Avian-type receptor-binding ability can increase influenza virus pathogenicity in macaques

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA-222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA-222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA-222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans.     

Structure and receptor-binding properties of an airborne transmissible avian influenza A virus hemagglutinin H5 (VN1203mut)

Avian influenza A virus continues to pose a global threat with occasional H5N1 human infections, which is emphasized by a recent severe human infection caused by avian-origin H7N9 in China. Luckily these viruses do not transmit efficiently in human populations. With a few amino acid substitutions of the hemagglutinin H5 protein in the laboratory, two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model. Here in this study one of the mutant H5 proteins developed by Kawaoka’s group (VN1203mut) was expressed in a baculovirus system and its receptor-binding properties were assessed. We herein show that the VN1203mut had a dramatically reduced binding affinity for the avian α2,3- linkage receptor compared to wild type but showed no detectable increase in affinity for the human α2,6-linkage receptor, using Surface Plasmon Resonance techonology. Further, the crystal structures of the VN1203mut and its complexes with either human or avian receptors demonstrate that the VN1203mut binds the human receptor in the same binding manner (cis conformation) as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses. Our receptor binding and crystallographic data shown here further confirm that the ability to bind the avian receptor has to decrease for a higher human receptor binding affinity. As the Q226L substitution is shown important for obtaining human receptor binding, we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.     

Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation.

The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to study the role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reverse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560 can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change from cysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail. This interpretation is further supported by experiments in which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted in infectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position 560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate a biological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, we cannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA--rather than altered palmitylation--are the determining factors for infectious-particle formation.     

Single-amino-acid substitution in an antigenic site of influenza virus hemagglutinin can alter the specificity of binding to cell membrane-associated gangliosides.

Antigenic variants of influenza virus A/Mem/1/71-Bel/42 (H3N1) selected with monoclonal antibodies and having single substitutions in their hemagglutinins were examined for their ability to hemagglutinate and hemolyse erythrocytes coated with different gangliosides. The majority of variants, including one with a substitution near the receptor-binding site (Asn-133----Lys), did not differ from the parent in specificity for receptor molecules. However, a substitution in HA1 at residue 205 (Ser----Tyr), which is distant from the receptor-binding site in antigenic site D, affected hemagglutination and hemolysis of erythrocytes coated with sialyl-paraglobosides. The variant preferentially recognized N-acetylneuraminic acid-alpha 2,6-galactose linkages to sialylparaglobosides, whereas the parent and other variants preferentially recognized N-acetylneuraminic acid-alpha 2,3-galactose linkages. In the trimeric hemagglutinin molecule, residue 205 is located across the subunit interface from the receptor-binding site. The bulky hydrophobic tyrosine in the variant may cause a conformational change in the receptor-binding pocket on the neighboring subunit and influence receptor binding.     

Oligosaccharide composition of an influenza virus hemagglutinin with host-determined binding properties

We have previously reported that the binding properties of the hemagglutinin (HA) of the WSN-F strain of influenza A are affected by the cells in which the virus is grown (Crecelius, D. M., Deom, C. M., and Schulze, I.T. (1984) Virology 139, 164-177); at 37 degrees C chick embryo fibroblast-grown F virus has a greater affinity for host cells than does the same virus grown in Madin-Darby bovine kidney (MDBK) cells. In an attempt to explain this host-determined property, we have characterized the carbohydrate put onto the viral HA by these two cells. Experiments using tunicamycin indicate that the HA made by MDBK cells contains about 4000 daltons of carbohydrate in excess of that on the HA from chick embryo fibroblast. Serial lectin affinity chromatography of the asparagine-linked oligosaccharides on the HA subunits, HA1 and HA2, detected a number of host-dependent differences in the complex oligosaccharides. Both HA1 and HA2 from MDBK cells contained more highly branched (i.e. tri- and tetraantennary) complex oligosaccharides than did the subunits from chick embryo fibroblasts. In addition, the HA subunits from the two sources differed in the amount of galactose-containing "bisected" complex oligosaccharides and in the presence of certain fucosylated triantennary oligosaccharides. Profiles of the asparagine-linked oligosaccharides from the host cells did not show these differences, indicating that the HA subunit profiles were not necessarily representative of the structures found on the cellular glycoproteins. The data support the conclusion that bulky oligosaccharides on the MDBK-HA subunits of WSN-F reduce the affinity of the virus for cellular receptors.     

