Sialic acid occupies the terminal position within glycan molecules on the surfaces of many vertebrate cells, where it functions in diverse cellular processes such as intercellular adhesion and cell signalling. Pathogenic bacteria have evolved to use this molecule beneficially in at least two different ways: they can coat themselves in sialic acid, providing resistance to components of the host's innate immune response, or they can use it as a nutrient. Sialic acid itself is either synthesized de novo by these bacteria or scavenged directly from the host. In this mini-review we will summarize recent findings relating to sialic acid transport, modification of sialic acid by O-acetylation, and the mechanisms of sialic acid-mediated complement resistance.

HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1-V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1-V2, showing an epitope comprising both high mannose-type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1-glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.

Polyoma virus (Py) and simian virus 40 (SV40) travel from the plasma membrane to the endoplasmic reticulum (ER) from where they enter the cytosol and then the nucleus to initiate infection. Here we demonstrate that specific gangliosides can serve as plasma membrane receptors for these viruses, GD1a and GT1b for Py and GM1 for SV40. Binding and flotation assays were used to show that addition of these gangliosides to phospholipid vesicles allowed specific binding of the respective viruses. The crystal structure of polyoma VP1 with a sialic acid-containing oligosaccharide was used to derive a model of how the two terminal sugars (sialic acid-alpha2,3-galactose) in one branch of GD1a and GT1b are recognized by the virus. A rat cell line deficient in ganglioside synthesis is poorly infectible by polyoma and SV40, but addition of the appropriate gangliosides greatly facilitates virus uptake, transport to the ER and infection. Lipid binding sites for polyoma are shown to be present in rough ER membranes, suggesting that the virus travel with the ganglioside(s) from the plasma membranes to the ER.

Myelin inhibitors of axonal regeneration, like Nogo and MAG, block regrowth after injury to the adult CNS. While a GPI-linked receptor for Nogo (NgR) has been identified, MAG's receptor is unknown. We show that MAG inhibits regeneration by interaction with NgR. Binding of and inhibition by MAG are lost if neuronal GPI-linked proteins are cleaved. Binding of MAG to NgR-expressing cells is GPI dependent and sialic acid independent. Conversely, NgR binds to MAG-expressing cells. MAG, but not a truncated MAG that binds neurons but does not inhibit regeneration, precipitates NgR from NgR-expressing cells, DRG, and cerebellar neurons. Importantly, NgR antibody, soluble NgR, or dominant-negative NgR each prevent inhibition of neurite outgrowth by MAG. Also, MAG and Nogo66 compete for binding to NgR. These results suggest redundancy in myelin inhibitors and indicate therapies for CNS injuries.

The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the alpha2,6(SAalpha2,6Gal) linkage, while equine viruses favor SAalpha2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] and N-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcalpha2,6Gal but not NeuAcalpha2,3Gal or NeuGcalpha2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcalpha2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcalpha2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.

An activity that is inhibitory to the properdin-stabilized amplification C3 convertase (C3b,Bb,P) was solubilized from human erythrocyte (E(hu)) membranes by Nonidet P-40 and purified to homogeneity. The inhibitory membrane glycoprotein had an apparent M(r) of 1-1.2x10(6) on gel filtration in the presence of Nonidet P-40. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis it presented a single stained band with an apparent M(r) of 205,000, with or without prior reduction of disulfides. The inhibitory protein of the E(hu) membrane produced a dose-related, first-order decay of C3b,Bb,P function on sheep erythrocytes (E(s)) and released (125)I-labeled Bb from these sites, indicating a mechanism of inhibition by decay-dissociation of the amplification C3 convertase. The 50% inhibitory dose of the E(hu) membrane protein was not altered by removal of sialic acid from the E(s) bearing C3b,Bb,P sites. E(hu) membrane protein also serves as a cofactor for C3b inactivator-induced cleavage of the alpha polypeptide chain of C3b. Thus, the inhibitory membrane protein can abrogate the activity of amplification convertase sites that have formed and also can prevent generation of such sites by augmenting irreversible inactivation of C3b.Discrimination between cells by the alternative complement pathway occurs after initial deposition of C3b and is related to the modulation by surface constituents of the capacity of bound C3b to function as a subunit of the amplification C3 convertase. The existence in the E(hu) membrane of a protein that can impair the functions of membrane-bound C3b and C3b,Bb,P could represent a molecular basis for preventing inappropriate self-recognition.

