Biomedical subjects

A Portner

Publications and source records attributed to A Portner.

At least 55 records · Page 3Linked to original sources

PubMed1991

The nucleoproteins of human parainfluenza virus type 1 and Sendai virus share amino acid sequences and antigenic and structural determinants.

The complete nucleotide sequence of the nucleoprotein (NP) gene of human parainfluenza virus type 1 (hPIV-1) was determined from a cDNA clone of mRNA. The mRNA is 1683 nucleotides long (excluding polyadenylic acid) and encodes a protein of 524 amino acids with a predicted Mr of 57,548. An amino acid identity of 83% was predicted between the NPs of the human pathogen hPIV-1 and the murine paramyxovirus, Sendai virus, compared to 72% similarity at the level of the nucleotide sequence. In contrast, the amino acid sequence identity between the NPs of hPIV-1 and hPIV-3 was 59%, suggesting a more distant evolutionary relationship. The NP amino acid sequences of hPIV-1 and Sendai virus were highly conserved in the amino-terminal half of the molecule, in which 395 of the first 420 amino acids were identical. Of 11 monoclonal antibodies (MAbs) targeted against the Sendai virus NP, five cross-reacted with the hPIV-1 NP. The MAbs that cross-reacted recognize epitopes within regions of high amino acid similarity between the NPs of the two viruses. Also, five of the eight MAbs raised against hPIV-1 NP cross-reacted with Sendai virus NP. Taken together, our observations suggest that the essential amino acid sequence determinants of the NP structures of hPIV-1 and Sendai virus are conserved despite changes in their nucleotide sequences during evolution. This implies that there was a selective pressure to maintain the important functional domains of the protein.

Amino Acid Sequence↗

PubMed1990

Separate domains of Sendai virus P protein are required for binding to viral nucleocapsids.

The role of Sendai virus P protein in viral RNA synthesis involves association with the nucleocapsid template. There is evidence that the carboxyl-terminal region of P protein is responsible for this association (K. W. Ryan and D. W. Kingsbury, 1988, Virology 167, 106-112). To define the P protein sequences involved more precisely, deletions were generated in a cDNA clone of the P gene. Proteins synthesized in vitro from these altered P genes were mixed with extracts from infected cells to determine if they could attach to nucleocapsids. Under conditions where full-size P protein was able to bind, a protein comprising the 95 carboxyl-terminal residues of P protein (Sendai virus X protein) did not bind. This indicated that other P protein residues were required, in addition to the 95 residues at the carboxyl-terminal end. To locate these other residues, P genes were constructed with overlapping deletions of sequences encoding the carboxyl-terminal 40% of the protein. Analysis of these deleted proteins revealed that the necessary residues were in two separate binding domains, amino acids 345 to 412 and 479 to 568 (the carboxyl-terminus). Deletion of the 66 residues between these regions did not affect attachment. Therefore, the formation of a functional binding site requires residues within two separate regions of P protein.

Amino Acid Sequence↗

PubMed1990

The hemagglutinin-neuraminidase glycoproteins of human parainfluenza virus type 1 and Sendai virus have high structure-function similarity with limited antigenic cross-reactivity.

