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A Portner

Publications and source records attributed to A Portner.

At least 73 records · Page 4Linked to original sources
PubMed1985

Expression of Sendai virus defective-interfering genomes with internal deletions.

Sendai virus strain 7 has been shown to contain four defective interfering (DI) RNA species in which both genome termini and various adjacent fragments of the 3'-terminal NP gene and 5'-terminal L gene are represented, but most or all internal genes and gene boundaries are deleted. Previous sequence analyses of these mutant RNAs suggested that all four possessed the transcription initiation signal of the NP gene and the transcription termination signal of the L gene. The supposition that these signals should specify transcripts has now been supported by oligo(dT) selection of four DI 7 specific RNA species that had apparent molecular weights slightly lower than each DI genome. DI RNA 7a, which contains the entire NP gene, except for two U residues at the end of the poly(A) initiation signal, appeared to be transcribed solely as a readthrough product. Since DI RNA 7a contains the entire NP protein-coding sequence and DI RNAs 7c and 7d contain fragments of it, whereas DI RNA 7b is devoid of it, only transcripts of RNAs 7c and 7d were expected to specify fusion proteins containing NP gene-specific sequences. A strain 7-induced protein that reacted with monoclonal antibodies against the NP protein had the 33,000 Mr size appropriate for the translation product predicted by the sequence of RNA 7d. Other proteins of lower molecular weight were seen only in cells infected by strain 7, but they did not react with NP-specific antibody and their translation in vitro was not blocked by hybridization to an NP gene-specific oligonucleotide. Therefore, at least some of these proteins may be cellular products induced by DI virus infection. These DI transcripts and translation products may influence interference with replication of the parental helper virus.

Antibodies, Monoclonal↗
PubMed1985

Monoclonal antibodies to the P protein of Sendai virus define its structure and role in transcription.

Four monoclonal antibodies specific for Sendai virus nucleocapsid protein P were used to examine both the antigenic structure of P and its role in transcription. Three distinct antigenic regions were delineated on P by competitive radioimmunoassays (RIAs), and through Western blot analysis all three sites were mapped to a 40,000-MW (40K) Staphylococcus aureus protease V8-digestion fragment, which remains associated with the neucleocapsid structure. To study the function of P, nucleocapsids were treated with saturating amounts of anti-P monoclonal antibodies and it was found that transcription in vitro was inhibited by 60-90%. Data, therefore, are consistent with the conclusion that the P protein is required for transcription and that the 40K protease-resistant core contains the functionally important portion of the molecule. Further analysis of the P structure showed that some of the 40K fragments were linked by disulfide bonds. These results suggest that the protease-resistant 40K fragment is in the carboxyl-terminal half of P, since the three cysteine residues of P are found there (C. Giorgi, B. M. Blumberg, and D. Kolakofsky (1983), Cell 35, 829-836).

Animals↗
PubMed1985

An alternative route of infection for viruses: entry by means of the asialoglycoprotein receptor of a Sendai virus mutant lacking its attachment protein.

During the first stage of infection, the paramyxovirus Sendai virus attaches to host cells by recognizing specific receptors on the cell surface. Productive virus-cell interactions result in membrane fusion between the viral envelope and the cell surface membrane. It has recently been shown that the ganglioside GD1a and its more complex homologs GT1b and GQ1b are cell surface receptors for Sendai virus. We report in this paper that the temperature-sensitive mutant ts271 of the Enders strain of Sendai virus lacks the viral attachment protein HN and the biological activities of hemagglutination and sialidase activity associated with it when the virus is grown at 38 degrees C. This HN- virus was unable to infect or agglutinate conventional host cells that contained receptor gangliosides and were readily infected by the parental wild-type virus. The HN- virus did, however, attach to and infect Hep G2 cells, a line of hepatoma cells that retains the asialoglycoprotein receptor (ASGP-R) upon continuous culture. This receptor is a mammalian lectin that recognizes galactose- or N-acetylgalactosamine-terminated proteins. In accordance with the known properties of this receptor, infection by the HN- virus was abolished by treatment of Hep G2 cells with sialidase, by the presence of Ca2+ chelators, and by competition with N-acetylgalactosamine, asialoorosomucoid, and antibody to the receptor. F, the only glycoprotein on the HN- virus, was shown to compete with the galactose-terminated protein asialoorosomucoid for the ASGP-R. The ability of the HN- virus to cause cell-cell fusion of Hep G2 cells indicated that attachment of this virus to the ASGP-R still permitted viral entry by its usual mode--i.e., membrane fusion at the cell surface. These results open up the possibility that enveloped viruses, which contain glycosylated proteins or lipids, may make use of naturally occurring lectins in addition to their normal receptors as a means of attachment to host cells.