Sheep erythrocytes in their native state did not activate the alternative complement pathway, as measured by lysis in dilutions of normal human serum containing [ethylenebis(oxyethylenenitrilo)] tetraacetic acid but acquired this capacity after membrane sialic acid residues had been removed (by sialidase) or modified (by NaIO(4)). Activation of the alternative pathway by sheep erythrocytes required removal or modification of at least 40% of the membrane sialic acid to reach threshold, and it increased proportionately when larger amounts of sialic acid had been affected. Studies with isolated proteins of the alternative pathway demonstrated that the altered erythrocyte membranes resembled natural activators in protecting bound C3b from inactivation by C3b inactivator and beta1H and protecting bound amplification C3 convertase (C3b,Bb) from decay-dissociation by beta1H. A 1% decrease in intact sialic acid was associated with a 1% decrease in beta1H activity in decay-dissociation of membrane bound C3b,Bb. Because removal of the C8 and C9 carbon atoms from the polyhydroxylated side chain of sialic acid by oxidation with NaIO(4) was functionally equivalent to removal of the entire sialic acid moiety, secondary effects of the latter reaction, such as diminution of the negative charge of the membrane or exposure of penultimate galactose residues, were not considered to be responsible for the altered activity of beta1H. These studies suggest that facilitation, by membrane sialic acid residues, of the interaction between bound C3b and beta1H is essential to prevent the particle from effectively activating the alternative pathway.

Factor H (fH), a key alternative complement pathway regulator, is a cofactor for factor I-mediated cleavage of C3b. fH consists of 20 short consensus repeat (SCR) domains. Sialic acid binding domains have previously been localized to fH SCRs 6-10 and 13. To examine fH binding on a sialylated microbial surface, we grew Neisseria gonorrhoeae in the presence of 5'-cytidinemonophospho-N-acetylneuraminic acid, which sialylates lipooligosaccharide and converts to serum resistance gonococci previously sensitive to nonimmune serum killing. fH domains necessary for binding sialylated gonococci were determined by incubating organisms with recombinant human fH (rH) and nine mutant rH molecules (deletions spanning the entire fH molecule). rH and all mutant rH molecules that contained SCRs 16-20 bound to the sialylated strain; no mutant molecule bound to serum-sensitive nonsialylated organisms. Sialic acid was demonstrated to be the fH target by flow cytometry that showed a fourfold increase in fH binding that was reversed by neuraminidase-mediated cleavage of sialic acid off gonococci. Functional specificity of fH was confirmed by decreased total C3 binding and almost complete conversion to iC3b on sialylated gonococci. Sialic acid can therefore bind fH uniquely through SCRs 16-20. This blocks complement pathway activation for N. gonorrhoeae at the level of C3.

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.

Sialic acids are a diverse family of monosaccharides widely expressed on all cell surfaces of vertebrates and so-called "higher" invertebrates, and on certain bacteria that interact with vertebrates. This overview surveys examples of biological roles of sialic acids in immunity, with emphasis on an evolutionary perspective. Given the breadth of the subject, the treatment of individual topics is brief. Subjects discussed include biophysical effects regulation of factor H; modulation of leukocyte trafficking via selectins; Siglecs in immune cell activation; sialic acids as ligands for microbes; impact of microbial and endogenous sialidases on immune cell responses; pathogen molecular mimicry of host sialic acids; Siglec recognition of sialylated pathogens; bacteriophage recognition of microbial sialic acids; polysialic acid modulation of immune cells; sialic acids as pathogen decoys or biological masks; modulation of immunity by sialic acid O-acetylation; sialic acids as antigens and xeno-autoantigens; antisialoglycan antibodies in reproductive incompatibility; and sialic-acid-based blood groups.

Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid alpha2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.

The carbohydrate binding specificities of the leukoagglutinating phytohemagglutinin (L-PHA) and erythroagglutinating phytohemagglutinin (E-PHA) lectins of the red kidney bean, Phaseolus vulgaris, have been investigated by lectin-agarose affinity chromatography of Asn-linked oligosaccharides. High affinity binding to E-PHA-agarose occurs only with biantennary glycopeptides containing 2 outer galactose residues and a residue of N-acetylglucosamine linked beta 1,4 to the beta-linked mannose residue in the core. This species is not retarded on L-PHA-agarose. In contrast, tri- and tetraanternnary glycopeptides containing outer galactose residues and an alpha-linked mannose residue substituted at positions C-2 and C-6 are specifically retarded on L-PHA-agarose. Triantennary glycopeptides containing outer galactose residues and an alpha-linked mannose residue substituted at positions C-2 and C-4 are not retarded on L-PHA-agarose. Additionally, the presence of outer sialic acid residues or a core fucose residue does not influence the behavior of complex glycopeptides on either of these lectin-agarose conjugates. This ability of E-PHA and L-PHA to discriminate between Asn-linked oligosaccharides with various branching patterns can be utilized in the fractionation of these glycopeptides (see paper following).