Human parainfluenza virus type 1 (hPIV-1) is closely related to Sendai virus on the basis of cross-reactivity of antisera. We examined this association further by using monoclonal antibodies to the Sendai virus hemagglutinin-neuraminidase (HN) glycoprotein to determine the relationship between overall protein structure and the hemagglutination and neuraminidase functions. Of 10 monoclonal antibodies representing four nonoverlapping antigenic sites on the HN of Sendai virus, only 4 from two sites cross-reacted with hPIV-1, indicating a limited conservation of epitopes. One of these four inhibited the hemagglutinating activity of hPIV-1 comparably to Sendai virus, but none appreciably inhibited the neuraminidase activity of hPIV-1. The ability of some of these monoclonal antibodies to inhibit only hemagglutinating or neuraminidase activity of either virus provided evidence for two separate active sites on the HN molecule. To determine the overall structural relationship of the HNs of hPIV-1 and Sendai virus, we cloned and sequenced the HN gene of hPIV-1. The HN clone was made from genomic RNA and was identified by hybrid-arrested in vitro translation of mRNA. The predicted HN protein sequence of hPIV-1 was identical in length to that of Sendai virus and had a shared identity of 72%. There was a marked conservation of structural elements (cysteines, prolines, and glycines), which would predict a similar molecular conformation. However, there were 10 potential glycosylation sites on the HN of hPIV-1, compared with 5 on Sendai virus. Some of these sites may be responsible for the inability of the Sendai virus monoclonal antibodies to cross-react. The results of our study support a close structure-function relationship between hPIV-1 and Sendai virus but suggest limited antigenic cross-reactivity.

Amino Acid Sequence↗

PubMed1990

Antibody response in children to antigen sites on human PIV-3 HN: correlation with known epitopes mapped by monoclonal antibodies.

The antibody response in children to known epitopes on the HN of human parainfluenza virus type 3 was investigated. Children's sera with Haemagglutination-Inhibition titres between 1/480 to 1/1280 were used. When tested by ELISA, this high-titre serum from each of five children blocked 7 of 17 specific anti-HN murine monoclonal antibodies by greater than 75% at 1 micrograms well-1 of antigen. However, four monoclonal antibodies were blocked less than or equal to 30%, while six were partially blocked between 50% and 75%. Antigen concentrations of 0.5, 1.5 and 2.0 micrograms well-1 did not substantially change this pattern. Comparison of our results with published antigenic maps indicated that antigenic site A on the HN protein was the site with the most significant antibody representation in the children's sera. These findings suggest that antigenic maps deduced using monoclonal antibodies need to be carefully interpreted before they are used in vaccine development. Murine monoclonal antibodies may not fully represent either qualitatively or quantitatively important antibody components of the human or murine immune response to human PIV-3 HN.

Antibodies, Monoclonal↗

PubMed1990

Localization of P protein binding sites on the Sendai virus nucleocapsid.

Previous studies have shown that the molecules of P protein associated with transcriptionally active Sendai virus nucleocapsids are arranged in discrete clusters. Our study investigates whether or not this localized distribution is due to the existence of only a few P protein binding sites on the nucleocapsid core. We used immunoelectron microscopy to examine whether additional P proteins could bind at locations between the groups of endogenous P proteins. To differentiate between endogenous and added proteins, we constructed a recombinant gene which instructs the in vitro synthesis of a chimeric protein containing the carboxyl-terminal nucleocapsid-binding region of P protein, fused to chloramphenicol acetyltransferase (CAT). Immunogold labelling, using an antibody to the CAT moiety, revealed at the electron microscope level, that the chimeric product bound to nucleocapsids at many sites located over the entire length of the nucleocapsid. This indicated that the localized distribution of P protein molecules is not due to a limited number of P protein binding sites on the nucleocapsid core.

Binding Sites↗

PubMed1989

A recent field isolate of Sendai virus has a temperature-sensitive HN glycoprotein.

A recent field isolate of Sendai virus was found to have a temperature-sensitive (ts) hemagglutinin-neuraminidase (HN) glycoprotein. The ts phenotype was manifested as a loss of cell binding, reduced replication, and virions that were lacking surface HN after growth at the nonpermissive temperature (38 degrees). Low neuraminidase activity and failure of the field isolate to remove sialic acid from receptors on the surface of erythrocytes indicated that rapid elution of the field isolate virions from erythrocytes at the nonpermissive temperature was not due to neuraminidase activity but to a proposed conformational change in the HN molecule. The specific amino acids responsible for the ts phenotype could not be determined due to the number of amino acid differences between the field isolate and Enders strain. Heat inactivation and monoclonal antibody inhibition of HN functions indicated that the HN protein of this isolate was, in addition to ts, an unstable molecule.

