Infection Of Chick Embryo Cells Research Articles

Transcriptional response of chicken embryo cells to Newcastle disease virus (D58 strain) infection

Newcastle disease virus (NDV), the causative agent of Newcastle disease (ND) in chicken causes significant economic loss for the poultry industry worldwide. The mechanism involved in host response to NDV infection is not well understood. For better understanding of the virus-host interaction; transcriptional profile of some genes of chicken embryo (CE) cells infected with NDV vaccine strain D58 was established using quantitative RT-PCR SYBR Green method. The relative standard curve method was used to measure the level of transcripts of the cellular genes against an endogenous control (β actin) gene. Among the genes studied, IFN α, IFN γ, MHC I and DDX 1 were up-regulated while IL 6 was down regulated. The expression of viral genes (M and F) in the infected CE cells was also confirmed by relative quantification. The host cellular genes involved in pro-inflammatory response, interferon-regulated proteins and the cellular immune response were affected by NDV infection, indicating involvement of complex signaling pathways of host cell responses to the infection. Thus, this study contributes to the understanding of the pathogenesis of ND and provides an insight into the virus-host interaction.

Apoptosis is induced by infectious bursal disease virus replication in productively infected cells as well as in antigen-negative cells in their vicinity.

The kinetics of infectious bursal disease virus (IBDV) replication and induction of apoptosis were investigated in vitro and in vivo. After infection of chicken embryo (CE) cells with IBDV strain Cu-1, the proportion of apoptotic cells increased from 5.8% at 4 h post-infection (p.i.) to 64.5% at 48 h p.i. The proportion of apoptotic cells correlated with IBDV replication. UV-inactivated IBDV particles did not induce apoptosis. Double labelling revealed that, early after infection, the majority of antigen-expressing cells were not apoptotic; double-labelled cells appeared more frequently at later times. Remarkably, apoptotic cells were frequently located in the vicinity of antigen-expressing cells. This indicated that an apoptosis-inducing factor(s) might be released by cells that replicate IBDV. Since interferon (IFN) production has been demonstrated after IBDV infection, IFN was considered to be one of several factors. However, supernatants of infected CE cells in which virus infectivity had been neutralized were not sufficient to induce apoptosis. Similar results were observed in the infected bursae of Fabricius: early after infection, most of the cells either showed virus antigens or were apoptotic. Again, double-labelled cells appeared more frequently late after infection. This suggests that indirect mechanisms might also be involved in the induction of apoptosis in vivo, contributing to the rapid depletion of cells in the IBDV-infected bursa.

Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins.

A large proportion of the Newcastle disease virus (NDV) matrix (M) protein is found in the nuclei of infected chicken embryo cells. Kinetic analysis indicated that much of the M protein enters the nucleus early in infection, concentrating in discrete regions of the nucleus and remaining there throughout infection. The M protein was found in localized regions of the nuclei of a variety of cell lines infected with NDV. Immunostaining for both M protein and nucleolar antigens indicated that most of these regions represent nucleoli. Moreover, this nucleolar localization of the M protein was observed in chicken embryo cells infected with 11 different strains of NDV. Only the M protein of strain HP displayed a modified pattern, concentrating in the nucleolus early in infection but in the cytoplasm late in infection. M protein transiently expressed in COS-1 cells also localized to the nucleus and nucleolus, indicating that the M protein does not require other NDV proteins for this localization.

Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions

The fusion activity of flaviviruses [tick-borne encephalitis (TBE) virus and Japanese encephalitis virus] was assessed by inducing fusion from without of C6/36 mosquito cells with purified virus preparations. Membrane fusion and polykaryocyte formation was observed only after incubating the viruses at acidic pH. Two groups of monoclonal antibodies reacting with distinct non-overlapping antigenic domains on the TBE virus protein E inhibited fusion from without. One of these domains contains the most highly conserved and putative fusion-active sequence of the flavivirus protein E. Of five TBE virus monoclonal antibody escape mutants, each defined by a single amino acid substitution in the envelope protein E, one revealed a reduced fusion activity and another one a lower pH threshold. TBE virus grown in the presence of ammonium chloride as well as Langat virus purified from the supernatant of infected chick embryo cells contained the precursor of protein M (prM) rather than M itself. These 'immature' virions did not cause fusion from without, suggesting that the proteolytic processing of prM may be necessary for the generation of fusion-competent virions.