The complement of sialyloligosaccharides present on the surface of human tracheal epithelium has been implicated as an important factor in the selection of hemagglutinin receptor specificity of human influenza A virus. Human strains of influenza A virus preferentially recognize host cell receptors bearing SA alpha 2,6Gal sequences, a sequence which is found on the surface of ciliated tracheal epithelium. A fluorescently-labelled H3 human virus strain bound avidly to the apical surface of human tracheal epithelium, while a fluorescently-labelled receptor variant strain, which preferentially binds SA alpha 2,3Gal sequences, showed little binding to the epithelial surface and localized primarily to intracellular mucin droplets. Extracts of human bronchial mucin, which is known to contain sialic acid primarily in the SA alpha 2,3Gal linkage, was a potent inhibitor of the binding of the receptor variant strain to trachea sections, while the binding of the parent strain was unaffected by the presence of mucin. Human bronchial mucin also inhibited the binding of the receptor variant strains, but not the parent virus strains, to human erythrocytes derivatized to contain SA alpha 2,6Gal sequences. These results suggest that a combination of selection pressures present in the respiratory tract environment have resulted in the evolution of a hemagglutinin receptor specificity in human influenza A virus strains which optimizes recognition of, binding to and infection of host cells.

Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cgamma2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542-549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin.

Adeno-associated virus serotype 4 (AAV4) and AAV5 have different tropisms compared to AAV2 and to each other. We recently reported that alpha 2--3 sialic acid is required for AAV5 binding and transduction. In this study, we characterized AAV4 binding and transduction and found it also binds sialic acid, but the specificity is significantly different from AAV5. AAV4 can hemagglutinate red blood cells from several species, whereas AAV5 hemagglutinates only rhesus monkey red blood cells. Treatment of red blood cells with trypsin inhibited hemagglutination for both AAV4 and AAV5, suggesting that the agglutinin is a protein. Treatment of Cos and red blood cells with neuraminidases also indicated that AAV4 bound alpha 2--3 sialic acid. However, resialylation experiments with neuraminidase-treated red blood cells demonstrated that AAV4 binding required alpha 2--3 O-linked sialic acid, whereas AAV5 required N-linked sialic acid. Similarly, resialylation of sialic acid-deficient CHO cells supported this same conclusion. The difference in linkage specificity for AAV4 and AAV5 was confirmed by binding and transduction experiments with cells incubated with either N-linked or O-linked inhibitors of glycosylation. Furthermore, AAV4 transduction was only blocked with soluble alpha 2-3 sialic acid, whereas AAV5 could be blocked with either alpha 2--3 or alpha 2-6 sialic acid. These results suggest that AAV4 and AAV5 require different sialic acid-containing glycoproteins for binding and transduction of target cells and they further explain the different tropism of AAV4 and AAV5.

A lethal synergism exists between influenza virus and Streptococcus pneumoniae, accounting for excess mortality during influenza epidemics. Using a model of viral-bacterial synergism, we assessed the role that the influenza virus neuraminidase (NA) has in priming mice for pneumococcal infection. Administration of the selective NA inhibitor oseltamivir improved survival, independent of viral replication and morbidity from influenza. Both pathologic examination of the lungs and live imaging of pneumonic lesions, using a bioluminescent pneumococcus, suggested that the effect of NA inhibition was to limit the extent of pneumococcal pneumonia during early infection. Adherence assays and immunohistochemical staining for sialic acids in lungs from infected mice demonstrated that the influenza virus NA potentiates development of pneumonia by stripping sialic acid from the lung, thus exposing receptors for pneumococcal adherence. Selective NA inhibitors may be useful clinically to interrupt this novel mechanism of synergism and to prevent excess mortality from secondary bacterial pneumonia.