HN Protein↗

PubMed1989

Crystallization of Sendai virus HN protein complexed with monoclonal antibody Fab fragments.

The hemagglutinin-neuraminidase (HN) protein of Sendai virus has been isolated from virus particles in a biologically active soluble form after removal by proteolytic digestion of the hydrophobic amino-terminal anchor sequence (S. D. Thompson, W. G. Laver, K. G. Murti, A. Portner, J. Virol., 62, 4653-4660, 1988). The soluble HN exists as both dimers and tetramers, and crystallization trials with each of these forms have so far yielded amorphous material. Dimers complexed with Fab fragments of a monoclonal antibody formed long needle crystals. So far, these are not suitable for X-ray diffraction analysis but the results suggest that HN molecules from paramyxoviruses, even if not crystallizable, may, when complexed with Fab fragments, in some cases yield crystals suitable for X-ray diffraction analysis.

Antibodies, Monoclonal↗

PubMed1989

The Sendai virus nucleocapsid exists in at least four different helical states.

Sendai virus nucleocapsids have been observed by electron microscopy to coexist in three different helical pitch conformations, 5.3, 6.8, and 37.5 nm. The 5.3- and 6.8-nm conformations are present both in uranyl acetate negatively stained preparations and in tantalum-tungsten metal-shadowed preparations, whereas the 37.5-nm conformation, which has not been previously reported, is present only in the shadowed preparations. The 5.3-nm pitch conformation appears to be a mixture of two discrete structural states, with a small difference in the twist of the structure between the two. We have used image reconstruction techniques on an averaged data set from eight negatively stained nucleocapsids to produce a three-dimensional reconstruction at 2.4-nm resolution of the structure in one of the 5.3-nm pitch states. There are 13.07 nucleocapsid protein (NP) subunits in each turn of the helix in this state. The helical repeat is 79.5 nm, containing 196 subunits in 15 turns of the left-handed 5.3-nm helix. The arrangement of subunits produces a 5.0-nm-diameter hollow core which forms an internal helical groove. The RNA accounts for about 3% of the mass of the nucleocapsid, and so its location is not conspicuous in the reconstruction. Because of the RNA remains associated with the NP subunits during mRNA transcription and genome replication, structural transitions in the nucleocapsid may determine the accessibility of the genome to polymerases. Alternatively, the large hollow core and internal helical groove we have reconstructed may allow access to the RNA even in the tightly coiled 5.3-nm pitch conformation.

Capsid↗

PubMed1988

Fusion of a Sendai mutant deficient in HN protein (ts271) with cardiolipin liposomes.

Sendai mutant ts271 contains less than 5% of the amount of HN glycoprotein found in wild-type Sendai. Fusion of this mutant with cardiolipin liposomes revealed no differences from the wild-type virus with regard to specific activity, pH dependence, or radiation inactivation. Target sizes of both mutant and wild-type viral proteins were determined by the radiation-induced disappearance of each band from an SDS-polyacrylamide gel and no differences were found. Of the viral proteins, only F had a target size corresponding to the monomer molecular weight, ca. 60 kDa, identical to the minimum unit previously determined by functional assay for Sendai virus-erythrocyte membrane fusion (K. Bundo-Morita, S. Gibson, and J. Lenard, Biochemistry 26, 6223-6227 (1987)). This provides additional evidence that F alone is the active protein mediating Sendai-erythrocyte fusion. It is concluded that the HN protein is unlikely to mediate any fusion reactions of the intact virions, either with biological membranes or with cardiolipin liposomes.

Cardiolipins↗

PubMed1988

Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy.

