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Contribution of matrix, fusion, hemagglutinin, and large protein genes of the CAM-70 measles virus vaccine strain to efficient growth in chicken embryonic fibroblasts.

ABSTRACT: Attenuated live vaccines of measles virus (MV) have been developed from clinical isolates by serial propagation in heterologous cells, mainly chicken embryonic cells. The safety and effectiveness of these vaccines have been well established. However, the molecular mechanism of their attenuation remains a subject of investigation. The CAM-70 MV vaccine strain was developed from the Tanabe strain by serial propagation in chicken embryonic cells. In the present study, we assessed the contribution of each gene in the CAM-70 strain to efficient growth in chicken embryonic fibroblasts (CEF). We used a cloned MV IC323 based on the wild-type IC-B strain and generated a series of IC323s that possess one or more of the CAM-70 genes. Then, we examined the infection of CEF and CEF expressing human signaling lymphocyte activation molecule with the recombinant MVs. Our results demonstrated that MV needs to adapt to CEF at both the entry and postentry steps and that the CAM-70 matrix protein gene plays an important role in adaptation to CEF at the early stage of the virus replication cycle. The CAM-70 large protein gene was responsible for the efficient transcription and replication in CEF, and the CAM-70 hemagglutinin and fusion protein genes were responsible for efficient entry. Investigations focusing on these genes might elucidate unknown molecular mechanisms underlying the attenuation of MV.

SUBMITTER: Sharma LB

PROVIDER: S-EPMC2772719 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Contribution of matrix, fusion, hemagglutinin, and large protein genes of the CAM-70 measles virus vaccine strain to efficient growth in chicken embryonic fibroblasts.

Sharma Luna Bhatta LB Ohgimoto Shinji S Kato Seiichi S Kurazono Sekiko S Ayata Minoru M Takeuchi Kaoru K Ihara Toshiaki T Ogura Hisashi H

Journal of virology 20090902 22

Attenuated live vaccines of measles virus (MV) have been developed from clinical isolates by serial propagation in heterologous cells, mainly chicken embryonic cells. The safety and effectiveness of these vaccines have been well established. However, the molecular mechanism of their attenuation remains a subject of investigation. The CAM-70 MV vaccine strain was developed from the Tanabe strain by serial propagation in chicken embryonic cells. In the present study, we assessed the contribution o ...[more]

PMID: 19726516

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Project description:Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV.IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by a fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE.

Project description:Paramyxoviruses include several insidious and ubiquitous pathogens of humans and animals, with measles virus (MeV) being a prominent one. The MeV membrane fusion apparatus consists of a receptor binding protein (hemagglutinin [H]) tetramer and a fusion (F) protein trimer. Four globular MeV H heads are connected to a tetrameric stalk through flexible linkers. We sought here to characterize the function of a 17-residue H-head segment proximal to the stalk that was unresolved in all five MeV H-head crystal or cocrystal structures. In particular, we assessed whether its primary sequence and length are critical for proper protein oligomerization and intracellular transport or for membrane fusion triggering. Extensive alanine substitutions had no effect on fusion triggering, suggesting that sequence identity is not critical for this function. Excessive shortening of this segment reduced or completely abrogated fusion trigger function, while length compensation restored it. We then characterized the mechanism of function loss. Mutated H proteins were efficiently transported to the cell surface, but certain alterations enhancing linker flexibility resulted in accumulation of high-molecular-weight H oligomers. Some oligomers had reduced fusion trigger capacity, while others retained this function. Thus, length and rigidity of the unresolved head segment favor proper H tetramerization and counteract interactions between subunits from different tetramers. The structurally unresolved H-head segment, together with the top of the stalk, may act as a leash to provide the right degree of freedom for the heads of individual tetramers to adopt a triggering-permissive conformation while avoiding improper contacts with heads of neighboring tetramers.Understanding the molecular mechanism of membrane fusion triggering may allow development of new antiviral strategies. The fusion apparatus of paramyxoviruses consists of a receptor binding tetramer and a fusion protein trimer. Structural analyses of the receptor binding hemagglutinin-neuraminidases of certain paramyxoviruses suggest that fusion triggering is preceded by relocation of its head domains, facilitated by flexible linkers. Having noted a structurally unresolved 17-residue segment linking the globular heads to the tetrameric stalk of the measles virus hemagglutinin (H), we asked whether and how it may facilitate membrane fusion triggering. We conclude that, together with the top of the stalk, the flexible linker keeps H heads on a leash long enough to adopt a triggering-permissive conformation but short enough to limit roaming and improper contacts with heads of neighboring tetramers. All morbillivirus H-protein heads appear to be connected to their stalks through a "leash," suggesting a conserved triggering mechanism.

Project description:The study of adipogenesis is one of the most important areas for not only regulating meat quality, but production efficiency associated with fat accretion in the poultry species. Current in vitro models for avian adipogenesis require adipogenic inducers including dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), fatty acids, or insulin. However, problems still remain in these models for testing/screening potential nutritional, hormonal, and pharmaceutical factors because of interfering/overriding effects of the inducing factors. Therefore, the purpose of this study was to develop a simple in vitro method for avian adipogenesis. In this study, chicken serum (CS) and fetal bovine serum (FBS) were compared for adipogenic potential using chicken embryonic fibroblasts (CEF). Oil-red O staining at 4 d in culture of CEF under CS revealed that lipid droplet formation was increased by CS in a dose-dependent manner (0 to 10%). On the contrary, all concentrations of FBS (0 to 10%) alone did not show lipid droplet formation. In accordance with the morphological data of CEF, mRNA expression of genes involved in adipocyte differentiation/determination, fatty acid uptake, and triacylglycerol (TAG) synthesis, were most significantly up-regulated by 10% CS at d 4 compared to 1 or 5% CS. In addition, embryonic cells isolated from quail (QEF) at E5, duck (DEF) at E6, and turkey (TEF) at E6, were tested for adipogenic differentiation by media containing the same concentrations of CS. Similar to the morphological data from CEF, quantitative data of the Oil-red O staining showed that lipid droplet formation in QEF, DEF, and TEF was increased by CS in a dose-dependent manner (0 to 10%). The current study demonstrates that CS alone can induce adipogenesis on embryonic fibroblasts of various poultry species. By providing a new simple in vitro method of avian adipogenesis, diverse nutritional, hormonal, and pharmaceutical factors can be broadly and easily tested for scientific and industrial purposes.

Dataset Information

Contribution of matrix, fusion, hemagglutinin, and large protein genes of the CAM-70 measles virus vaccine strain to efficient growth in chicken embryonic fibroblasts.

Publications

Contribution of matrix, fusion, hemagglutinin, and large protein genes of the CAM-70 measles virus vaccine strain to efficient growth in chicken embryonic fibroblasts.

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