Synthetic peptide immunogens eliciting antibodies to Plasmodium falciparum sporozoite and merozoite surface antigens in H-2b and H-2k mice

Peptides representing conserved (MSA2/1A and MSA2/1B) and variant (MSA2/2, MSA2/6 and MSA2/7) regions of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum (FCQ-27/PNG isolate) were coupled to either peptide NP(NANP)5NA or peptide C(NANP)6 both of which contained the core sequence (NANP)n. The coupling was done via the N-terminus of one peptide and a cysteine residue on either terminus of the other. BL/10 (H-2b) and B10.BR (H-2k) mice were immunized with these MSA2-(NANP)n conjugates. The mice were also immunized with the unconjugated MSA2 peptides and with NP(NANP)5NA and C(NANP)6. Antibody responses were evaluated by 1) ELISA, in which the MSA2 peptides and C(NANP)6 were used as Ag; 2) immunofluorescence assays (IFAT) against intact sporozoites and merozoites; and 3) immunoblotting experiments against solubilized P. falciparum blood stage proteins. High titer antibodies to (NANP)n were elicited in both BL/10 and B10.BR mice after immunization with all the conjugates except MSA2/7-(NANP)n which gave only a very limited response in B10.BR mice. These antibodies recognized unfixed sporozoites. The conjugates also elicited antibodies to MSA2 as shown by ELISA, IFAT, and immunoblotting except for mice immunized with MSA2/1B-(NANP)n where an anti-MSA2 response was only detectable by immunoblotting. Immunization with unconjugated MSA2 peptides showed that MSA2/2 was immunogenic in both BL/10 and BR.10 mice, with MSA2/6 and MSA2/7 being immunogenic only in BL/10 mice. The antibodies elicited recognized both merozoites and the MSA2 protein. However, the antibody titers were lower overall than those seen when these peptides were used in the conjugated form. No anti-MSA2 antibodies were detected after immunization with MSA2/1A and MSA2/1B. Immunization of mice with the peptide NP(NANP)5NA produced antibodies in BL/10 (H-2b) mice only, and the immunogenicity of this preparation was poor. In contrast, C(NANP)6 produced a strong antibody response in both mouse strains. The antibodies elicited by NP(NANP)5NA and C(NANP)6 recognised sporozoites in IFAT. The MSA2 peptides studied (or their derivatives) were previously shown to be recognized by human T cells. Their immunogenic potential shows promise in that complex anti-P. falciparum responses can be elicited with simple synthetic immunogens based on these peptides.     

The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines

The immunogenicity of a novel synthetic peptide consisting of an average of 40 (Asn-Ala-Asn-Pro) repeats of the circumsporozoite protein of Plasmodium falciparum, (NANP)40, was studied in mice without using any carrier proteins. First, high titers of anti-(NANP)40 antibodies could be obtained after immunization of C57BL/6 mice. These antibodies also reacted with an extract of mosquitoes infected with P. falciparum sporozoites. C57BL/6 nu/nu mice did not produce antibodies against (NANP)40. Secondly, when 14 strains of mice with nine different H-2 haplotypes were immunized with (NANP)40 without carrier, only H-2b mice were found to produce anti-(NANP)40 antibodies, whereas all non-H-2b mice were consistently unresponsive. This response was demonstrated to be I-A-linked by using recombinant and mutant mice. I-Ab [B10.A(5R)] mice produced anti-(NANP)40 antibodies as well as H-2b inbred mice. B6CH-2bm12 I-Ab-mutant mice showed only a very low response. Third, the antibody response against (NANP)40 could be induced in nonresponder mice by immunization with the peptide coupled to a carrier protein. In view of the existence of such an exceptional H-2b restriction in the response to sporozoite synthetic peptides in mice, the triggering of peptide-specific T cell responses in humans receiving sporozoite malaria vaccines might be difficult to achieve.     