Mucins are high-molecular weight epithelial glycoproteins with a high content of clustered oligosaccharides O-glycosidically linked to tandem repeat peptides rich in threonine, serine, and proline. There are two structurally and functionally distinct classes of mucins: secreted gel-forming mucins (MUC2, MUC5AC, MUC5B, and MUC6) and transmembrane mucins (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC17), although the products of some MUC genes do not fit well into either class (MUC7, MUC8, MUC9, MUC13, MUC15, MUC16). MUC1 mucin, as detected immunologically, is increased in expression in colon cancers, which correlates with a worse prognosis. Expression of MUC2 secreted gel-forming mucin is generally decreased in colorectal adenocarcinoma, but preserved in mucinous carcinomas, a distinct subtype of colon cancer associated with microsatellite instability. Another secreted gel-forming mucin, MUC5AC, a product of normal gastric mucosa, is absent from normal colon, but frequently present in colorectal adenomas and colon cancers. The O-glycosidically linked oligosaccharides of mucins can be described in terms of core type, backbone type, and peripheral structures. Colon cancer mucins have differences in both core carbohydrates and in peripheral carbohydrate structures that are being investigated as diagnostic and prognostic markers, and also as targets for cancer vaccines. Colon cancer mucins typically have increases in three core structures: Tn antigen (GalNAcalphaThr/Ser), TF antigen (Galbeta3GalNAc) and sialyl Tn (NeuAcalpha6GalNAc). The type 3 core (GlcNAcbeta3Ga1NAc) predominant in normal colonic mucin is lacking in colon cancer mucins. There are cancer-associated alterations in the peripheral carbohydrates of colonic mucins including a decrease in O-acetyl-sialic acid and a decrease in sulfation. There are, however, cancer-associated increases in sialyl LeX and related structures on mucins and other glycoproteins that can serve as ligands for selectins, increasing the metastatic capacity of colon cancer cells. The endogenous galactoside-binding protein galectin-3, which is expressed at higher levels in colon cancers than normal colon, binds to colon cancer mucin as well as other glycoproteins. Interference of the binding of selectins and galectin-3 to mucin may show therapeutic or preventative promise for colon cancer.

The alternative or properdin pathway of complement is primarily controlled by the endopeptidase C3b inactivator (C3bINA) and the nonproteolytic glycoprotein beta1H. The molecular mechanisms of control were investigated by performing binding studies of radiolabeled complement proteins to C3b bearing sheep erythrocytes (E(S)C3b). C3b was found to have distinct binding sites for beta1H, C3bINA, Factor B, and properdin. beta1H binding increased C3bINA binding 30-fold, while Factor B binding prevented C3bINA action on C3b and was competitive with beta1H binding. Properdin binding, which facilitates Factor B interaction with C3b, had no effect on the beta1H and C3bINA sites. Activators such as rabbit erythrocytes (E(R)) have previously been shown to interfere with the effectiveness of the control by C3bINA and beta1H, thereby allowing unrestricted formation of C3 convertase. Such restriction of control does not occur on the surface of E(S), a nonactivator of the alternative pathway. On the basis of comparative binding studies, restriction of control is explained entirely by reduced binding of beta1H to E(R)C3b relative to E(S)C3b. Access of properdin, Factor B, C3bINA, and the Fab fragment of anti-C3 to the two cell types was unrestricted. Restriction of beta1H control could be generated on the surface of E(S) by removal of cell-surface sialic acid with neuraminidase (acylneuraminyl hydrolase; EC 3.2.1.18). This enzymatic treatment converted E(S) from a nonactivator to an activator of the alternative pathway.

The different sialic acid (serogroups B, C, Y, and W-135) and nonsialic acid (serogroup A) capsular polysaccharides expressed by Neisseria meningitidis are major virulence factors and are used as epidemiologic markers and vaccine targets. However, the identification of meningococcal isolates with similar genetic markers but expressing different capsular polysaccharides suggests that meningococcal clones can switch the type of capsule they express. We identified, except for capsule, isogenic serogroups B [(alpha2-->8)-linked polysialic acid] and C [(alpha2-->9)-linked polysialic acid] meningococcal isolates from an outbreak of meningococcal disease in the U. S. Pacific Northwest. We used these isolates and prototype serogroup A, B, C, Y, and W-135 strains to define the capsular biosynthetic and transport operons of the major meningococcal serogroups and to show that switching from the B to C capsule in the outbreak strain was the result of allelic exchange of the polysialyltransferase. Capsule switching was probably the result of transformation and horizontal DNA exchange in vivo of a serogroup C capsule biosynthetic operon. These findings indicate that closely related virulent meningococcal clones may not be recognized by traditional serogroup-based surveillance and can escape vaccine-induced or natural protective immunity by capsule switching. Capsule switching may be an important virulence mechanism of meningococci and other encapsulated bacterial pathogens. As vaccine development progresses and broader immunization with capsular polysaccharide conjugate vaccines becomes a reality, the ability to switch capsular types may have important implications for the impact of these vaccines.