Reversible stages of the low-pH-triggered conformational change in influenza virus hemagglutinin

The refolding of the prototypic fusogenic protein hemagglutinin (HA) at the pH of fusion is considered to be a concerted and irreversible discharge of a loaded spring, with no distinct intermediates between the initial and final conformations. Here, we show that HA refolding involves reversible conformations with a lifetime of minutes. After reneutralization, low pH-activated HA returns from the conformations wherein both the fusion peptide and the kinked loop of the HA2 subunit are exposed, but the HA1 subunits have not yet dissociated, to a structure indistinguishable from the initial one in functional, biochemical and immunological characteristics. The rate of the transition from reversible conformations to irreversible refolding depends on the pH and on the presence of target membrane. Importantly, recovery of the initial conformation is blocked by the interactions between adjacent HA trimers. The existence of the identified reversible stage of refolding can be crucial for allowing multiple copies of HA to synchronize their release of conformational energy, as required for fusion.     

New insights into the spring-loaded conformational change of influenza virus hemagglutinin

Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix "spring-loaded" conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only approximately 50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.     

Infectivity studies of influenza virus hemagglutinin receptor binding site mutants in mice

The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.     

Electrostatic energy calculation on the pH-induced conformational change of influenza virus hemagglutinin

The pH-induced conformational change of influenza virus hemagglutinin (HA) has been investigated by calculating the change of electrostatic energy of the fragment of HA2 upon pH change. The average charge and electrostatic free energy are calculated as a function of pH for the fusion peptide (residues 1-20 of HA2) and the polypeptide of residues 54-77 of HA2 by using the finite difference Poisson-Boltzmann method. It is found that as pH decreases from 8 to 5, the electrostatic free energy of the fusogenic state is lowered by approximately 2 kcal/mol and the fusogenic state is less ionized compared to that of the native state for both polypeptides. For the fusion peptide at the fusogenic state, most of ionizable residues are neutral at acidic pH except Glu-11. For the polypeptide of residues 54-77 at the fusogenic state, most of residues except Glu-74 and His-64 are fully charged between pH 5 and pH 8.     

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.     

Conformational change of influenza virus hemagglutinin is sensitive to ionic concentration

The homotrimeric spike glycoprotein hemagglutinin (HA) of influenza virus undergoes a low pH-mediated conformational change which mediates the fusion of the viral envelope with the target membrane. Previous approaches predict that the interplay of electrostatic interactions between and within HA subunits, HA 1 and HA2, are essential for the metastability of the HA ectodomain. Here, we show that suspension media of low ionic concentration promote fusion of fluorescent labelled influenza virus X31 with erythrocyte ghosts and with ganglioside containing liposomes. By measuring the low pH mediated inactivation of the fusion competence of HA and the Proteinase K sensitivity of low pH incubated HA we show that the conformational change is promoted by low ionic concentration. We surmise that electrostatic attraction within the HA ectodomain is weakened by lowering the ionic concentration facilitating the conformational change at low pH. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Heptapeptide ligands against receptor-binding sites of influenza hemagglutinin toward anti-influenza therapy

The initial attachment of influenza virus to cells is the binding of hemagglutinin (HA) to the sialyloligosaccharide receptor; therefore, the small molecules that inhibit the sugar–protein interaction are promising as HA inhibitors to prevent the infection. We herein demonstrate that sialic acid-mimic heptapeptides are identified through a selection from a primary library against influenza virus HA. In order to obtain lead peptides, an affinity selection from a phage-displayed random heptapeptide library was performed with the HAs of the H1 and H3 strains, and two kinds of the HA-binding peptides were identified. The binding of the peptides to HAs was inhibited in the presence of sialic acid, and plaque assays indicated that the corresponding N-stearoyl peptide strongly inhibited infections by the A/Aichi/2/68 (H3N2) strain of the virus. Alanine scanning of the peptides indicated that arginine and proline were responsible for binding. The affinities of several mutant peptides with single-amino-acid substitutions against H3 HA were determined, and corresponding docking studies were performed. A Spearman analysis revealed a correlation between the affinity of the peptides and the docking study. These results provide a practicable method to design of peptide-based HA inhibitors that are promising as anti-influenza drugs.     