Animals↗
PubMed1985

The role of haemagglutinin-neuraminidase glycoprotein cell surface expression in the survival of Sendai virus-infected BHK-21 cells.

A temperature-sensitive mutant of Sendai virus with a lesion in the haemagglutinin-neuraminidase protein (HN) (ts 271) was used to study the effect of HN cell surface expression on the fate of infected BHK-21 cells. The total amount of HN was reduced in ts 271 virus-infected cells at the non-permissive temperature (38 degrees C) presumably due to degradation of the protein. At this temperature, neither HN nor a modified form of HN were found expressed at the surface of the infected cells. BHK-21 cells infected with ts 271 were nevertheless killed by the infection at 38 degrees C as well as at 30 degrees C. These results ruled out the hypothesis that the lack of HN cell surface expression could be the unique requirement allowing BHK cell survival.

Animals↗
PubMed1985

Three variations in the cell surface expression of the haemagglutinin-neuraminidase glycoprotein of Sendai virus.

The fate of the haemagglutinin-neuraminidase glycoprotein (HN) of Sendai virus in three types of infection was studied by measuring its sensitivity to endoglycosidase H and its rate of appearance and turnover at the cell surface. HN behaved differently in the three types of infection. When highly expressed at the surface, as in a lytic standard virus infection, HN accumulated at the surface in a stable form (half-life of disappearance from the surface much greater than 10 h). When moderately expressed, as in a non-lytic standard virus plus defective interfering virus infection, HN reached the membrane normally, but turned over rapidly (half-life about 2 h) and was re-internalized. When poorly expressed, as in long-term persistent infection, HN did not reach the cell surface and appeared to be degraded before reaching it. In contrast to HN, the other viral glycoprotein, F0, exhibited a similar turnover rate at the cell surface in the three situations. However, when compared to surface expression in standard virus-infected cells under standardized conditions, F0 surface expression in persistently infected cells was reduced. This reduction correlates with a decreased maturation rate in these cells.

Animals↗
PubMed1984

Restriction of cell surface expression of Sendai virus hemagglutinin-neuraminidase glycoprotein correlates with its higher instability in persistently and standard plus defective interfering virus infected BHK-21 cells.

To gain an understanding of the mechanism(s) by which Sendai virus generates a persistent infection, the expression of the hemagglutinin-neuraminidase (HN) and fusion (Fo) glycoproteins at the surfaces of BHK-21 cells infected with standard virus, a mixture of standard and defective interfering (DI) particles (mixed virus infection), and during persistent infection was investigated. The expression of HN and Fo was measured on the surfaces of infected cells by the binding of anti-HN and anti-Fo monoclonal antibodies. The results show that HN expression was restricted relative to Fo during mixed virus and persistent infections. The decreased levels of HN were investigated further by pulse-chase experiments which revealed that HN has an increased turnover rate in persistently infected cells and, to a lesser extent, in mixed virus infected cells. In analyzing the [35S]methionine-labeled protein composition of virus particles produced during the pulse-chase experiments, the increased turnover of newly synthesized HN was found to correlate with its decreased incorporation into virus particles. Interestingly, the poor HN incorporation also correlates with less efficient incorporation of the matrix M protein into virus particles.

Animals↗
PubMed1984

Structural and functional analysis of Sendai virus nucleocapsid protein NP with monoclonal antibodies.

