Biomedical subjects

A Portner

Publications and source records attributed to A Portner.

At least 37 records · Page 2Linked to original sources

PubMed1994

Viral cross-reactivity and antigenic determinants recognized by human parainfluenza virus type 1-specific cytotoxic T-cells.

To obtain information relevant to vaccination against human parainfluenza virus type 1 (hPIV-1), cytotoxic T-lymphocyte (CTL) responses to individual viral components were tested. The CD8-positive T-cell fraction was first enriched from human, adult PBL and grown for several passages in the presence of hPIV-1-infected stimulator cells. T-cell lines were then tested for CTL activity toward hPIV-1 and toward the related viruses hPIV-3 and Sendai virus (the murine parainfluenza type 1 virus). All tested cultures which responded to hPIV-1 also responded to hPIV-3 and Sendai virus, demonstrating sequence conservation between all three viruses among major antigenic determinants for CTL. Specificity for particular viral components was defined using recombinant vaccinia viruses expressing individual proteins from either mouse or human parainfluenza type 1 viruses. Strong CTL responses toward hemagglutinin-neuraminidase, phosphoprotein, and nucleoprotein (NP) were demonstrated. The testing of vaccinia constructs expressing truncated proteins then showed that there were multiple CTL determinants within NP. Several T-cell lines from one donor recognized an NP peptide (amino acids 321-336) conserved between the hPIV-1 and Sendai virus. In total, the results demonstrated that the human CTL response is directed to multiple determinants within several distinct hPIV-1 proteins.

Adult↗

PubMed1994

Age-related development of human memory T-helper and B-cell responses toward parainfluenza virus type-1.

Human parainfluenza-1 virus (hPIV-1) infections are a major cause of respiratory illness in young children. While children and adults are each susceptible to hPIV-1 infection, the clinical symptoms in adults are mild and hospitalizations are rare. One explanation for the differences in disease severity is that immune memory responses are simply inferior in children as compared to adults and cannot counter virus growth. Alternatively, it has been suggested that immune (particularly T-helper (TH) cell) responses toward respiratory viruses are superior in children versus older individuals, and that these responses contribute to, rather than protect from, disease symptoms. As a test of these possibilities, we analyzed hPIV-1-specific T-helper (TH) and B-cell memory responses among individuals of various ages, including children hospitalized with hPIV-1-induced croup. Experiments revealed: (1) hPIV-1-specific B-cell and class-II restricted TH-cell proliferative responses were present in all tested adults. (2) TH-cells responded to internal viral proteins as well as to the external glycoprotein, hemagglutinin-neuraminidase. (3) Immune responses were highly cross-reactive with Sendai virus. (4) Memory B-cell and TH-cell responses were extremely poor in young children, inclusive of children tested upon hospital entry for hPIV-1-induced croup. In total, results did not support the theory that naturally induced hPIV-specific memory responses cause respiratory illness. Rather, results showed a correlation between memory and a good clinical outcome and highlighted Sendai virus as a strong candidate for an hPIV-1 vaccine.

Adult↗

PubMed1994

The human parainfluenza virus type-1 prototypic strain contains a heat-labile hemagglutinin-neuraminidase protein.

The virus yield of human parainfluenza virus type-1 (hPIV-1) in cultured cells at 38 degrees C is reduced more than 100-fold compared to 34 degrees C, while the virus yield of Sendai virus (SV, Enders strain), a murine parainfluenza virus type-1 with high homology to hPIV-1 was almost equal at both temperatures. To understand the basis for the differences in the temperature growth characteristics of the two viruses, we examined the heat-stability of hPIV-1 and SV glycoproteins expressed from cDNAs by pulse-chase experiments. The hemagglutinin-neuraminidase (HN) protein of hPIV-1 was stable after a 6-h chase at 34 degrees C, while at 38 degrees C prominent protein degradation was observed starting at 3 h chase and by 6 h HN was reduced by 65%. In contrast, SV HN protein was stable at both 34 and 38 degrees C. The other hPIV-1 glycoprotein, the fusion (F) protein was stable at both temperatures. To identify the amino acids which are responsible for the heat-lability of hPIV-1 HN, mutant HN proteins were constructed by site-directed mutagenesis. Mutant hPIV-1 HN which had substitutions at positions 461 and 462 became heat-stable at 38 degrees C. These data indicate amino acids around 461 are responsible for the heat-lability of the wild type hPIV-1 HN protein and the reduced yield of the virus at 38 degrees C.