Transcription of proto-oncogenes in Rous sarcoma virus infected and transformed chicken embryo cells.

To evaluate an effect of infection by RSV on the transcriptional activity of proto-oncogenes, we tested 19 cellular oncogenes and the beta-actin gene performing "nuclear run on transcription assays" using nuclei isolated from RSV infected or transformed chick embryo cells (CEC). A drastic increase in the transcriptional activity of the c-fgr proto-oncogene and a slight increase of c-fos and c-myc could be observed in uninfected compared to RSV infected CEC. To distinguish between infection- and transformation-dependent alterations we used the transformation-defective temperature-sensitive RSV mutant strain NY 68. Ts NY 68 infected CEC maintained at the permissive temperature of 36 degrees C and exhibiting a transformed phenotype, displayed no significant differences in the transcriptional activity of the 19 proto-oncogenes when compared to ts NY 68 infected CEC maintained at the nonpermissive temperature of 42 degrees C and exhibiting a normal phenotype. We conclude that the alterations observed were caused by the viral infection and are not due to the process of transformation. No significant changes in the transcription rate of the investigated genes could be observed after shifting ts NY 68 infected CEC from 42 degrees C to 36 degrees C. In contrast, a shift of ts NY 68 infected CEC from 36 degrees C to 42 degrees C results, after 2 hours, in a transient increase of the c-fos transcription.

Temperature-sensitive mutants of Newcastle disease virus altered in HN glycoprotein size, stability, or antigenic maturity

It has been suggested that the 11 group B, C, and BC temperature-sensitive (ts) mutants of Newcastle disease virus (NDV), strain Australia-Victoria (AV-WT), have lesions in the gene for the hemagglutinin/neuraminidase glycoprotein (HN), and that complementation between groups B and C is intracistronic. Virions produced by these mutants even at permissive temperature contain greatly reduced amounts of HN, and the accompanying hemagglutinating and neuraminidase functions. To explore the basis for decreased HN incorporation into virions and the temperature sensitivity of these mutants, infected chick embryo cells were examined for changes in HN characteristics. The HN of two of the mutants was clearly altered in electrophoretic migration rates in both virions and infected cells. The migrational differences were not due to differences in glycosylation because altered migration rates were also observed in the presence of tunicamycin. In all cases, cells infected by these mutants produced as much HN as did AV-WT-infected cells, but the HN of six of these mutants was metabolically unstable. All of the mutants, including those with metabolically stable HN, exhibited greatly restricted ability to convert HN to an antigenically reactive form, indicating an early block in processing. For most of these mutants, the neuraminidase activities of infected cells were somewhat temperature sensitive, but the production of hemadsorbing activities on cell surfaces was not temperature sensitive. In contrast, the hemadsorbing and neuraminidase activities of cells infected by one mutant, BC2, were temperature sensitive, probably a reflection of the previously described extreme thermolability of the HN of this mutant. The relationship between these mutant characteristics, their temperature sensitivity and the virion phenotypes, is discussed. The data presented here confirm the assignment of these 11 group B, C, and BC mutants to defects in HN and begin to separate them into groups with different characteristics.

Interference between influenza A viruses with a cleavable and a noncleavable hemagglutinin; pH-stability after mixed infection

The infectivity of influenza A viruses like fowl plague virus (FPV) with a cleaved hemagglutinin (HA) is highly sensitive to treatment at pH 5, while strains like PR 8 or virus N with a noncleaved HA survive under this condition. After double infection of chick embryo cells with FPV and PR 8 or virus N, the yield of virus with the HA gene of FPV is greatly reduced. However, it can now survive treatment at pH 5, and the surviving FPV particles form plaques only in the presence of trypsin, indicating that they were coated by the HA of PR 8 or virus N, depending on the coinfecting virus. The results are discussed with respect to the build-up and maintenance of a large reservoir of nonpathogenic influenza A viruses with noncleavable HA in water fowl.