Antibodies against the L protein of Sendai virus were made by immunizing rabbits with a synthetic peptide representing a carboxyl-terminal region of the protein predicted from the base sequence of its gene. These antibodies were used to localize the L protein in viral nucleocapsids by electron microscopy. Immunogold labeling revealed that L protein molecules were distributed in clusters along nucleocapsids, suggesting that L molecules act cooperatively in viral RNA synthesis. Immunogold double-labeling showed that all L clusters were associated with clusters of P molecules. We believe that this morphological association reflects the functional cooperation of the L and P proteins in viral RNA synthesis.

Antibodies, Viral↗

PubMed1988

Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus.

As a first step in establishing the three-dimensional structure of the Sendai virus hemagglutinin-neuraminidase (HN), we have isolated and characterized a potentially crystallizable form of the molecule. The sequence of HN, a surface glycoprotein, predicts a protein with an uncharged hydrophobic region near the amino terminus which is responsible for anchorage in the viral envelope. To avoid rosette formation (aggregation), which would preclude crystallization, this hydrophobic tail was removed from a membrane-free form of HN by proteolytic digestion. This digestion resulted in a single product with a molecular weight of about 10,000 less than native HN. N-terminal amino acid sequence analysis of cleaved HN (C-HN) indicated a single cleavage site at amino acid residue 131, resulting in a product consisting of the carboxyl-terminal 444 amino acids of HN. Functional analyses revealed that C-HN retained full neuraminidase activity and was able to bind erythrocytes, indicating that the N-terminal 131 residues were not necessary for these biological activities. Furthermore, this cleavage product retained the antigenic structure of intact HN, since monoclonal antibodies still bound to C-HN in enzyme-linked immunosorbent assay and Western (immuno-) blot analysis. Viewed by electron microscopy, the dimeric and tetrameric forms of intact HN form rosettes while C-HN maintains the oligomeric structure but no longer aggregates. Furthermore, the electron micrographs revealed a C-HN tetramer strikingly similar to the influenza virus neuraminidase in both size and gross structural features.

Animals↗

PubMed1988

Mapping of antigenic domains of Sendai virus nucleocapsid protein expressed in Escherichia coli.

Several nonoverlapping epitopes were mapped on the primary sequence of the Sendai virus NP protein. After a complete cDNA clone of the Sendai virus NP gene was expressed in Escherichia coli, deletion constructs were used to generate a series of overlapping NP fragments deleted at their C termini. Immunoblot analyses with 11 monoclonal antibodies identified four antigenic sites. All of these sites resided in the C-terminal half of NP and were also the only sites detected with a polyclonal serum. These findings confirm and extend the evidence that the C terminus of the NP protein represents the domain exposed on the surface of the nucleocapsid. One of the monoclonal antibodies reacted with a site, comprising only 6 amino acids, lying with a hinge between an alpha-helix and a beta-strand in the predicted secondary structure of NP. Since this antibody is a potent inhibitor of in vitro viral RNA synthesis (K. L. Deshpande and A. Portner, Virology 139:32-42, 1984), the epitope may be critical to the flexibility of the NP molecule that makes the RNA template accessible during RNA synthesis.

Antibodies, Monoclonal↗

PubMed1987

Localization of functional sites on the hemagglutinin-neuraminidase glycoprotein of Sendai virus by sequence analysis of antigenic and temperature-sensitive mutants.