Surface properties of membrane systems. Transport of staphylococcal delta-toxin from aqueous to membrane phase

Hemolytic delta-toxin from Staphylococcus aureus was soluble in either water, methanol or chloroform/methanol (2 : 1, v/v). The toxin spread readily from distilled water into films with pressures (pi) of 10 dynes/cm on water and 30 dynes/cm on 6 M urea; from chloroform/methanol it produced 40 dynes/cm pressure on distilled water. The toxin adsorbed barely from water (pi = 1 dyne/ cm) but it did rapidly from 6 M urea (pi = 35 dynes/cm). The protein films had unusually high surface potentials, which increased with the film pressure and decreased with increasing both pH and urea concentration in the aqueous phase. The fluorescence of 1-aniline 8-naphthalene sulfonate with delta-toxin was much greater than that with RNAase and dipalmitoyl phosphatidylcholine itself, indicating probably a marked lipid-binding character of the toxin. By circular dichroism the alpha-helix content of delta-toxin was 42% in water, 45% in methanol, 24% in 6 M urea. Infrared spectroscopy showed predominant alpha-helix in both 2H2O and deuterated chloroform/methanol as well as in films spread from either solvent on 2H2O. In spreading from 6 M [2H]urea, in which the major infrared absorption was that of [2H]urea with peaks at 1600 and 1480 cm(-1), the delta-toxin film showed prevalently non-alpha-helix structures with major peak intensities at 1633 cm(-1) > 1680 cm(-1), indicating the appearance of new beta-aggregated and beta-antiparallel pleated sheet structures in the film. The data prove that (1) high pressure protein films can consist of alpha-helix as well as non-alpha-helix structures and, differently from another cytolytic protein, melittin, delta-toxin does not resume the alpha-helix conformation in going into the film phase from the extended chain in 6 M urea; (2) conformational changes are important in the transport of proteins from aqueous to lipid or membrane phase; (3) delta-toxin is by far more versatile in structural dynamics and more surface active than alpha-toxin.     

流感病毒H1N1血凝素基因和神经氨酸酶基因在酵母菌中的表达

在成功克隆流感病毒H1N1全长HA(Hemagglulinin,HA)、NA(Neuramidinase,NA) 基因并测序的基础上,将部分基因序列克隆到表达载体pMETA上,构建了重组表达质粒pMETA/HA(52~1 557 bp)、pMETA/NA(121~1 263 bp),电转化真核酵母菌pMAD16,甲醇诱导表达,利用Ni2+亲和层析柱对重组蛋白进行纯化,并用Western blotting和ELISA方法检测其抗原性。SDS-PAGE显示重组蛋白在酵母菌中可以高效表达,蛋白纯度占总蛋白的95％以上,ELISA和Western blotting实验证实,重组蛋白具有良好的抗原性。成功克隆和表达了流感病毒H1N1 HA、NA基因序列,为流感病毒H1N1诊断试剂和疫苗的开发等进一步的研究提供了参考。     

Structure of a malaria parasite antigenic determinant displayed on filamentous bacteriophage determined by NMR spectroscopy: implications for the structure of continuous peptide epitopes of proteins

The NANP repeating sequence of the circumsporozoite protein of Plasmodium falciparum was displayed on the surface of fd filamentous bacteriophage as a 12-residue insert (NANP)(3) in the N-terminal region of the major coat protein (pVIII). The structure of the epitope determined by multidimensional solution NMR spectroscopy of the modified pVIII protein in lipid micelles was shown to be a twofold repeat of an extended and non-hydrogen-bonded loop based on the sequence NPNA, demonstrating that the repeating sequence is NPNA, not NANP. Further, high resolution solid-state NMR spectra of intact hybrid virions containing the modified pVIII proteins demonstrate that the peptides displayed on the surface of the virion adopt a single, stable conformation; this is consistent with their pronounced immunogenicity as well as their ability to mimic the antigenicity of their native parent proteins.     

Construction of immunogens for synthetic malaria vaccines

The immunogenicity of a peptide consisting of eight repeats of the tetrapeptide sequence NANP (Asn-Ala-Asn-Pro) contained in the circumsporozoite protein of Plasmodium falciparum was investigated in mice under different modes of presentation. This peptide was able to produce biologically active antibodies when administered with adjuvant and linked to a protein carrier. However, a (NANP) peptide polymerized by carbodiimide was found to be immunogenic in the absence of protein carrier in H-2b mice. In contrast, the (NANP)8 peptide polymerized by glutaraldehyde was not immunogenic in the same strain. Furthermore, the efficacy of murabutide in saline, as an immunological adjuvant, was compared to the efficacy of Freund's complete adjuvant.     