The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses

In an attempt to assess the importance of the nucleoprotein (NP) in the determination of host specificity, a series of experiments was performed on influenza A viruses of the H3N2 subtype. We have examined rescue of mutants of A/FPV/Rostock/34 with temperature-sensitive (ts) lesions in the nucleoprotein (NP) gene by double infection of chick embryo cells with H3N2 strains isolated from different species. The is mutants could be rescued by all avian H3N2 strains but not by any of the human H3N2 isolates. Only two of the swine H3N2 strains tested were able to rescue our mutants. The NP gene of these two swine isolates resembled the NP gene of the avian strains genetically in the hybridization test. However, their NPs reacted differently with a set of monoclonal antibodies when compared with NPs of avian H3N2 strains. Concerning multiplication in ducks they behaved like the other swine and human strains. The phosphopeptide fingerprints of all swine isolates tested were alike and were different from those of human or avian origin. Our observations are compatible with the idea that human H3N2 strains might not be able to cross the species barrier to birds directly, and possibly also not the other way around, without prior reassortment in pigs, which seem to have a broader host range concerning the compatibility of the NP gene in reassortants.

Location of influenza virus M, NP and NS1 proteins in microinjected cells.

When microinjected as cloned DNA, the nucleoprotein (NP) of influenza virus A/NT/60/68 (H3N2) accumulated in the nuclei of Xenopus laevis oocytes, and cultured cells of rodent and primate origin. This accumulation appeared to be specific and a property of the NP itself (or conceivably NP in association with unknown cellular constituents) since no other influenza virus components were present in DNA-injected cells. In the oocyte nucleus, clonally derived NP achieved an eightfold concentration over that in the cytoplasm. Such NP was full-length as judged by its mobility during PAGE and had the native conformation of H3N2 virus NP according to its reaction with a panel of monoclonal antibodies. NP appeared to be in the soluble fraction of the nucleus as it did not sediment under conditions which removed particulate matter from nuclear extracts. Microinjection of extracts of chick embryo fibroblast cells infected with A/FPV/Rostock/34 (H7N1) showed that exogenous NP had an affinity for the nucleus similar to that synthesized intracellularly from cloned NP DNA. This conclusion was supported by an experiment in which cloned NP from the oocyte nucleus re-entered the nucleus after injection into the cytoplasm of fresh oocytes. Injection of mRNA, extracted from chick embryo fibroblast cells infected with A/FPV/Rostock/34, into oocytes directed the synthesis of the viral proteins M (Mr 28 000), and NS1 (Mr 27 000) as well as NP (Mr 56 000). While NP from this source concentrated in the nucleus as before, M merely associated with the nucleus without exceeding the cytoplasmic level. Even more remarkable was NS1; although in injected cells this protein is concentrated in nucleoli, in microinjected oocytes its nuclear concentration was threefold less than that in the cytoplasm, despite the very large number (greater than 1500) of nucleoli present in Xenopus oocytes. It seems likely that the karyophilic nature of M and NS1, unlike that of NP, is a property not of the proteins themselves, but of a complex which they form with some other product of the infected cell. These findings were repeated when extracts from infected chick embryo cells containing NP, M and NS1 proteins radiolabelled in vivo, were injected into the cytoplasm of oocytes.

A minimal structural requirement for proteolytic cleavability of HA and for pathogenicity of avian influenza viruses

The nucleotide sequences of the connecting peptide regions on the HA of pathogenic avian strain A/fowl/Victoria/75 and of three variant strains were determined by dideoxy sequencing of reverse transcripts of virion RNA primed with synthetic oligonucleotides. The deduced amino acid sequence of the connecting peptide of the wild type HA is Lys-Lys-Arg-Glu-Lys-Ary-Gly (with Gly representing the NHZ-terminal residue of the HA2 subunit). All three variant strains, obtained by diluted serial passages in chicken embryo cells (CEC), showed the same single base change in the connecting peptide. The resulting amino acid change, which was unexpectedly located C-terminal to Glu, rendered the HA of the variants noncleavable in infected CEC and the viruses were no longer pathogenic for chickens. The biochemical/biological implications of these results are discussed. 143

Demonstration of Endocytosis during Newcastle Disease Virus Infection of Chicken Embryo Cell Cultures