Amino Acid Sequence↗

PubMed1994

Human parainfluenza virus type 1 evolution combines cocirculation of strains and development of geographically restricted lineages.

The hemagglutinin neuraminidase (HN) glycoprotein of human parainfluenza virus type 1 (HPIV-1) mediates attachment to the host cell and is the target of protective antibody. Since the efficacy of a potential vaccine depends on antigenic constancy, the antigenic and genetic stability of the HPIV-1 HN glycoprotein was examined for 13 isolates obtained between 1981 and 1989. Antigenic analysis with a panel of 11 monoclonal antibodies demonstrated a single change among 3 isolates from 1989 that distinguished them from all other isolates. The HN genes from all 13 isolates and 13 previously published HN gene sequences shared > 95% homology. Evolutionary analysis demonstrated cocirculation of strains, without a dominant lineage. The 1989 isolates and the previously proposed subtype A isolates occupied distinct evolutionary branches, indicating geographically limited evolution. The slow rate of evolution and HN homogeneity may allow development of a single vaccine formulation for the prevention of disease.

Antigens, Viral↗

PubMed1994

Analysis of the primary T-cell response to Sendai virus infection in C57BL/6 mice: CD4+ T-cell recognition is directed predominantly to the hemagglutinin-neuraminidase glycoprotein.

Sendai virus infection of C57BL/6 mice elicits a strong CD4+ and CD8+ T-cell response in the respiratory tract. To investigate the specificity of the CD4+ T-cell response, a panel of hybridomas was generated from cells recovered from the respiratory tracts of infected mice. Using vaccinia virus recombinants expressing individual Sendai virus proteins, we found that the majority of these hybridomas (34 of 37) were specific for the hemagglutinin-neuraminidase (HN) glycoprotein. The hybridomas were then analyzed for reactivity to a set of overlapping peptides spanning the entire length of the hemagglutinin-neuraminidase glycoprotein. At least five H-2 I-Ab-restricted epitopes were defined in HN. The strong bias toward recognition of class II epitopes derived from a single viral protein contrasts with T-cell recognition of epitopes of several proteins in influenza A virus as found previously by others.

Amino Acid Sequence↗

PubMed1993

Crystals of hemagglutinin-neuraminidase of parainfluenza virus contain triple-stranded helices.

When purified dimers of hemagglutinin-neuraminidase molecules released by protease digestion from three strains of human parainfluenza virus 1 were used in crystallization trials, long thin needle crystals formed. Electron microscopic analysis of these needle crystals revealed that they are composed of stacks of triple-stranded helices with each strand of the helix made up of subunits of hemagglutinin-neuraminidase. To our knowledge, this is the first direct demonstration of the assembly of protein subunits into large triple-stranded helices. An understanding of the organization of these triple helices may shed light on the structural properties of the hemagglutinin-neuraminidase molecules that cause them to form these helices.

Crystallization↗

PubMed1993

Antibodies to paramyxovirus nucleoproteins define regions important for immunogenicity and nucleocapsid assembly.