Use of synthetic carriers and adjuvants for increasing the immunogenicity of a synthetic peptide from the CS-protein of Plasmodium falciparum]

In order to increase immunogenicity of the peptide (NANP)3, we have prepared a large set of fully synthetic constructions based on the peptide, glycopeptide adjuvant GMDP and some synthetic carriers. Immunogenicity of these constructions was tested on mice (line C57B1/6) responding to the peptide polymer (NANP)40 without carrier and on mice (line BALB/c) not responding to this antigen. Immunogenic constructions based on synthetic polytuftsin induced as high titres of anti-(NANP)3 antibodies as the standard conjugate KLH--(NANP)3. The chimeric peptide consisting of (NANP)3 and tuftsin dimer induced anti-(NANP)3 antibodies in both lines of mice as well. The GMDP covalent attachment to the immunogenic constructions increased the antipeptide antibodies titre. The results are discussed in terms of an approach to synthetic vaccines.     

Glycan binding and specificity of viral influenza neuraminidases by classical molecular dynamics and replica exchange molecular dynamics simulations

Two important glycoproteins on the influenza virus membrane, hemagglutinin (HA) and neuraminidase (NA), are relevant to virus replication. As previously reported, HA has a substrate specificity towards SIA-2,3-GAL-1,4-NAG (3SL) and SIA-2,6-GAL-1,4-NAG (6SL) glycans, while NA can cleave both types of linkages. However, the substrate binding into NA and its preference are not well understood. In this work, the glycan binding and specificity of human and avian NAs were evaluated by classical molecular dynamics (MD) simulations, whilst the conformational diversity of 3SL avian and 6SL human glycans in an unbound state was investigated by replica exchange MD simulations. The results indicated that the 3SL avian receptor fits well in the binding cavity of all NAs and does not require a conformational change for such binding compared to the flexible shape of the 6SL human receptor. From the QM/MM-GBSA binding free energy and decomposition free energy data, 6SL showed a much stronger binding towards human NAs (H1N1, H2N2 and H3N2) than to avian NAs (H5N1 and H7N9). This suggests that influenza NAs have a substrate specificity corresponding to their HA, indicating the functional balance between the two important glycoproteins. Both linkages show distinct glycan topologies when complexed with NAs, while the flexibility of torsion angles between GAL and NAG in 6SL results in the various shapes of glycan and different binding patterns. Lower conformational diversities of both glycans when bound to NA compared to the unbound state were found, and were required in order to be accommodated within the NA cavity.

Communicated by Ramaswamy H. Sarma     

Display and release of the Plasmodium falciparum circumsporozoite protein using the autotransporter MisL of Salmonella enterica

The Salmonella enterica MisL (protein of membrane insertion and secretion) is an autotransporter with high homology to AIDA-I (adhesin involved in diffuse adherence) of enteropathogenic Escherichia coli. Considering that it has been reported that the MisL beta translocator domain is able to display heterologous passenger peptides to the bacterial surface, we developed a system to display proteins and release them to the external environment by means of proteolytic cleavage. Plasmids were constructed encoding 8 or 53 repeats of the NANP (Asp-Ala-Asp-Pro) tetrapeptide, which is the main B cell epitope of the Plasmodium falciparum circumsporozoitic protein (CSP), fused to the the MisL beta-domain and including the recognition cleavage sequence from the E. coli OmpT surface protease. E. coli XL-10Gold and BL21(DE3) (OmpT positive and negative, respectively) and Salmonella enterica serovar Typhimurium SL3261 (Aro A(-)) were transformed with the plasmids and, both expression and localization of the fusion proteins were assessed by Western blot, indirect immunofluorescence, and flow cytometry, using a monoclonal antibody against (NANP)(3). Higher expression of the (NANP)(8) and (NANP)(53) fusion proteins was demonstrated on the bacterial surface of the OmpT negative E. coli strains and the (NANP)(53) in the culture supernatant of E. coli XL-10Gold indicating a protease mediated cleavage. The flow cytometry analysis suggested 71 and 98% cleavage efficiency for the (NANP)(8) and (NANP)(53), respectively, in E. coli XL-10Gold. Similar results were obtained in S. enterica serovar Typhimurium SL3261, suggesting the involvement of other proteases related to OmpT. These results demonstrate that MisL may be used for the autodisplay and release of passenger proteins in attenuated Salmonella or E. coli strains, which may have several applications in vaccine design.     