A study utilizing thin sectioned cells and electron microscopy demonstrated that Newcastle Disease virus (NDV) penetrates chicken embryo cells by adsorptive endocytosis. The penetrated virus was then observed to localize in lysosomal vacuoles. It was found that treatment of infected cells with 500 Ag/ml of ammonium chloride (NH4C1, lysosomotropic agent) caused 87% inhibition of progeny synthesis and inhibition of cellular hemagglutinin synthesis, while not interfering with viral adsorption to cells. Electron microscopic studies indicated that NH4C1 treatment of infected cells interfered with NDV uncoating in lysosomal vacuoles. Viruses replicate in host cells by diverting the biosynthetic pathways of cells to production of virus progeny. To infect a cell, a virus must transfer its genome from the extracellular space to the cytoplasm. This process requires transport through one or more membrane barriers. In contrast to the detailed knowledge of the later stages of viral infection, little is known about the entry of animal viruses into host cells. For enveloped viruses, two pathways of entry are generally observed by electron microscopy: a) fusion between the viral membrane and the host cell plasmalemma, where the viral genome is delivered into the cytoplasm; and b) an endocytotic route, where the phagocytized or viroplexed virions are taken into the cytoplasm in endocytotic vesicles and delivered to intracellular organelles. The present report is a study to ascertain the mode of entry of Newcastle Disease (NDV) upon infection of chicken embryo cells as determined by electron microscopy of thin sectioned cells. The study also included the effect of ammonium chloride (NH4C1), a lysosomotropic agent, on NDV infection of chicken embryo cells. The events studied were virion binding, cellular penetration, virion localization in cellular lysosomes and progeny synthesis.

Induction of stress proteins in Sindbis virus- and vesicular stomatitis virus-infected cells

Two proteins which are related to certain proteins induced by hyperthermia (heat shock proteins; hsp) are synthesized during lytic infection of chick embryo (CE) cells by Sindbis virus or vesicular stomatitis virus (VSV). Incubation of the infected cells at elevated temperature further increased the rate of synthesis of these proteins. The stress proteins induced by Sindbis virus had different mobilities on SDS-polyacrylamide gels compared to related stress proteins induced in mock-infected CE cells. Induction of the stress proteins in Sindbis virus- and VSV-infected CE cells was actinomycin D sensitive. Kinetic studies indicated that induction of the stress proteins is an early event during infection. The lytic virus-induced selective termination of host protein synthesis did not affect the synthesis of these proteins. Furthermore, the synthesis of these virus-induced stress proteins was resistant, relative to the synthesis of most host proteins, to alterations in the intracellular concentrations of Na + and K +. The synthesis of a protein related to a major low-molecular-weight hsp of CE cells was not induced after Sindbis virus or VSV infection. Immunoprecipitation experiments and sedimentation analyses demonstrated that significant levels of the capsid protein (C) of Sindbis virus and nucleocapsid protein (N) of VSV are physically associated with a hsp in lysates of infected CE cells.

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.

Characterization of the virus isolated from HeLa cells persistently infected with Sendai virus (HVJ).

