Analysis of the relationship between cleavability of a paramyxovirus fusion protein and length of the connecting peptide.

The relationship between the length of the connecting peptide in a paramyxovirus F0 protein and cleavage of F0 into the F1 and F2 subunits has been examined by constructing a series of mutant F proteins via site-directed mutagenesis of a cDNA clone encoding the simian virus 5 F protein. The mutant F proteins had one to five arginine residues deleted from the connecting peptide. The minimum number of arginine residues required for cleavage-activation of the simian virus 5 F0 protein by host cell proteases was found to be four. F proteins with two or three arginine residues in the connecting peptide were not cleaved by host cell proteases but could be cleaved by exogenously added trypsin. The mutant F protein possessing a connecting peptide consisting of one arginine residue was not cleaved by trypsin. The altered F proteins were all transported to the infected-cell plasma membrane as shown by cell surface immunofluorescence or cell surface trypsinization. However, the only mutant F protein found to be biologically active as detected by syncytium formation was the F protein which has four arginine residues at the cleavage site. The results presented here suggest that in the paramyxovirus F protein the number of basic amino acid residues in the connecting peptide is important for cleavage of the precursor protein by host cell proteases but is not the only structural feature involved. In addition, the data indicate that cleavage of F0 into F1 and F2 does not necessarily result in biological activity and that the connecting peptide may affect the local conformation of the F polypeptide.     

Structural features influencing hemagglutinin cleavability in a human influenza A virus.

The cleavability of the hemagglutinin (HA) molecule is related to the virulence of avian influenza A viruses, but its influence on human influenza virus strains is unknown. Two structural features are involved in the cleavage of avian influenza A virus HAs: a series of basic amino acids at the cleavage site and an oligosaccharide side chain in the near vicinity. The importance of these properties in the cleavability of a human influenza A virus (A/Aichi/2/68) HA was investigated by using mutants that contained or lacked an oligosaccharide side chain and had either four or six basic amino acids. All mutants except the one that contains a single mutation at the glycosylation site were cleaved, although not completely, demonstrating that a series of basic amino acids confers susceptibility to cellular cleavage enzymes among human influenza virus HAs. The mutants containing six basic amino acids at the cleavage site showed limited polykaryon formation upon exposure to low pH, indicating that cleavage was adequate to impart fusion activity to the HA. Deletion of the potential glycosylation site had no effect on the cleavability of these mutants; hence, the oligosaccharide side chain appears to have no role in human influenza virus HA cleavage. The inability to induce high cleavability in a human influenza A virus HA by insertion of a series of basic amino acids at the cleavage site indicates that other, as yet unidentified structural features are needed to enhance the susceptibility of these HAs to cellular proteases.     

Biologic importance of neuraminidase stalk length in influenza A virus.

To investigate the biologic importance of the neuraminidase (NA) stalk of influenza A virus, we generated mutant viruses of A/WSN/33 (H1N1) with stalks of various lengths (0 to 52 amino acids), by using the recently developed reverse genetics system. These mutant viruses, including one that lacked the entire stalk, replicated in tissue culture to the level of the parent virus, whose NA stalk contains 24 amino acid residues. In eggs, however, the length of the stalk was correlated with the efficiency of virus replication: the longer the stalk, the better the replication. This finding indicates that the length of the NA stalk affects the host range of influenza A viruses. The NA stalkless mutant was highly attenuated in mice; none of the animals died even after intranasal inoculation of 10(6) PFU of the virus (the dose of the parent virus required to kill 50% of mice was 10(2.5) PFU). Moreover, the stalkless mutant replicated only in the respiratory organs, whereas the parent virus caused systemic infection in mice. Thus, attenuation of the virus with the deletion of the entire NA stalk raises the possibility of its use as live vaccines.     

Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus.

To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses.     

Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety.

The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.     

Removal of carbohydrate from influenza A virus and its hemagglutinin and the effect on biological activities.