Changing the N-terminal sequence protects recombinant <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> circumsporozoite protein from degradation in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Proteolytic degradation is the primary obstacle in the use of the yeast Pichia pastoris for the expression of recombinant proteins. During the production of a recombinant Plasmodium falciparum circumsporozoite protein in this system, the (NANP) _n repeats region at the N-terminus were completely proteolytically degraded. To remove the potential proteolytic site within the recombinant protein, different strategies were tried, including adjusting the cultivation conditions and mutating the sequence at the junction of the repeat domain and C-terminal region, but the degradation continued. However, modification of the N-terminal sequence by adding an epitope-based peptide to the N-terminus not only protected the repeat domain from cleavage by native proteases during longer induction in the yeast host and purification process, but also stabilized this recombinant protein emulsified with adjuvant ISA720 for at least 6 months. The results showed that proteolytic degradation of the recombinant circumsporozoite protein produced in P. pastoris was amino acid sequence (NANP)-specific, and that this effect was likely dependent on the conformation of the recombinant protein.     

Expression of an exogenous peptide epitope genetically engineered in the variable domain of an immunoglobulin: implications for antibody and peptide folding.

Immunoglobulins bind antigens and express individual antigenic specificities mainly through residues located in hypervariable loops of their N-terminal domains. Hypervariable loops are kept in place by a molecular scaffold organized in a sandwich-like structure with two beta-sheets stabilized by a disulfide bridge (the immunoglobulin fold). This structural feature, together with the possibility of obtaining high level expression, extracellular secretion, easy purification and stability of the protein product, render immunoglobulin an ideal 'molecular vehicle' for the expression of exogenous peptides. Here we report on the engineering of an immunoglobulin expressing an exogenous epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP)3. By recombinant DNA techniques, we inserted three copies of the tetrapeptide (NANP)3 in the third hypervariable loop (D region) of an immunoglobulin heavy chain variable domain. We show that the engineered antibody was properly assembled and secreted. A panel of polyclonal and monoclonal antibodies, including anti-synthetic peptides and anti-(NANP)n antibodies, were used to study the molecular configuration of the engineered domain's surface. The results indicate that (i) the exogenous sequence did not appreciably alter the overall fold of the variable domain; and (ii) the inserted epitope folded with a configuration immunologically similar to the one assumed in the native protein, suggesting that short- and medium- rather than long-range interactions stabilized the structure of the (NANP)3 peptide in the folded protein. We propose this system for the expression of peptidic sequences, and their structural and functional analysis.     

Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms

The NH2 terminus of the F1 subunit of the paramyxovirus SV5 fusion protein (fusion related external domain; FRED) is a hydrophobic domain that is implicated as being involved in mediating membrane fusion. We have examined the ability of the FRED to function as a combined signal/anchor domain by substituting it for the natural NH2-terminal signal/anchor domain of a model type II integral membrane protein: the hybrid protein (NAF) was expressed in eukaryotic cells. The FRED was shown to act as a signal sequence, targeting NAF to the lumen of the ER, by the fact that NAF acquired N-linked carbohydrate chains. Alkali fractionation of microsomes indicated that NAF is a soluble protein in the lumen of the ER, and the results of NH2-terminal sequence analysis showed that the FRED is cleaved at a site predicted to be recognized by signal peptidase. NAF was found to be efficiently secreted (t1/2 approximately 90 min) from the cell. By using a combination of sedimentation velocity centrifugation and immunoprecipitation assays using polyclonal and conformation-specific monoclonal antibodies it was found that extracellular NAF consisted of a mixture of monomers, disulfide-linked dimers, and tetramers. The majority of the extracellular NAF molecules were not reactive with the conformation- specific monoclonal antibodies, suggesting they were not folded in a native form and that only the NAF tetramers had matured to a native conformation such that they exhibited NA activity. The available data indicate that NAF is transported intracellularly in multiple oligomeric and conformational forms.     

Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, Raman, CD spectroscopy and molecular modeling.

The synthesis and biological evaluation of penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs and comparison with their proline(7)-oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen(1),Pen(6)]-oxytocin (1, pA(2) = 8.22, EC(50) = 6.0 nM) and [dPen(1),Pen(6),5-tBuPro(7)]-oxytocin (2, pA(2) = 8.19, EC(50) = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using (1)H NMR, laser Raman and CD techniques. In H(2)O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln(4) and Pen(6) in the major trans-conformer of 1 as well as for Gln(4) in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 +/- 0.2 kcal/mol) for isomerization of the amide N-terminal to 5-tBuPro(7) relative to OT was calculated from measuring the coalescence temperature of the Gly(9) backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C(beta)-S-S-C(beta) dihedral angles of +110-115 degrees and +/-90 degrees , respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a chi(CS-SC) dihedral angle value close to 115 degrees was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a +/-90 degrees angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a +/-90 degrees chi(CS-SC) dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114 degrees and +116 degrees . Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104 degrees . In sum, the conformation about the disulfide in [dPen(1),Pen(6)]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen(1),Pen(6),5-tBuPro(7)]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro(7) (5R)-tert-butyl group, the Leu(8) isopropyl sidechain and the Pen(6)beta-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110 degrees may be a prerequisite for their binding at the receptor.     