Microbiology and ImmunologyVolume 27, Issue 2 p. 207-211 NoteFree Access Characterization of the Virus Isolated from HeLa Cells Persistently Infected with Sendai Virus (HVJ) Tetsuya Yoshida, Tetsuya Yoshida Department of Bacteriology, Nagoya, Cancer Research Institute, Aichi, 466Search for more papers by this authorMichinari Hamaguchi, Michinari Hamaguchi Department of Virology and Cell Biology, Cancer Research Institute, Aichi, Nagoya, 466Search for more papers by this authorHiroshi Naruse, Hiroshi Naruse Department of Pediatrics, Nagoya University School of Medicine, Aichi, Nagoya, 466Search for more papers by this authorKazuo Nishikawa, Kazuo Nishikawa Department of Pediatrics, Nagoya University School of Medicine, Aichi, Nagoya, 466Search for more papers by this authorYoshiyuki Nagai, Yoshiyuki Nagai Department of Virology and Cell Biology, Cancer Research Institute, Aichi, Nagoya, 466Search for more papers by this author Tetsuya Yoshida, Tetsuya Yoshida Department of Bacteriology, Nagoya, Cancer Research Institute, Aichi, 466Search for more papers by this authorMichinari Hamaguchi, Michinari Hamaguchi Department of Virology and Cell Biology, Cancer Research Institute, Aichi, Nagoya, 466Search for more papers by this authorHiroshi Naruse, Hiroshi Naruse Department of Pediatrics, Nagoya University School of Medicine, Aichi, Nagoya, 466Search for more papers by this authorKazuo Nishikawa, Kazuo Nishikawa Department of Pediatrics, Nagoya University School of Medicine, Aichi, Nagoya, 466Search for more papers by this authorYoshiyuki Nagai, Yoshiyuki Nagai Department of Virology and Cell Biology, Cancer Research Institute, Aichi, Nagoya, 466Search for more papers by this author First published: February 1983 https://doi.org/10.1111/j.1348-0421.1983.tb03575.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1Kimura, Y., Ito, Y., Shimokata, K., Nishiyama, Y., Nagata, I., and Kitoh, J. 1975. Temperature-sensitive virus derived from BHK cells persistently infected with HVJ (Sendai virus). J. Virol. 15: 55– 63. 2Maeno, K., Yoshii, S., Nagata, I., and Matsumoto, T. 1966. Growth of Newcastle disease virus in a HVJ carrier culture of HeLa cells. Virology 29: 255– 263. 3Naruse, H., Nagai, Y., Yoshida, T., Hamaguchi, M., Matsumoto, T., Isomura, S., and Suzuki, S. 1981. The polypeptides of munps virus and their synthesis in infected chick embryo cells. Virology 112: 119– 130. 4Ogura, H., Sato, H., and Hatano, M. 1981. Temperature-sensitive HVJ (Sendai virus) with altered P polypeptide derived from persistently infected cell lines. J. Gen. Virol. 55: 469– 473. 5Roux, L., and Holland, J.J. 1979. Role of defective interfering particles of Sendai virus in persistent infections. Virology 93: 91– 103. 6Sato, H., Ogura, H., and Hatano, M. 1981. An intracellular interaction between a temperature- sensitive mutant and the original wild-type HVJ (Sendai virus) is responsible for the establishment and maintenance of HVJ persistent infection. J. Gen. Virol. 55: 459– 468. 7Yoshida, T., Hamaguchi, M., Naruse, H., and Nagai, Y. 1982. Persistent infection by a temperature-sensitive mutant isolated from a Sendai virus (HVJ) carrier culture: Its initiation and maintenance without aid of defective interfering particles. Virology 120: 329– 339. 8Yoshida, T., Nagai, Y., Maeno, K., Iinuma, M., Hamaguchi, M., Matsumoto, T., Nagayoshi, S., and Hoshino, M. 1979. Studies on the role of M protein in virus assembly using a ts mutant of HVJ (Sendai virus). Virology 92: 139– 154. Citing Literature Volume27, Issue2February 1983Pages 207-211 ReferencesRelatedInformation

Inhibition of formation of complex oligosaccharides by the glucosidase inhibitor bromoconduritol.

The alpha-glucosidase inhibitor bromoconduritol (6-bromo-3,4,5-trihydroxycyclohex-1-ene) inhibits trimming of the innermost glucose residue from the Glc3Man9GlcNAc2 precursor of high-mannose and complex oligosaccharides. This inhibition occurs both in intact cells and with a microsomal enzyme preparation. The formation of lipid-linked oligosaccharides was increased in glucosidase-inhibited cells. Inhibition of transfer of high-mannose oligosaccharides to protein was not observed. In bromoconduritol-treated virus-infected cells, trimming of mannose can occur despite incomplete removal of glucose. The glucosylated high-mannose oligosaccharides GlcMan9GlcNAc, GlcMan8GlcNAc, and GlcMan7GlcNAc were released from viral glycoproteins after digestion with Pronase and endo-beta-N-acetylglucosaminidase H. The formation of complex oligosaccharides was concomitantly inhibited. The release of infectious fowl plague virus particles (an influenza virus) was inhibited from bromoconduritol-treated infected chicken-embryo cells.

