed that the receptor binding specificity, but not the origin of the hemagglutinin subtype, is responsible for this differential inhibitory effect. Changing the binding preference of hemagglutinin from α2,6-linked sialic acid to α2,3-linked sialic acid can make the virus resistant to the anti-fibronectin antibody treatment and vice versa. Our further characterizations indicate that anti-fibronectin antibody acts on the early phase of viral replication cycle, but it has no effect on the initial binding of influenza A virus to cell surface. Our subsequent investigations further show that anti-fibronectin antibody can block the postattachment entry of influenza virus. Overall, these results indicate that the sialic acid binding preference of influenza viral hemagglutinin can modulate the preferences of viral entry pathways, suggesting that there are subtle differences between the virus entries of human and avian influenza viruses.     

Influenza virus receptors in the human airway

Avian influenza A (H5N1) virus infections have resulted in more than 100 human deaths; yet, human-to-human transmission is rare. We demonstrated that the epithelial cells in the upper respiratory tract of humans mainly possess sialic acid linked to galactose by alpha 2,6 linkages (SA alpha 2,6Gal), a molecule preferentially recognized by human viruses. However, many cells in the respiratory bronchioles and alveoli possess SA alpha 2,3Gal, which is preferentially recognized by avian viruses. These facts are consistent with the observation that H5N1 viruses can be directly transmitted from birds to humans and cause serious lower respiratory tract damage in humans. Furthermore, this anatomical difference in receptor prevalence may explain why the spread of H5N1 viruses among humans is limited. However, since some H5N1 viruses isolated from humans recognize human virus receptors, additional changes must be required for these viruses to acquire the ability for efficient human-to-human transmission.     

Infection of differentiated porcine airway epithelial cells by influenza virus: differential susceptibility to infection by porcine and avian viruses

Background

Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses.

Methodology/Principal Findings

To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses.

Conclusions/Significance

Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine.     

Enterovirus 70 binds to different glycoconjugates containing alpha2,3-linked sialic acid on different cell lines

Enterovirus 70 (EV70), the causative agent of acute hemorrhagic conjunctivitis, exhibits a restricted tropism for conjunctival and corneal cells in vivo but infects a wide spectrum of mammalian cells in culture. Previously, we demonstrated that human CD55 is a receptor for EV70 on HeLa cells but that EV70 also binds to sialic acid-containing receptors on a variety of other human cell lines. Virus recognition of sialic acid attached to underlying glycans by a particular glycosidic linkage may contribute to host range, tissue tropism, and pathogenesis. Therefore, we tested the possibility that EV70 binds to alpha2,3-linked sialic acid, like other viruses associated with ocular infections. Through the use of linkage-specific sialidases, sialyltransferases, and lectins, we show that EV70 recognizes alpha2,3-linked sialic acid on human corneal epithelial cells and on U-937 cells. Virus attachment to both cell lines is CD55 independent and sensitive to benzyl N-acetyl-alpha-D-galactosaminide, an inhibitor of O-linked glycosylation. Virus binding to corneal cells, but not U-937 cells, is inhibited by proteinase K, but not by phosphatidylinositol-specific phospholipase C treatment. These results are consistent with the idea that a major EV70 receptor on corneal epithelial cells is an O-glycosylated, non-glycosyl phosphatidylinositol-anchored membrane glycoprotein containing alpha2,3-linked sialic acid, while sialylated receptors on U-937 cells are not proteinaceous.     

Comparative distribution of human and avian type sialic acid influenza receptors in the pig

Background  

A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid α2,3-galactose (SAα2,3-Gal) linked receptors, whereas human strains bind to sialic acid α2,6-galactose (SAα2,6-Gal) linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAα2,3-GalG(1-3)GalNAc and SAα2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II) and Sambucus nigra agglutinin (SNA) respectively.     

Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity

Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.