Treatment of influenza virus and its purified hemagglutining with glycosidases from Diplococcus pneumoniae, which included beta-galactosidase, beta-N-acetylglucosminidase, and endoglycosidase D, released amino and neutral sugars from the virus and these as well as large oligosaccharides from the purified hemagglutinin. The released glucosamine-containing oligosaccharides were of one discrete size. Large oligosaccharides not removed by the glycosidases were found on the virus as well as the hemagglutinin. Some oligosaccharides on the virus were inaccessible to the enzymes, since they could be removed only from the purified hemagglutinin. Approximately 50% of the hemagglutinin carbohydrates could be removed without effect on hemagglutinating activity. Similarly, removal of 20 to 25% of the carbohydrates from intact virus particles did not alter infectivity.     

Interplay between influenza A virus and the innate immune signaling

Pathogens such as influenza A viruses (IAV) have to overcome a number of barriers defined and maintained by the host, to successfully establish an infection. One of the initial barriers is collectively characterized as the innate immune system. This is a broad anti-pathogen defense program that ranges from the action of natural killer cells to the induction of an antiviral cytokine response. In this article we will focus on new developments and discoveries concerning the interaction of IAV with the cellular innate immune signaling. We discuss new mechanisms of interference of IAV with the pathogen recognition receptor RIG-I and the type I IFN antagonist NS1 in the background of already known and established concepts. Further we summarize progress related to recently identified IFN induced proteins and the role of RNA interference in the context of IAV infection.     

Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin.

Influenza virus hemagglutinin (HA) fuses membranes at endosomal pH by a process which involves extrusion of the NH2-terminal region of HA2, the fusion peptide, from its buried location in the native trimer. We have examined the amino acid sequence requirements for a functional fusion peptide by determining the fusion capacities of site-specific mutant HAs expressed by using vaccinia virus recombinants and of synthetic peptide analogs of the mutant fusion peptides. The results indicate that for efficient fusion, alanine can to some extent substitute for the NH2-terminal glycine of the wild-type fusion peptide but that serine, histidine, leucine, isoleucine, or phenylalanine cannot. In addition, mutants containing shorter fusion peptides as a result of single amino acid deletions are inactive, as is a mutant containing an alanine instead of a glycine at HA2 residue 8. Substitution of the glycine at HA2 residue 4 with an alanine increases the pH of fusion, and valine-for-glutamate substitutions at HA2 residues 11 and 15 are without effect. We confirm previous reports on the need for specific HAo cleavage to generate functional HAs, and we show that both inappropriately cleaved HA and mutant HAs, irrespective of their fusion capacities, upon incubation at low pH undergo the structural transition required for fusion.     

Three-dimensional structure determines the pattern of CD4+ T-cell epitope dominance in influenza virus hemagglutinin

The structural context of a CD4⁺ T-cell epitope is known to influence immunodominance at the level of antigen processing, but general rules have not emerged. Dominant epitopes of influenza virus hemagglutinin are found to be localized to the C-terminal flanks of conformationally stable segments identified by low crystallographic B-factors or high COREX residue stabilities. The bias toward C-terminal flanks is distinctive for antigens from the influenza virus. Dominant epitopes in antigens/allergens from other sources also localize to the flanks of stable segments but are found on either N- or C-terminal flanks. Thus, dominance arises from preferential endoproteolytic nicking between stable segments followed by loading of fragment terminal regions into antigen-presenting proteins. This mechanism probably arose in order to direct CD4⁺ responses onto sequences that are conserved for structure and function. Structure-guided presentation could enhance protection against genetically drifting influenza virus variants but most likely reduces protection against new viral subtypes.     

Purification of the influenza hemagglutinin glycoprotein and characterization of its carbohydrate components.