Architecture of the influenza hemagglutinin membrane fusion site

The mechanism of influenza hemagglutinin (HA) mediated membrane fusion has been intensively studied for over 20 years after the bromelain-released ectodomain of HA at neutral pH was first crystallized. Nearly 10 years ago, the low-pH-induced "spring coiled" conformational change of HA was predicted from peptide chemistry and confirmed by crystallography. Other work has yielded a wealth of knowledge on the observed changes in HA fusion/hemifusion phenotypes as a function of site-specific mutations of HA, or added amphipathic molecules or particular IgGs. It is becoming clear that the conformational changes predicted by the crystallography are necessary to cause fusion and that interfering with these changes can block fusion or reduce it to hemifusion. What is not known is how the conformational changes cause fusion. In particular, while it is generally agreed that fusion requires an aggregate of HAs, how the aggregate may act to transduce the energy of the HA conformational changes to creating the initial fusion defect is not known. We have used a comprehensive mass action kinetic model of HA-mediated fusion to carry out a "meta-analysis" of several key data sets, using HA-expressing cells and using virions. The consensus result of these detailed kinetic studies was that the fusion site of influenza hemagglutinin (HA) is an aggregate with at least eight HAs. The high-energy conformational change of only two of these HAs within the aggregate permits the formation of the first fusion pore. This "8 and 2" result was required to best fit all the data. We review these studies and how this kinetic result can guide and constrain HA fusion models. The kinetic analysis suggests that the sequence of fusion intermediates starts with protein control and ends with lipid control, which makes sense. While curvature intermediates, e.g. the lipid stalk, are almost certainly within the fusion sequence, the "8 and 2" result does not suggest that they are the first step after HA aggregation. The stabilized hydrophobic defect model we have proposed as a precursor to the lipid stalk can form and is consistent with the "8 and 2" result.     

Architecture of the influenza hemagglutinin membrane fusion site

The mechanism of influenza hemagglutinin (HA) mediated membrane fusion has been intensively studied for over 20 years after the bromelain-released ectodomain of HA at neutral pH was first crystallized. Nearly 10 years ago, the low-pH-induced “spring coiled” conformational change of HA was predicted from peptide chemistry and confirmed by crystallography. Other work has yielded a wealth of knowledge on the observed changes in HA fusion/hemifusion phenotypes as a function of site-specific mutations of HA, or added amphipathic molecules or particular IgGs. It is becoming clear that the conformational changes predicted by the crystallography are necessary to cause fusion and that interfering with these changes can block fusion or reduce it to hemifusion. What is not known is how the conformational changes cause fusion. In particular, while it is generally agreed that fusion requires an aggregate of HAs, how the aggregate may act to transduce the energy of the HA conformational changes to creating the initial fusion defect is not known. We have used a comprehensive mass action kinetic model of HA-mediated fusion to carry out a “meta-analysis” of several key data sets, using HA-expressing cells and using virions. The consensus result of these detailed kinetic studies was that the fusion site of influenza hemagglutinin (HA) is an aggregate with at least eight HAs. The high-energy conformational change of only two of these HAs within the aggregate permits the formation of the first fusion pore. This “8 and 2” result was required to best fit all the data. We review these studies and how this kinetic result can guide and constrain HA fusion models. The kinetic analysis suggests that the sequence of fusion intermediates starts with protein control and ends with lipid control, which makes sense. While curvature intermediates, e.g. the lipid stalk, are almost certainly within the fusion sequence, the “8 and 2” result does not suggest that they are the first step after HA aggregation. The stabilized hydrophobic defect model we have proposed as a precursor to the lipid stalk can form and is consistent with the “8 and 2” result.     