To help illuminate the surface topography of paramyxovirus nucleocapsids, epitopes recognized by monoclonal antibodies have been mapped on the primary structure of human parainfluenza virus type 1 (hPIV1) nucleoprotein (NP). Full-size NP (524 amino acids) was used, as well as a series of truncated proteins with segments resected from either their carboxyl or their amino termini. Immunoprecipitation by three anti-hPIV1 NP monoclonal antibodies required the presence of amino acids within the carboxyl-terminal 23% of NP. This was consistent with an earlier study of the closely related Sendai virus (SV) NP which mapped all epitopes to regions near the carboxyl terminus. However, in contrast to those results, we found that three other antibodies specific for hPIV1 NP recognized epitopes in the amino-terminal 30% of the molecule. Two of these antibodies also cross-reacted with SV NP and with SV nucleocapsid complexes, showing that the same epitopes were present in the SV protein and were accessible on the nucleocapsid surface. Differences in immunogenicity of these epitopes in the hPIV1 and SV nucleoproteins may reflect sequence differences elsewhere in each NP molecule. In addition, two antibodies to epitopes near the NP carboxyl terminus caused P protein to be released from SV nucleocapsid complexes and prevented binding of exogenous P protein to nucleocapsids. Antibody inhibition of P protein binding helps to locate the NP domains important for attachment of P protein during nucleocapsid assembly.

Antibodies, Monoclonal↗

PubMed1992

Sequence characterization and expression of the matrix protein gene of human parainfluenza virus type 1.

The nucleotide sequence of the M gene of human parainfluenza virus type 1 (hPIV1) was determined from genomic RNA and cDNA copies of the entire gene. The M gene contained 1173 nucleotides. It had one large open reading frame capable of encoding a protein of 348 amino acids (M(r) = 38,404). The predicted amino acid sequence of the hPIV1 M protein is highly basic (+20 at neutral pH). A pGEM-1 expression vector containing the M gene was used for cell-free transcription and translation. The resultant protein was confirmed to be M by electrophoretic mobility and immunoprecipitation. Among other paramyxoviridae the hPIV1 M amino acid sequence was most closely related to the Sendai virus M sequence (87% identity). The pattern of M gene relatedness observed from the alignment of 16 paramyxoviridae M protein amino acid sequences was not predicted by the viruses' taxonomic classification.

Amino Acid Sequence↗

PubMed1992

Expression of cDNA encoding the Sendai virus hemagglutinin-neuraminidase gene: characterization of wild-type and mutant gene products.

Cloned cDNA encoding the Sendai virus (SV) hemagglutinin-neuraminidase (HN) envelope glycoprotein was expressed in cultured cells in two ways: (I) infection with HN-expressing recombinant vaccinia virus, or (II) transfection with a plasmid with T7 promoter and termination sequences flanking the HN gene, with intracellular T7 RNA polymerase supplied by coinfection with recombinant vaccinia virus that expresses the enzyme. The HN expressed was indistinguishable from the authentic SV protein in antigenicity, cell surface location, and formation of oligomeric structures. In addition, HN expressed from cDNA functioned normally in both hemadsorption and neuraminidase activities. The usefulness of cDNA expression for analyzing HN structure and function was evaluated by mutating the HN cDNA and observing the consequences for HN protein activity. Since previous work indicated that the lysine residue at position 461 is important for the neuraminidase activity of HN, we used site-directed mutation to produce HN protein with this lysine residue changed to glutamic acid. The mutated HN had neuraminidase activity with significantly increased thermal stability, indicating that residue 461 may be essential to the protein's conformation.

Antigens, Surface↗

PubMed1992

The P genes of human parainfluenza virus type 1 clinical isolates are polycistronic and microheterogeneous.

The nucleotide sequence of the P gene of human parainfluenza virus type 1 (hPIV1) strain C35 was determined directly from genomic viral RNA and by molecular cloning. The gene contained 1893 nucleotides. Four open reading frames (ORF) capable of encoding a P protein (568 amino acids; M(r) = 64,784), a C' protein (219 amino acids; M(r) = 25,997), a C protein (204 amino acids; M(r) = 24,237), and a Y1 protein (182 amino acids; M(r) = 21,471) were identified. The latter three ORFs are in a +1 reading frame relative to P. The sequencing data are consistent with the hPIV1 C' protein being initiated at a GUG codon (nt 68-70), in contrast to the ACG initiation of the Sendai virus (SV) C' protein. Unlike SV, there is no evidence of a hPIV1 ORF capable of encoding a cysteine-rich V protein. Also, there is no ORF capable of encoding a protein analogous to the SV Y2 protein. In vitro transcription, translation, and immunoprecipitation showed that the hPIV1 P gene is polycistronic. Comparison of the P gene with those of two other distinct clinical isolates confirmed the coding potential of the hPIV1 P gene but also revealed genetic heterogeneity among the isolates. Our results indicate that the hPIV1 P gene uses some coding strategies similar to and others that are different from those of other paramyxovirus P genes.