To locate the various functions associated with the hemagglutinin-neuraminidase (HN) glycoprotein of Sendai virus in the primary structure of the protein, a temperature-sensitive (ts) mutant and seven antigenic mutants were sequenced. The ts mutant was defective in its ability to agglutinate erythrocytes and infect host cells, while its neuraminidase activity was normal. Its sequence revealed two closely spaced amino acid substitutions (residues 262 and 264) and one distant substitution (residue 461). Revertants could not be isolated, suggesting that more than one of the substitutions is responsible for the defective hemagglutinating activity. The antigenic mutants were selected with monoclonal antibodies that delineate four nonoverlapping antigenic sites (I-IV) and separately inhibit hemagglutinating, neuraminidase, and hemolysis activities. Mutants selected with antibodies to antigenic sites I-III were used to map these functions on the primary sequence of HN. Each antigenic mutant had a single point mutation in the HN gene that resulted in an amino acid substitution in the protein. A site II mutant selected with an antibody which inhibits hemolysin activity had a substitution at amino acid 420, while a mutant selected with antibody that inhibits only erythrocyte binding (site III) had a substitution at amino acid 541. Two antigenic mutants selected with an antibody that inhibits hemagglutination and neuraminidase activities (site I) had amino acid substitutions in close proximity (residues 277 and 279) to the two closely spaced substitutions of the ts mutant. These findings suggest that the region defined by the ts mutant and these two antigenic mutants is involved in host cell binding. Antigenic mutants selected with another site I antibody had amino acid changes at residue 184, indicating that antigenic site I is discontinuous in the primary sequence. This antibody blocks only hemagglutination, but mutants selected with it had a decreased neuraminidase activity. This finding supports the idea that the neuraminidase site is close to, but distinct from, the hemagglutination site.

Antibodies, Monoclonal↗

PubMed1987

Distinct functions of antigenic sites of the HN glycoprotein of Sendai virus.

Monoclonal antibodies specific for the hemagglutinin-neuraminidase (HN) glycoprotein of Sendai virus were used to examine the antigenic structure of HN and its role in the initiation of infection and immunity. Using 10 anti-HN antibodies, four distinct antigenic sites designated I-IV were topographically mapped on the HN molecule by competitive-binding assays. To relate the biological functions of HN to its antigenic structure, anti-HN antibodies were analyzed for their inhibitory activity in neuraminidase, hemagglutination, and hemolysis inhibition tests. Antibodies to antigenic site I inhibited hemagglutination and one of these antibodies also inhibited neuraminidase activity. Antibodies to site II inhibited neither activity. However, hemolysis an F protein activity was inhibited, suggesting that these antibodies which bind to HN interfere with F-mediated fusion. Antigenic sites III and IV had different effects on the hemagglutinating and neuraminidase functions of HN: Site III antibodies inhibited hemagglutination while antibodies to site IV only inhibited neuraminidase activity. Antibodies to each antigenic site inhibited virus production. Since antibodies to sites I and III inhibited hemagglutination, it is likely that they block virus adsorption. Antibodies to HN site II only inhibited hemolysis, and therefore, may prevent virus penetration. Antibodies reacting with site IV inhibited virus production after virus penetration. Since neuraminidase activity was the only function inhibited, the viral enzyme may be involved in virus release. The fact that site IV antibodies inhibited neuraminidase but not hemagglutination suggests that these sites are distinct.

Animals↗

PubMed1987

The fusion glycoprotein of Sendai virus: sequence analysis of an epitope involved in fusion and virus neutralization.

To localize the amino acid residues on the F glycoprotein that are involved in Sendai virus fusion and virus neutralization, an anti-F monoclonal antibody which inhibits these functions was used to select three antigenic variants. Sequence analysis of the entire F gene of the three variants identified a single mutation that was responsible for the loss of antibody binding. The mutation, a proline to glutamine substitution at residue 399, was at a position in the primary sequence far removed from the hydrophobic F1-NH2 terminus thought to be directly involved in fusion. A synthetic peptide, comprising amino acid sequences in the region of the mutation, bound to the antibody used to select the variants, suggesting that the site of mutation is also the site of antibody binding. This information suggests that in the three-dimensional structure of the F molecule the amino acid residues around proline 399 are located close to the F1-NH2 terminus, and that fusion is directly inhibited by antibody binding. Other less likely alternatives are discussed.

Amino Acid Sequence↗

PubMed1986

Localization of P, NP, and M proteins on Sendai virus nucleocapsid using immunogold labeling.