The receptor-binding domain of influenza virus hemagglutinin produced in Escherichia coli folds into its native, immunogenic structure

The hemagglutinin (HA) surface glycoprotein promotes influenza virus entry and is the key protective antigen in natural immunity and vaccines. The HA protein is a trimeric envelope glycoprotein consisting of a globular receptor-binding domain (HA-RBD) that is inserted into a membrane fusion-mediating stalk domain. Similar to other class I viral fusion proteins, the fusogenic stalk domain spontaneously refolds into its postfusion conformation when expressed in isolation, consistent with this domain being trapped in a metastable conformation. Using X-ray crystallography, we show that the influenza virus HA-RBD refolds spontaneously into its native, immunogenic structure even when expressed in an unglycosylated form in Escherichia coli. In the 2.10-Å structure of the HA-RBD, the receptor-binding pocket is intact and its conformational epitopes are preserved. Recombinant HA-RBD is immunogenic and protective in ferrets, and the protein also binds with specificity to sera from influenza virus-infected humans. Overall, the data provide a structural basis for the rapid production of influenza vaccines in E. coli. From an evolutionary standpoint, the ability of the HA-RBD to refold spontaneously into its native conformation suggests that influenza virus acquired this domain as an insertion into an ancestral membrane-fusion domain. The insertion of independently folding domains into fusogenic stalk domains may be a common feature of class I viral fusion proteins.The genetic drift of seasonal influenza viruses and the occasional emergence of pandemic strains represent a continuing and serious burden on human health. Pandemic influenza viruses arise at irregular intervals, can infect up to 50% or more of the population, and vary in disease severity. Most notably, the H1N1 Spanish influenza pandemic of 1918 killed an estimated 20 to 50 million people worldwide, and the 1957 H2N2 Asian flu and 1968 H3N2 Hong Kong flu pandemics killed between 0.5 and 1 million people in the United States alone (30). The ongoing danger of influenza was recently emphasized by the emergence of the novel H1N1 pandemic virus from Mexico in April of 2009. The urgent need to speed up vaccine production was highlighted by this outbreak because over 340,000 confirmed cases and 4,100 deaths had occurred worldwide during the 6 months that were necessary to produce a vaccine using current procedures (39).As the major surface antigen of influenza A viruses, the hemagglutinin (HA) envelope glycoprotein is the primary source of natural immunity and the key target in vaccination. However, changes in the antigenic sites of the HA protein due to antigenic drift result in lost or diminished immunity acquired from previous infection or vaccination (35). This necessitates the production of new vaccines against seasonal influenza viruses each year. The HA protein also plays a central role in the emergence of human pandemic influenza viruses. There are 16 known antigenic subtypes of HA proteins in influenza A viruses (H1 through H16), and a pandemic occurs when an influenza virus that has an HA protein to which most of the population lacks immunity acquires the ability to be efficiently transmitted from person to person.The HA protein has multiple roles in the virus life cycle, notably receptor binding and membrane fusion. The protein is synthesized as a single precursor protein, HA₀, that trimerizes and becomes glycosylated in the endoplasmic reticulum as it traffics to the cell surface (33). The HA protein contains multiple disulfide bonds and is cleaved into a mature form consisting of two subunits, HA₁ and HA₂ (9, 18). HA₂ and the N- and C-terminal portions of HA₁ form a membrane-proximal stalk that mediates membrane fusion during viral entry (40). A receptor-binding domain (HA-RBD) forms the distal head of the molecule and is inserted into the HA₁ subunit. During virus entry, the HA-RBD engages sialic acid-containing receptors on the surface of the host cell, and the virion is subsequently internalized by endocytosis (33). Structurally and functionally, the HA-RBD is a member of the lectin superfamily, and the specificity of the binding pocket contributes to the host range of influenza viruses. For example, α(2,6)-containing sialosides are typically preferred by the HA protein from human viruses and α(2,3) sialosides by the HA proteins from avian viruses (13, 28). Upon triggering by the low-pH environment of endosomes, the HA protein undergoes an irreversible conformational change (6, 40) during which the intact HA-RBDs dissociate from the stalk of the trimer (3, 14, 19, 21). This observation, together with the manner in which the lectin-like domain is inserted as a folded module into the full-length HA protein, led us to hypothesize that the HA-RBD is able to adopt its native structure in isolation. Proper folding of the isolated HA-RBD into its native immunogenic structure has important therapeutic implications because the domain contains all of the known HA antigenic epitopes responsible for antibody recognition (5), and producing a protein-based influenza vaccine composed of isolated HA-RBD would dramatically speed up vaccine development during the early stages of a pandemic.In a recently published report, a construct of the 2009 pandemic H1N1 HA protein that encompasses the HA-RBD, designated HA_63-286-RBD, was expressed in Escherichia coli as inclusion bodies, refolded and purified, and used as a vaccine to produce immunity in ferrets (2). In this report, we show that this construct behaves as a stable, structured protein in solution, can be readily crystallized, and indeed adopts a structure that is virtually indistinguishable from that in the H1N1 HA protein ectodomain (41).     