Amino Acid Sequence↗

PubMed1992

Comparison of IgA versus IgG monoclonal antibodies for passive immunization of the murine respiratory tract.

The protective efficacy of anti-Sendai virus IgA was compared to that of IgG after topical application of monoclonal antibodies (MAb) to the respiratory tract of mice. BALB/c mice were passively intranasally immunized with 50 microliters ascites containing equivalent ELISA titers of MAb 1 h before and 4 and 24 h after intranasal challenge with Sendai virus. Lung viral titers were determined by plaque assay 3 days following challenge. In most instances IgA MAb afforded equivalent protection to IgG MAb in that there was no significant difference in virus recovery from the lungs of animals treated with either IgA or IgG MAb, including subclasses of IgG. When IgA MAb was fractionated into monomers and oligomers, there was no inherent advantage to the oligomeric form with respect to passive protection against viral challenge. The data indicate that IgA and IgG antibodies are equally efficacious in protecting the airways from viral infection. The experiments suggest that the advantage of IgA for protecting mucosal surfaces, such as the respiratory tract, relates to the presence of a specialized mechanism for transporting oligomeric IgA across epithelial surfaces. The results also support the rationale for active mucosal immunization protocols designed to generate an IgA response.

Animals↗

PubMed1992

A plasmid that improves the efficiency of foreign gene expression by intracellular T7 RNA polymerase.

To facilitate the construction of recombinant plasmids for expressing cloned genes with T7 RNA polymerase supplied by recombinant vaccinia virus, a plasmid expression vector was designed by combining parts of plasmids pTZ18R, pBluescript II KS+, and pAR2529. The 3043-bp plasmid pTF1 has a T7 RNA polymerase promoter, multiple cloning site for insertion of foreign genes, and a T7-specific transcription termination signal. Plasmid pTF1 had several advantages compared with the reference plasmid pAR2529, including more efficient replication in bacteria, greater flexibility in the insertion and subcloning of foreign genes, and increased efficiency of liposome-mediated introduction into cultured cells for expression of the foreign gene.

Base Sequence↗

PubMed1992

Crystallization of biologically active hemagglutinin-neuraminidase glycoprotein dimers proteolytically cleaved from human parainfluenza virus type 1.

We isolated, purified, and characterized the hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 1, with the ultimate goal of producing crystals suitable for three-dimensional X-ray structure analysis. Pronase was used to cleave the globular head of the HN molecule directly from virus particles, forming HN monomers and dimers. The purified dimers retained neuraminidase and hemadsorption activity and were recognized by 14 anti-HN monoclonal antibodies, demonstrating intact HN antigenic structure and function. N-terminal sequence analysis of the dimers showed that cleavage had occurred at amino acid 136 or 137, freeing the C-terminal 438 or 439 amino acids. On electron micrography, the dimer appeared as two box-shaped structures, each approximately 5 by 5 nm. When the purified HN dimers were crystallized in hanging drops by vapor diffusion against 20% polyethylene glycol 3350, they formed both rectangular plates and needlelike crystals. The rectangular crystals diffracted X-rays, indicating an ordered atomic structure. However, the resolution was approximately 10 A (1 nm), insufficient for three-dimensional structural analysis. Experiments to improve the resolution by increasing the size and quality of the crystals are in progress.

Amino Acid Sequence↗

PubMed1991

Two noncontiguous regions of Sendai virus P protein combine to form a single nucleocapsid binding domain.