The increase in hormone-stimulated adenylate cyclase activity following Rous sarcoma virus transformation.

Rous sarcoma virus (RSV)-infected chicken embryo cells were used to study the effect of viral transformation on the hormone-stimulated synthesis of cyclic AMP. Transformation by RSV greatly increased the cells' ability to synthesize and accumulate cyclic AMP in response to the beta-adrenergic agonist isoproterenol as compared to untransformed cells. This enhancement was observed in both intact cells and in membranes prepared from these cells. The inclusion of guanosine 5'-0-(3-thiotriphosphate), a nonhydrolyzable analogue of GTP, in assays of adenylate cyclase activity did not abolish the quantitative differences between the transformed and normal cell membranes. Infection of cells by Rous-associated virus, which lacks the oncogene src, did not induce this hyperresponsiveness thus indicating the probable involvement of the src gene product in this phenomenon. The duration of the isoproterenol-induced cyclic AMP elevation was longer in the transformed than in the untransformed cells; transformed cells, unlike untransformed cells, required at least 120 min before full desensitization became established. Membranes prepared from transformed cells specifically bound more than 5 times the quantity of the beta-adrenergic radiolabeled antagonist (-)3H-dihydroalprenolol and 125I-iodocyanopindolol compared to the untransformed cell membranes. Thus, it appears that major differences between the transformed and normal phenotypes reside in the concentration of membrane beta-adrenergic receptors and the inability of RSV-transformed cells to self-limit their response to specific external stimuli.

Kinetics of synthesis of influenza virus ribonucleoprotein structures.

The synthesis of influenza virus ribonucleoprotein structures (RNPs) in infected chick embryo cells was analysed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium deoxycholate which resolves the RNPs into five size classes. A relatively small proportion of total RNPs accumulated in the nucleus but free NP protein was found there in large amounts over the period 1.5 to 4 h post-infection. In contrast, by 4 h post-infection, all cytoplasmic NP was complexed into RNP structures. At early times, during at 15 min pulse of (35S]methionine, nearly all the newly synthesized NP was incorporated into RNPs but by 4 h the majority of pulse-labelled NP was present as free protein. However, the proportion of free NP: NP in RNPs remained constant over the 1.5 to 4 h post-infection period, indicating that there was a delay before the NP synthesized later in infection was assembled into RNP structures. Individual RNP size classes were predominantly cytoplasmic and accumulated at similar rates but were not produced in equimolar amounts. The rates of synthesis of individual RNPs were in general agreement with their rates of accumulation with the remarkable exception of RNP d (containing RNA 7, the matrix protein gene). This was synthesized nearly 10-fold faster but accumulated at the same rate as the other RNPs. Possibly RNP d is more rapidly degraded than the other RNPs.

Detection and quantitation of Newcastle disease virus proteins in infected chicken embryo cells.

A technique was analyzed by which Newcastle disease virus (NDV) proteins could be quantitatively detected in the presence of chicken embryo cellular proteins in NDV-infected cells. The technique involved removal of electropho-proteins from a sodium dodecyl sulfate-polyacrylamide-agarose gel matrix by chemical cleavage of the acrylamide gel cross-linker. The proteins were subsequently transferred and covalently bound to diazobenzyloxymethyl paper. By incubating the paper with unlabeled antisera and 125I-labeled Staphylococcus aureus protein A, the specificity of the antisera and the sensitivity of this method of quantitative antigen detection were tested. The results demonstrated that as little as 1 ng of an individual NDV protein could be detected. Furthermore, this technique can simultaneously quantitate the synthesis of multiple NDV proteins under experimental conditions in which immunofluorescence, hemadsorption, and plaque assays failed to show virus protein synthesis or the formation of virus progeny.

Productive infection of chick embryo cells by influenza viruses tightly bound on substratum.

To test whether penetration of influenza viruses could occur at the plasma membrane of host cells, virus particles were tightly bound on Concanavalin A-coated substratum of plastic culture plates and then overlayed with embryo cells. Under these conditions, endocytosis of the viruses was prevented but the cells were found to be effectively infected. The results indicate, that infection by influenza viruses can occur through fusion between the viral membrane and the host cell plasma membrane.