Influenza viruses are causative agents of an acute febrile respiratory disease called influenza (commonly known as "flu") and belong to the Orthomyxoviridae family. These viruses possess segmented, negative stranded RNA genomes (vRNA) and are enveloped, usually spherical and bud from the plasma membrane (more specifically, the apical plasma membrane of polarized epithelial cells). Complete virus particles, therefore, are not found inside infected cells. Virus particles consist of three major subviral components, namely the viral envelope, matrix protein (M1), and core (viral ribonucleocapsid [vRNP]). The viral envelope surrounding the vRNP consists of a lipid bilayer containing spikes composed of viral glycoproteins (HA, NA, and M2) on the outer side and M1 on the inner side. Viral lipids, derived from the host plasma membrane, are selectively enriched in cholesterol and glycosphingolipids. M1 forms the bridge between the viral envelope and the core. The viral core consists of helical vRNP containing vRNA (minus strand) and NP along with minor amounts of NEP and polymerase complex (PA, PB1, and PB2). For viral morphogenesis to occur, all three viral components, namely the viral envelope (containing lipids and transmembrane proteins), M1, and the vRNP must be brought to the assembly site, i.e. the apical plasma membrane in polarized epithelial cells. Finally, buds must be formed at the assembly site and virus particles released with the closure of buds. Transmembrane viral proteins are transported to the assembly site on the plasma membrane via the exocytic pathway. Both HA and NA possess apical sorting signals and use lipid rafts for cell surface transport and apical sorting. These lipid rafts are enriched in cholesterol, glycosphingolipids and are relatively resistant to neutral detergent extraction at low temperature. M1 is synthesized on free cytosolic polyribosomes. vRNPs are made inside the host nucleus and are exported into the cytoplasm through the nuclear pore with the help of M1 and NEP. How M1 and vRNPs are directed to the assembly site on the plasma membrane remains unclear. The likely possibilities are that they use a piggy-back mechanism on viral glycoproteins or cytoskeletal elements. Alternatively, they may possess apical determinants or diffuse to the assembly site, or a combination of these pathways. Interactions of M1 with M1, M1 with vRNP, and M1 with HA and NA facilitate concentration of viral components and exclusion of host proteins from the budding site. M1 interacts with the cytoplasmic tail (CT) and transmembrane domain (TMD) of glycoproteins, and thereby functions as a bridge between the viral envelope and vRNP. Lipid rafts function as microdomains for concentrating viral glycoproteins and may serve as a platform for virus budding. Virus bud formation requires membrane bending at the budding site. A combination of factors including concentration of and interaction among viral components, increased viscosity and asymmetry of the lipid bilayer of the lipid raft as well as pulling and pushing forces of viral and host components are likely to cause outward curvature of the plasma membrane at the assembly site leading to bud formation. Eventually, virus release requires completion of the bud due to fusion of the apposing membranes, leading to the closure of the bud, separation of the virus particle from the host plasma membrane and release of the virus particle into the extracellular environment. Among the viral components, M1 contains an L domain motif and plays a critical role in budding. Bud completion requires not only viral components but also host components. However, how host components facilitate bud completion remains unclear. In addition to bud completion, influenza virus requires NA to release virus particles from sialic acid residues on the cell surface and spread from cell to cell. Elucidation of both viral and host factors involved in viral morphogenesis and budding may lead to the development of drugs interfering with the steps of viral morphogenesis and in disease progression.

Cell surface oligosaccharides can be engineered to display unusual functional groups for the selective chemical remodeling of cell surfaces. An unnatural derivative of N-acetyl-mannosamine, which has a ketone group, was converted to the corresponding sialic acid and incorporated into cell surface oligosaccharides metabolically, resulting in the cell surface display of ketone groups. The ketone group on the cell surface can then be covalently ligated under physiological conditions with molecules carrying a complementary reactive functional group such as the hydrazide. Cell surface reactions of this kind should prove useful in the introduction of new recognition epitopes, such as peptides, oligosaccharides, or small organic molecules, onto cell surfaces and in the subsequent modulation of cell-cell or cell-small molecule binding events. The versatility of this technology was demonstrated by an example of selective drug delivery. Cells were decorated with biotin through selective conjugation to ketone groups, and selectively killed in the presence of a ricin A chain-avidin conjugate.