Binding of Sendai virus P protein to viral nucleocapsids requires amino acids in two separate regions of P protein. Both required regions are near the carboxyl terminus, and they are separated by a region which is expendable for binding (K. W. Ryan and A. Portner, 1990, Virology 174, 515-521). To examine the topography of these regions in the folded P protein molecule we mapped the epitopes present in several undenatured P proteins with overlaping deletions near their carboxyl termini. The epitopes recognized by two monoclonal antibodies were each composed of both protein regions necessary for binding, indicating that these two regions are each required at some point during the folding of P protein. To determine if these protein regions interact directly in forming the nucleocapsid binding domain, we constructed a deleted P gene which encodes a protein comprising only these two regions with all other P protein sequences deleted. This protein was able to bind to nucleocapsids, demonstrating that these two regions alone are sufficient to form the nucleocapsid-binding domain. In addition, this protein formed the folded epitopes comprising the two nucleocapsid-binding regions, indicating that the two regions interact directly with each other to form a single folded structure. The involvement of this binding domain in viral mRNA synthesis was examined by testing the ability of each monoclonal antibody to inhibit the in vitro transcription activity of full-size P protein. Several antibodies to epitopes near the binding domain were found to be potent inhibitors of viral transcription, showing that these regions contribute to P protein's role in mRNA synthesis.

Animals↗

PubMed1991

Identification of amino acid positions associated with neuraminidase activity of the hemagglutinin-neuraminidase glycoprotein of Sendai virus.

Identification of amino acid positions associated with neuraminidase activity on the hemagglutinin-neuraminidase (HN) glycoprotein of paramyxoviruses has been difficult because neuraminidase-inhibiting antibodies are not neutralizing and thus, escape mutants have not been isolated. Instead, many investigators have correlated an altered neuraminidase (NA) activity of natural virus variants, such as plaque-size variants, with sequence changes in the HN protein. To identify regions on the HN glycoprotein of Sendai virus (SV) that are associated with NA activity, we investigated NA activity of three plaque-size variants which potentially differed from the standard SV (SV/std). NA activity was measured by the ability of virus to elute from chicken erythrocytes as a result of cleaving sialic acid receptors, and by the ability of virus to cleave sialic acid from the small trisaccharide neuraminlactose and the larger substrate fetuin in an in vitro assay. Virions purified from each of the isolated plaques had a HN content and hemagglutinating activity similar to that of SV/std, yet each variant eluted much more rapidly from chicken erythrocytes than SV/std. In vitro NA activity of the plaque-size variants was 1.6 to 3.8 times greater than that of SV/std, providing supporting evidence for the elution data. Although all plaque-size variants showed elevated NA activity, there was no correlation of activity with plaque size. Sequence analysis showed that one of the variants had an amino acid change from glutamic acid to valine at position 165 and from lysine to glutamic acid at position 461, while a second variant had only the change at position 461. A third variant had a nearby change at position 468, from threonine to lysine. Taken together, these data support the conclusion that the amino acid residues at positions 461-468 and 165 are involved in neuraminidase activity of SV.

Amino Acids↗

PubMed1991

Glycosylation of the hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 1 affects its functional but not its antigenic properties.

The hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza virus type 1 (hPIV-1) has been shown to be similar in predicted protein sequence and structure to those of Sendai virus, but it is more highly glycosylated. Because glycosylation can modify protein structure and function, we investigated the effect of glycosylation on the antigenic structure and biological function of the HN of hPIV-1. Antigenic and functional analyses were carried out with purified hPIV-1 virions treated with Endoglycosidase F, which removes carbohydrate moieties, because treatment of hPIV-1-infected LLC-MK2 cells with an inhibitor of glycosylation resulted in virions which were deficient in both HN and F surface glycoproteins. No change in the antigenic structure of the HN of hPIV-1 was detected after carbohydrate removal; epitope recognition by a panel of 7 hPIV-1 HN monoclonal antibodies (MAbs) was unchanged compared to untreated virions. Moreover, there was no change in the cross-reactivity of 8 of 10 Sendai virus HN MAbs, and only a slight change in the remaining 2. Nor did carbohydrate removal appear to affect hemagglutinating or neuraminidase activities; hemagglutination titers with chicken erythrocytes (cRBC) were unchanged, and in vitro neuraminidase activity with a small substrate (N-acetylneuraminlactose) showed only a 20% reduction. However, elution of deglycosylated hPIV-1 from agglutinated cRBC as a result of neuraminidase activity was reduced by 80%. These results suggest that the enzymatic activity of hPIV-1 HN was not directly affected by carbohydrate removal but that the reduction in elution was due to a change in the interaction of the HN with the host receptor. This was further supported by a 2- to 16-fold reduction in the ability of all 7 hPIV-1 HN MAbs to inhibit hemagglutination of deglycosylated hPIV-1 virus. Such a change in HN-host receptor interaction was found to involve a change in receptor specificity because deglycosylated virus was able to fully agglutinate cRBC stripped of receptors required by the native, glycosylated virus. We propose the following model for our results: deglycosylation of the HN of hPIV-1 causes the hemagglutinating portion of the molecule to recognize a new receptor which is not susceptible to enzymatic cleavage by the neuraminidase.

Antibodies, Monoclonal↗

PubMed1991

Neutralizing epitopes of human parainfluenza virus type 3 are conformational and cannot be imitated by synthetic peptides.

The possibility that linear epitopes on the haemagglutinin-neuraminidase (HN) surface glycoprotein of human parainfluenza virus type 3 (PIV-3) might induce neutralizing antibodies after virus infection was investigated. Thirty-seven peptides, representing 64% of the extramembranous portion of the HN molecule of PIV-3, were synthesized. Their ability to bind to 14 neutralizing murine monoclonal antibodies (mAbs) specific for HN or 26 high-titre human serum samples were tested in a direct enzyme-linked immunosorbent assay (ELISA) and in an indirect competition ELISA. None of the synthetic peptides reacted with any of the mAbs or serum samples in the direct test and none of 11 synthetic peptides tested blocked mAbs from binding to HN in the competition ELISA. These findings suggest that synthetic peptides cannot be used to imitate the known neutralizing epitopes on the HN. Analyses of reduced and non-reduced HN in ELISA and immunoblot assays confirmed that protein folding and tertiary structure are essential for epitope formation in these neutralizing sites. However, some children's sera analysed by immunoblotting contained antibodies to an uncharacterized linear epitope(s) not recognized by our panel of mAbs, raising the possibility that a neutralizing linear epitope does exist on the HN of PIV-3.

Amino Acid Sequence↗

PubMed1991

Location of amino acid residues important for the structure and biological function of the haemagglutinin-neuraminidase glycoprotein of Sendai virus by analysis of escape mutants.

To locate sites important for the structure and function of the haemagglutinin-neuraminidase glycoprotein (HN) of Sendai virus, the biological characteristics of antibody-selected escape mutants were correlated with mutations in the primary HN amino acid sequence. An escape mutant virus deficient only in neuraminidase function but with an HN content equal to that of the wild-type virus had an amino acid change at residue 184, implying that this position may be important for maintaining a functionally active enzymic site. In contrast, other escape mutant viruses with reductions in haemagglutination (eightfold) and neuraminidase activities (70 to 80%) had a sharply diminished HN content and substitutions either at residue 375, or double mutations at residues 279 and 461. The loss of biological activity with the concomitant loss of HN content suggests that these sites may be important for the processing and transport of HN, or in maintaining a structure resistant to proteolytic degradation; residue 451 was shown to have an undefined role in fusion activity. The monoclonal antibodies (MAbs) used to isolate the mutant viruses included those of the IgA and IgG classes and were divided into four operational groups based on their haemagglutination-inhibition pattern against the selected mutants. MAbs of the IgA class recognized epitopes overlapping with (group A) as well as epitopes distinct from (groups C and D) those recognized by the IgG class; group B included only IgG antibodies. The epitopes recognized by IgA antibodies may identify residues important for the secretory immune response to the HN molecule.

Animals↗