Hemagglutinin from influenza A/PR8 virus was purified after treatment of the virus with sodium deoxycholate followed by extraction with tri-n-butyl phosphate. This fully disrupted the virus while preserving hemagglutinating activity. The hemagglutinin was obtained in the form of small aggregates that could be separated from other viral components. Purified hemagglutinin was hydrolyzed to determine carbohydrate composition and digested with Pronase to analyze oligosaccharide structures. Sugars present in the hemagglutinin were galactose, mannose, fucose, and glucosamine in molar rates of about 6:11:2:5, and these comprised 16% of the hemagglutinin glycoprotein. Oligosaccharides obtained from virus included a major component of a molecular weight of 2,800, composed of glucosamine, galactose, mannose, and fucose, and a minor heterogenous component of a molecular weight of 1,500 to 2,000, containing predominantly mannose. The 2,800-molecular-weight oligosaccharide was a constituent of the hemagglutinin, and treatment of this large oligosaccharide with specific exo-glycosidases demonstrated the presence of terminal galactose and fucose and allowed the deduction of a general structure for this component.     

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.     

New water-soluble analog of the fusion peptide of influenza virus hemagglutinin: synthesis and properties

A water-soluble analogue F32 of the fusion peptide from the influenza virus hemagglutinin was synthesized. It consisted of 32 aa residues and retained the ability to interact with lipid membranes; its N-terminal sequence 1-24 coincided with that of the fusion protein from hemagglutinin (strain A/PR/8/34), whereas residues 25-32 (GGGKKKKK) provided its solubility in water. The peptide induced the conductivity fluctuations in planar bilayer lipid membranes characteristic of active fusion peptides. Conditions were found using CD spectroscopy under which the structure of F32 inside detergent micelles, where it can be studied by high-resolution 1H NMR spectroscopy, is close to the structure of the peptide during its interaction with phospholipid liposomes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

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。     

Genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin.

The immune response to a synthetic peptide, H3 HA1(305-328), representing the C'-terminal 24 amino acid residues of the HA1 chain of the hemagglutinin of the H3 subtype of influenza virus is controlled by genes in the I region of the major histocompatibility complex. Mice of the H-2d haplotype are high responders and produce antibody for several months after a single injection of peptide without carrier. Mice of the H-2b, H-2k, and H-2q haplotypes are low antibody responders. Investigation of recombinant and congenic mouse strains revealed that high responsiveness requires the genes that encode the I-Ed molecule. Immunoassays, involving direct binding to analogs of this peptide and inhibition by both these analogs and synthetic epitopes, were used to analyze the specificity of the polyclonal response. In BALB/c mice, the primary antibody response is directed principally against the antigenic site 314-LKLAT-318, whereas the secondary response after a boost is predominantly directed to a distinct site, 320-MRNVPEKQT-328. The T-cell response to the peptide H3 HA1(305-328), as measured by antigen-induced proliferation of primed T cells in vitro, is also I-Ed restricted in high-responder H-2d mice and is directed against an antigenic site that does not require the four C-terminal residues unique to the H3 influenza subtype. A different epitope appears to be recognized by T cells from CBA (H-2k) mice, which proliferate to a moderate extent on exposure to the peptide but, nevertheless, do not provide help for an antibody response.     

Testing topological models for the membrane penetration of the fusion peptide of influenza virus hemagglutinin

Low pH-induced binding of the bromelain-solubilized form of influenza virus hemagglutinin (BHA) to membranes occurs through the fusion peptide. From asymmetric hydrophobic photolabeling of membranes, evidence was obtained that this peptide penetrates only one leaflet of the bilayer. The asymmetrical labeling was achieved by employing a photoreactive analogue of a fatty acid whose transbilayer distribution can be manipulated by a membrane proton gradient.     

Morphological changes and fusogenic activity of influenza virus hemagglutinin.