Monoclonal antibodies specific for Sendai virus nucleocapsid protein NP were used to map the antigenic structure of NP and to investigate the role of NP in transcription. Using nine anti-NP antibodies in competitive-binding (CB) assays, it was found that the NP molecule contained at least two topographically distinct antigenic sites. By Western blot analysis, one of the NP epitopes belonging to antigenic site I was localized to a Mr 34,000 (34K) trypsin digest fragment, and another to a Mr 48,000 (48K) fragment which remained associated with the nucleocapsid. The other antibodies which define antigenic site I did not react with either fragment; however, the results of CB would indicate that their epitopes were in a region on the tertiary structure of the NP molecule that is closely proximal to these fragments. The 48K and 34K fragments on the published NP amino acid sequence have been tentatively identified. Since the 34K and 48K fragments bind antibody, it appears that nucleocapsid-bound NP may be folded into a configuration which places at least some of these sequences on the surface of the nucleocapsid structure. Six antibodies representing both antigenic sites were purified for functional studies. All the antibodies inhibited nucleocapsid transcription in vitro to the same extent (greater than 90%); however, they differed in the amount of antibody required to produce the same effect. Within site I, antibodies producing maximum inhibition were divided into three groups: three antibodies inhibited at relatively low concentrations (0.17 microgram), one antibody inhibited at an intermediate range (0.43 microgram), and another required a 10-fold higher concentration (1.73 microgram) to produce the same effect. The antibody which detected the 48K trypsin digest fragment was the one which fell into the intermediate range for transcription inhibition, while the antibody that detected the 34K fragment was in the low range. Thus, antigenic site I, defined by CB and trypsin digestion studies, can be defined further into three subsites which appear to differ in their involvement in the transcription process. Antigenic site II was defined by a single antibody which also inhibited transcription by greater than 90%.

Antibodies, Monoclonal↗
PubMed1982

Synthesis of message and genome RNAs in vitro by Sendai virus-infected cell nucleocapsids.

Purified Sendai virus nucleocapsids isolated from infected cells were used to programme a transcription system in vitro to study virus-specific RNA synthesis. The RNA products were analysed for size by centrifugation before and after denaturation with formamide or glyoxal. The polarity of the products [message (+) or genome (-) strands] was analysed by RNA-RNA hybridization. The non-denatured RNA products sedimented in three groups: 7S to 22S single-stranded RNA transcripts and two partially ribonuclease-resistant complexes. One complex, representing 12% of the total product, sedimented at 26S to 36S. After denaturing the 26S to 36S complex to single-stranded molecules, about half of the RNAs sedimented at 25S to 54S and about half at 6S to 24S. The second complex, representing about 13% of the total RNA product, sedimented at 42S to 52S. After denaturing, about 10% of the single-stranded RNAs sedimented at 38S to 52S and about 90% sedimented at 6S to 19S. In hybridization studies, single-stranded RNAs that sedimented at less than 19S were predominantly of message sense (+ strand), whereas RNAs that sedimented at 25S to 54S were a mixture of genome and anti-genome type. These results show that transcription and replication activities in vitro were associated with Sendai virus nucleocapsids obtained from infected cells and that some of the reaction products approached genome size.

Animals↗
PubMed1979

Topography of a flexible ribonucleoprotein helix: protein-protein contacts in Sendai virus nucleocapsids.

Contacts among the three polypeptide species in the flexible helical nucleocapsids of a paramyxovirus were examined with bifunctional protein cross-linking reagents. Polypeptides L and P, minor components of Sendai virus nucleocapsids implicated in viral RNA polymerase activity, were efficiently cross-linked into large complexes, indicating that they enjoy abundant contacts with neighboring protein molecules in the helix. Less reactivity was found in the case of the major structural polypeptide, NP; about half of all molecules of NP formed large cross-linked complexes, most of the rest remaining as monomers along with a small proportion of homodimers and low-order oligomers. Marked heterogeneity in the cross-linking reactivity of NP molecules, which may reflect the conformational quasi-equivalence inherent in a flexible helix, was indicated by the production of several conformers of homodimers and other low-order oligomers of NP, and by failure of the kinetics of NP cross-linking to conform to a simple statistical model of random polmerization. The validity of the statistical model was shown by cross-linking experiments with the rigid helical virus, tobacco mosaic virus.

Capsid↗
PubMed1976

Synthesis of Sendai virus polypeptides by a cell-free extract from wheat germ.

The '18S' RNA population from Sendai virus-infected cells efficiently directed the synthesis of three virus-specific polypeptides P, NP and M, in wheat germ cell-free extracts. In agreement with previous results obtained with a reticulocyte extract, there was little or no production of virus glycopolypeptides. Analyses of tryptic peptides revealed close correspondence between the primary structures of NP and M made in vitro and the authentic virus polypeptides.

Cell-Free System↗