Transesterification of Moz-Asn-Leu-Gly-OEt in methanol. Confirmation of Ca2+ mediated catalysis

During the recrystallization of the crude protected tripeptide ester Moz-Asn-Leu-Gly-OEt (obtained by an enzymatic coupling reaction) in methanol/water, the transesterification of this compound to methyl ester was observed. The involvement of Ca2+ in this process was indicated by the results obtained in the following experiments: 1) incubation of crude Moz-Asn-Leu-Gly-OEt in methanol solutions of o-phenanthroline and EDTA; 2) recrystallization of the crude Moz-Asn-Leu-Gly-OEt in ethanol/water followed by incubation in methanol; 3) determination of the Ca2+ content of the crude Moz-Asn-Leu-Gly-OEt. After recrystallization Moz-Asn-Leu-Gly-OEt lost the ability to be transesterified in methanol. However, in the presence of crude Moz-Asn-Leu-Gly-OEt, or calcium acetate, or a mixture of calcium chloride/sodium acetate, the compound was transesterified, suggesting that the transesterification of crude and recrystallized compounds occurs through different mechanisms. On the basis of these results, and of the conformational data obtained for these peptides by 1H-NMR, we suggest models for the transesterification reactions.     

Complete nucleotide sequence of the neuraminidase gene of human influenza virus A/WSN/33.

The complete nucleotide sequence of the neuraminidase (NA) gene of WSN/33 (H1N1) virus was determined. The entire sequence was derived from the insert of cDNA clones, except the last 20 nucleotides, which were determined by primer extension. The WSN NA gene contained 1,409 nucleotides beginning at the 5' end (sense strand), with an untranslated region of 19 nucleotides followed by 1,359 nucleotides coding for 453 amino acids and finally ending with a 31-nucleotide sequence of untranslated region at the 3' termini. The amino acid sequence of WSN NA, as deduced from the DNA sequence, showed the presence of a stretch of 29 amino acids (7 to 35) enriched in hydrophobic amino acids, which may anchor the protein into the viral or cellular membrane. When compared with the PR8 NA sequence, WSN NA appeared to possess a similar structure, including the identical location of all cysteine and proline residues. However, WSN NA contained only three of the five potential glycosylation sites present in PR8 NA. Additionally, WSN NA contained a substitution of a five-amino acid sequence for a six-amino acid sequence in PR8 NA. The possible significance of these sequence changes in the primary structure of WSN NA in the unique role of WSN NA as a virulence factor in mouse brain and MDBK cells is discussed.     

Synthetic Plasmodium falciparum circumsporozoite peptides elicit heterogenous L3T4+ T cell proliferative responses in H-2b mice

The ability of synthetic P. falciparum (NANP)n circumsporozoite peptides to elicit murine T cell proliferative responses was studied. When C57BL/6, C3H, and DBA/2 mice were injected with (NANP)40, only C57BL/6 (H-2b)-immune lymph node cells proliferated on restimulation in vitro with the same peptide. By using anti-I-A monoclonal antibodies or spleen cells from congenic H-2b mice as a source of antigen-presenting cells, the T cell proliferative response was shown to be restricted to the I-Ab region of the C57BL/6 haplotype. These results are in agreement with previous experiments which demonstrated that the anti-(NANP)40 antibody response was uniquely restricted to C57BL/6 (H-2b) mice. Several C57BL/6 long-term (NANP)n-specific T cell lines and clones were derived. All of the clones exhibited the L3T4 helper T cell phenotype. A considerable heterogeneity of T cell responses was observed when the lines and clones were stimulated with different concentrations of the various peptides studied. The results, together with the observed genetic restriction for both antibody and T cell responses, suggest that perhaps not all individuals who receive a similar repetitive tetrapeptide sporozoite malaria vaccine will develop T cell and or antibody responses.     