The distribution of NP, P, and M proteins on Sendai virus nucleocapsids purified from cells and virions were studied by immunogold staining using monoclonal antibodies. NP molecules were found uniformly along the entire length of both cytosol and virion derived nucleocapsids. This observation is in accord with the earlier proposals that NP molecules maintained the structural integrity of the nucleocapsid. The distribution of P in nucleocapsids derived from the cytosol differed from the distribution in those originating from virions. In nucleocapsids derived from the cytosol, P molecules occurred in 4 to 10 discreet clusters at varying locations along the length of the nucleocapsid. In contrast, on nucleocapsids derived from virions, P molecules were uniformly distributed over the entire length of the nucleocapsid. These observations suggest that the distribution of P depends on the functional state of the nucleocapsid. The occurrence of P clusters at different locations on intracellular nucleocapsids indicates that P is a mobile molecule; this suggestion is consistent with P's role in viral RNA synthesis. The distribution of the matrix (M) protein also depended on where the nucleocapsids were derived from. Large quantities of M protein were found along the entire length of nucleocapsids derived from the cytosol, while in virion nucleocapsids, many fewer molecules of M were observed. The large amounts of M on the nucleocapsids originating from the cytosol supports the hypothesis that M protein mediates the recognition between the nucleocapsid and the envelope glycoproteins.

Animals↗

PubMed1986

Localization and characterization of Sendai virus nonstructural C and C' proteins by antibodies against synthetic peptides.

Antibodies were raised in rabbits against two synthetic peptides, each 30 residues in length, one corresponding to the predicted common carboxyl termini of the nonstructural C and C' proteins of Sendai virus and the other to the unique amino terminus of the larger C protein. Each peptide was inoculated as a covalent complex with tetanus toxoid or in uncomplexed form. Only antibodies to the free carboxyl-terminal peptide precipitated both C and C' proteins made by in vitro translation of viral mRNA and reacted with the C protein from infected cells. These results confirm that the C and C' proteins are carboxyl-coterminal. Contrasting with the reported colocalization of intracellular measles virus C proteins with nucleocapsid inclusions, immunofluorescence studies revealed that Sendai virus C proteins were uniformly distributed in the cytoplasm whereas the viral P protein was present in inclusions that were mainly perinuclear. Since almost all P protein molecules are associated with viral nucleocapsids, these observations suggested that Sendai virus C protein molecules may be both nucleocapsid-associated and free in the cytoplasm. This interpretation was supported when the C and C' proteins were found in both nucleocapsid and free protein fractions of cell lysates. Anti-C antibodies did not inhibit viral RNA synthesis when added to an extract of infected cells. This result was consistent with the conclusion that the C proteins have no direct role in viral transcription, since virions lack C proteins but are transcriptionally active. Therefore, the functions of the C proteins remain undefined.

Animals↗

PubMed1986

Drastic immunoreactivity changes between the immature and mature forms of the Sendai virus HN and F0 glycoproteins.

The immunoreactivity of the Sendai virus HN and F0 glycoproteins was shown to mature before reaching the final form exhibited by the native mature proteins. The maturation process differed for the two proteins. The native F0 immunoreactivity was shown to be defined cotranslationally, and the addition of high-mannose sugar residues may represent the final step in defining the maturation of immunoreactivity. On the other hand, native HN immunoreactivity was slowly fashioned during the hour after the completion of protein synthesis. Although addition of high-mannose sugar could constitute a necessary step in this slow maturation process, it was shown not to be sufficient. Processing of high-mannose sugars and HN self-association in homodimers and homotetramers were investigated as possible steps involved in the slow maturation of HN immunoreactivity. They were found not to play a significant role. On the other hand, conformational changes presumably took place during the maturation of HN immunoreactivity. Drastic immunoreactivity differences were also demonstrated between the native and denatured forms of the glycoproteins. Possible implications of these results in defining the pathways of glycoprotein synthesis are discussed.

Animals↗