Immunogenic structure of the influenza virus hemagglutinin

We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenic determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of its pieces.     

Receptor properties of the hemagglutinin of influenza virus and its polypeptide fragments

The receptor properties of influenza virus A/Kiev/59/79 R (H1N1) and a number of its polypeptide fragments containing the aminoacids (from the 1st to 272d) of the heavy chain were studied. Two kinds of radioimmunoassay were used to test hemagglutinin or its polypeptide fragment interactions with cellular receptors. The studied polypeptides and hemagglutinin are shown to be capable of specific interactions with the receptors on the cell surface. The main linear fragment of hemagglutinin recognizing cellular receptors is localized within a polypeptide fragment including 1st-272d aminoacids of the heavy chain of hemagglutinin. The breaks of all the the S-S linkages including the ones linearly and spatially close to the receptor "pocket" of the bridge 95-135 do not affect significantly the receptor properties of the polypeptide.     

流感病毒血凝素蛋白裂解机制的研究进展

血凝素(Hemagglutinin,HA)是流感病毒的主要表面抗原之一,诱导机体产生中和抗体,介导病毒囊膜与靶细胞膜融合,从而启动病毒对宿主细胞的感染过程。HA蛋白以前体形式合成,需经宿主蛋白酶水解为HA1、HA2两个亚单位,并以二硫键连接,病毒才获得感染性。研究表明宿主蛋白酶的分布与流感病毒感染后的致病力和组织嗜性有直接关系。潜在的裂解酶及其抑制因子的发现为流感的防治提供了新的思路,成为干预治疗的新潜在靶点。就当前国内外关于流感病毒血凝素的结构与功能、裂解机制及其应用的研究进展进行综述。     

Role of receptor-binding activity of the viral hemagglutinin molecule in the presentation of influenza virus antigens to helper T cells.

The concentration of antigen required to stimulate influenza virus-specific helper T cells was observed to be dependent upon the antigenic form bearing the relevant determinant: intact, nonreplicative virus was needed only in picomolar amounts, while denatured proteins, protein fragments, or synthetic peptides were required in micromolar concentrations for a threshold level of stimulation. Antigenic efficiency of intact virus was found to result from the attachment of virus to sialic acid residues on the surface of the antigen-presenting cell since spikeless viral particles lacking the hemagglutinin molecule were much less efficient antigens for helper T cells and continuous presence of hemagglutination-inhibiting antihemagglutinin antibodies reduced efficiency of stimulation by intact virus approximately 100-fold for both hemagglutinin and internal virion proteins. Influenza virus associated rapidly with antigen-presenting cells; less than 10 min at 20 degrees C was sufficient to introduce virus for a maximal level of T-cell stimulation. This rapid attachment was blocked by antibodies to the hemagglutinin or by pretreatment of the antigen-presenting cells with neuraminidase to remove the cellular virus receptor. Following viral adsorption by antigen-presenting cells, a lag period of 30 min at 37 degrees C was required for the expression of helper T-cell determinants. One early event identified was the movement of the virus to a neuraminidase-insensitive compartment, which can occur at 10 degrees C, but which was not equivalent to expression of helper T-cell determinants. Preincubation of cells with virus at 10 degrees C for 4 h reduced the lag period of helper T-cell determinant expression to 15 min when these cells were shifted to 37 degrees C, suggesting that transition of the virus to a neuraminidase-resistant state is a required step in presentation of T-cell antigenic determinants.