The kinetics of low-pH induced fusion of influenza virus with liposomes have been compared to changes in the morphology of influenza hemagglutinin (HA). At pH 4.9 and 30 degrees C, the fusion of influenza A/PR/8/34 virus with ganglioside-bearing liposomes was complete within 6 min. Virus preincubated at pH 4.9 and 30 degrees C in the absence of liposomes for 2 or 10 min retained most of its fusion activity. However, fusion activity was dramatically reduced after 30 min, and virtually abolished after a 60-min preincubation. Cryo-electron microscopy showed that the hemagglutinin spikes of virions exposed to pH 4.9 at 30 degrees C for 10 min underwent no major morphological changes. After 30 min, however, the spike morphology changed dramatically, and further changes occurred for up to 60 min after exposure to low pH. Because the morphological changes occur at a rate corresponding to the loss of fusion activity, and because these changes are much slower than the rate at which fusion occurs, we conclude that the morphologically altered HA is inactive with respect to fusion-promoting activity. Molecular modeling studies indicate that the formation of an extended coiled coil within the HA trimer, as proposed for HA at low pH, requires a major conformational change in HA, and that the morphological changes we observe are consistent with the formation of an extended coiled coil. These results imply that the crystallographically determined low-pH form of HA does occur in the intact virus, but that this form is not a precursor of viral fusion. It is speculated that the motion to the low-pH form may be responsible for the membrane destabilization leading to fusion.     

Protonation and stability of the globular domain of influenza virus hemagglutinin.

A partial dissociation of the HA1 subunits of influenza virus hemagglutinin (HA) is considered to be the initial step of conformational changes of the HA ectodomain leading to a membrane fusion active conformation (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufman, E. Suchanek, C. Pabo, J.J. Skehel, D.C. Wiley, and S. Wharton, 1992, Cell 68:635-645; G.W. Kemble, D.L.Bodian, J. Rose, I.A. Wilson, and J.M. White, 1992, J. Virol. 66:4940-4950). Here, we explore a mechanism that provides an understanding of the physical and chemical basis for such dissociation and relies on two essential observations. First, based on the x-ray structure of HA from X31 (I.A. Wilson, J.J. Skehel, and D.C. Wiley, 1981, Nature 289:366-373), and by employing techniques of molecular modeling, we show that the protonation of the HA1 subunits is enhanced at the conditions known to trigger conformational changes of the HA ectodomain. Second, we found that the dependence of the calculated relative degree of protonation of the HA1 domain on temperature and pH is similar to that observed experimentally for the conformational change of HA assessed by proteinase K sensitivity. We suggest that at the pH-temperature conditions typical for the conformational change of HA and membrane fusion, dissociation of the HA1 subunits is caused by the enhanced protonation of the HA1 subunits leading to an increase in the positive net charge of these subunits and, in turn, to a weakened attraction between them.     

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.     

Minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin.

Synthetic peptides of increasing length and corresponding in sequence to the C-terminal end of the HA1 molecule of influenza virus were constructed and examined for their immunogenic and antigenic properties. Peptides containing at least the four C-terminal amino acids, when coupled to keyhole limpet hemocyanin, were capable of eliciting antibody in BALB/c mice that bound to the 24-residue parent peptide H3 HA1 (305 to 328). In the absence of a carrier, the C-terminal decapeptide was the shortest peptide capable of eliciting antibody. The specificity of this antibody was indistinguishable from that of a monoclonal antibody to the parent peptide which recognizes an epitope encompassed by the C-terminal seven residues. All peptides containing at least the C-terminal four residues were able to inhibit completely the binding of this monoclonal antibody to the parent peptide. Taken together, these results indicate that (i) the tetrapeptide is capable of eliciting specific antibody when coupled to a carrier, (ii) this tetrapeptide possesses all of the antigenic information necessary to occupy the paratope of a monoclonal antibody elicited by the longer parent peptide, and (iii) the decapeptide contains all of the information necessary to elicit a specific immune response and therefore carries an epitope recognized by T cells as well as one recognized by B cells.