Effect of mutations and deletions in a bicistronic mRNA on the synthesis of influenza B virus NB and NA glycoproteins.

The mRNA derived from influenza B virus RNA segment 6 is functionally bicistronic and encodes the NB and NA glycoproteins in different, overlapping reading frames. NB protein synthesis is initiated at the 5'-proximal AUG codon, and 4 nucleotides downstream there is a second AUG codon which is used to initiate NA protein synthesis. The nucleotide sequence context of the first AUG codon conforms closely with the established 5'-CC(A/G)CCAUGG-3' consensus sequence (M. Kozak, Nucleic Acids Res. 15:8125-8148, 1987), which should favor initiation of NB protein synthesis at this site, yet NB and NA are found to accumulate in approximately equal amounts in infected cells. To determine the features important for allowing initiation at the second 5'-proximal AUG codon, we made changes in the 5'-terminal region of the mRNA, including deletions, insertions, and site-specific mutations. The recombinant DNA molecules were expressed in eucaryotic cells, and the accumulation of NB and NA was quantitated. The data indicate that changes in the immediate sequence around the first AUG codon do not make a large difference in the amounts of NB and NA that accumulate, but that when the first AUG codon is displaced from its normal position it is now quite efficient at preventing downstream initiation events. In addition, the data indicate that an element of the B/NB/NA mRNA 5' untranslated leader region acts in cis to enhance the expression of NB and NA.     

Steps in maturation of influenza A virus neuraminidase.

We have studied the maturation of the influenza A virus neuraminidase (NA), using monoclonal antibodies (MAbs) with different conformational specificities against the head domains of the N8 NA. The results obtained with radioimmunoprecipitation, together with previously published information, suggest the following steps in maturation of this molecule. First, the folding of the nascent NA leads to formation of the epitope recognized by MAb N8-10, a step that depends on the formation of intramolecular disulfide bonds. Second, monomers form dimers by an intermolecular disulfide linkage in the stalk, with a t1/2 of 2.5 min. Third, the epitope recognized by MAb N8-82 appears after dimerization, suggesting that oligomeric NAs may undergo conformational change with a t1/2 of 8 min. Finally, a tetramer-specific epitope recognized by MAb N8-4 appears on the NA with a t1/2 of 13 min. Epitope detection by MAb N8-4 was inhibited by tunicamycin treatment, suggesting that glycosylation of this molecule is required for proper tetramerization. Each of these proposed steps occurs in the endoplasmic reticulum of host cells, as demonstrated by treatment of virus-infected cells with brefeldin A or carbonyl cyanide m-chlorophenylhydrazine; subsequently, tetrameric NA is transported to the Golgi apparatus, where oligosaccharide processing is completed. Our findings also provide a possible explanation--lack of a functionally active conformation--for the absence of enzymatic function by NA monomers.     

Human influenza viral neuraminidases augment cell-mediated cytotoxicity in vitro

Previously, we reported that influenza virus-induced cell-mediated cytotoxicity (CMC) was largely due to its glycoproteins, hemagglutinin and neuraminidase (NA). These observations were based on the use of a single influenza virus strain, the A/Port Chalmers/3/73 (H3N2), and these were considered insufficient to generalize that all human influenza virus NAs augment CMC. Therefore, antigenically different NAs of human influenza strains were used to study whether (a) all NAs possess the potential to stimulate NK activity and (b) does the enzymatic activity of NA play a role in the CMC stimulation. Biologically active preparations of N1 subtype NA (A/USSR/90/77 (H1N1) and N2 subtype NAs (A/Aichi/2/68 (H3N2) and A/Port Chalmers) were evaluated for NK activity stimulation in an overnight radiolabeled chromium-release assay consisting of human peripheral blood lymphocytes and K562 target cells. The level of CMC stimulation was the same at equivalent protein concentrations with all the NAs tested. The addition of homologous NA-monospecific antibody almost completely reduced the CMC stimulation, while the addition of homosubtypic antibody reduced the CMC by 56-75%. However, in the presence of heterosubtypic monospecific antibody, NA-augmented CMC was reduced by 27-47% in most experiments. The results suggest that the CMC stimulation site is probably the same in all NAs tested. This putative site is thermo-resistant and is independent of the conformational change of the NA molecule. Furthermore, it is distinct from the enzymatic and probably from the antigenic sites.