scholarly journals Adsorptive Endocytosis of California Encephalitis Virus into Mosquito and Mammalian Cells: A Role for G1

Virology ◽  
1997 ◽  
Vol 235 (1) ◽  
pp. 40-47 ◽  
Author(s):  
Jill K. Hacker ◽  
James L. Hardy
Keyword(s):  
Mammalian Cells ◽  
Encephalitis Virus ◽  
Adsorptive Endocytosis

St. Louis encephalitis virus infection of a mosquito vector

1972 ◽  
Vol 30 ◽  
pp. 312-313
Author(s):  
Sylvia G. Whitfield ◽  
Frederick A. Murphy ◽  
W. Daniel Sudia
Keyword(s):  
Mammalian Cells ◽  
Culex Pipiens ◽  
Ultrastructural Level ◽  
The United States ◽  
Encephalitis Virus ◽  
Mosquito Vector ◽  
Plasma Viremia ◽  
Equine Encephalitis Virus ◽  
Group A ◽  
Group B

The course of arbovirus infections in the arthropods necessary for their transmission in nature has been studied extensively, but in only a few instances at the ultrastructural level. In the present study a model system was established for the infection of Culex mosquitoes with St. Louis encephalitis (SLE) virus (arbovirus group B ), the most important mosquito-bome disease in the United States. Results were compared with those of a previous study of Eastern equine encephalitis virus (arbovirus group A) in Aedes mosquitoes (this Proceedings, 1969, pp. 378-379) since differences in sites of maturation in mammalian cells have been found between the two groups.Culex pipiens mosquitoes were infected by feeding upon suckling mice infected with SLE virus (plasma viremia titer 105.4 SMLD50/ml). Mosquitoes were then held at 27°C; they were tested for capacity to transmit virus via bite, and specimens of salivary glands and other organs were collected at 18 intervals during 32 days.

scholarly journals Expression and Self-Assembly of Norwalk Virus Capsid Protein from Venezuelan Equine Encephalitis Virus Replicons

2002 ◽  
Vol 76 (6) ◽  
pp. 3023-3030 ◽  
Author(s):  
Ralph S. Baric ◽  
Boyd Yount ◽  
Lisa Lindesmith ◽  
Patrick R. Harrington ◽  
Shermalyn R. Greene ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Norwalk Virus ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Large Numbers ◽  
Virus Capsid Protein

ABSTRACT The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.

scholarly journals Nuclear Localization of Japanese Encephalitis Virus Core Protein Enhances Viral Replication

2005 ◽  
Vol 79 (6) ◽  
pp. 3448-3458 ◽  
Author(s):  
Yoshio Mori ◽  
Tamaki Okabayashi ◽  
Tetsuo Yamashita ◽  
Zijiang Zhao ◽  
Takaji Wakita ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Cell Lines ◽  
Japanese Encephalitis ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Core Protein ◽  
Encephalitis Virus ◽  
Wild Type ◽  
The Core

ABSTRACT Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly42 and Pro43 in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly42 and Pro43 were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.

scholarly journals A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts

2015 ◽  
Vol 90 (5) ◽  
pp. 2676-2689 ◽  
Author(s):  
Mélissanne de Wispelaere ◽  
Cécile Khou ◽  
Marie-Pascale Frenkiel ◽  
Philippe Desprès ◽  
Nathalie Pardigon
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Encephalitis Virus ◽  
Mutant Virus ◽  
M Protein ◽  
Single Amino Acid ◽  
Progeny Virus

ABSTRACTJapanese encephalitis virus (JEV) membrane (M) protein plays important structural roles in the processes of fusion and maturation of progeny virus during cellular infection. The M protein is anchored in the viral membrane, and its ectodomain is composed of a flexible N-terminal loop and a perimembrane helix. In this study, we performed site-directed mutagenesis on residue 36 of JEV M protein and showed that the resulting mutation had little or no effect on the entry process but greatly affected virus assembly in mammalian cells. Interestingly, this mutant virus had a host-dependent phenotype and could develop a wild-type infection in insect cells. Experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant expresses structural proteins but fails to form infectious particles in mammalian cells. Using a mouse model for JEV pathogenesis, we showed that the mutation conferred complete attenuationin vivo. The production of JEV neutralizing antibodies in challenged mice was indicative of the immunogenicity of the mutant virusin vivo. Together, our results indicate that the introduction of a single mutation in the M protein, while being tolerated in insect cells, strongly impacts JEV infection in mammalian hosts.IMPORTANCEJEV is a mosquito-transmitted flavivirus and is a medically important pathogen in Asia. The M protein is thought to be important for accommodating the structural rearrangements undergone by the virion during viral assembly and may play additional roles in the JEV infectious cycle. In the present study, we show that a sole mutation in the M protein impairs the JEV infection cycle in mammalian hosts but not in mosquito cells. This finding highlights differences in flavivirus assembly pathways among hosts. Moreover, infection of mice indicated that the mutant was completely attenuated and triggered a strong immune response to JEV, thus providing new insights for further development of JEV vaccines.

scholarly journals Regulated IRE1-dependent decay pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication

2014 ◽  
Vol 95 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Sankar Bhattacharyya ◽  
Utsav Sen ◽  
Sudhanshu Vrati
Keyword(s):  
Japanese Encephalitis Virus ◽  
Unfolded Protein Response ◽  
Japanese Encephalitis ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Encephalitis Virus ◽  
Host Cells ◽  
Rnase Activity ◽  
Unfolded Protein ◽  
Protein Response

Japanese encephalitis virus (JEV) infection-induced encephalitis causes extensive death or long-term neurological damage, especially among children, in south and south-east Asia. Infection of mammalian cells has shown induction of an unfolded protein response (UPR), presumably leading to programmed cell death or apoptosis of the host cells. UPR, a cellular response to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, is initiated by three ER-lumen-resident sensors (PERK, IRE1 and ATF6), and involves transcriptional and translational regulation of the expression of several genes. The sensor IRE1 possesses an intrinsic RNase activity, activated through homo-dimerization and autophosphorylation during UPR. Activated IRE1 performs cytoplasmic cleavage of Xbp1u transcripts, thus facilitating synthesis of XBP1S transcription factor, in addition to cleavage of a cohort of cellular transcripts, the later initiating the regulated IRE1-dependent decay (RIDD) pathway. In this study, we report the initiation of the RIDD pathway in JEV-infected mouse neuroblastoma cells (Neuro2a) and its effect on viral infection. Activation of the RIDD pathway led to degradation of known mouse cell target transcripts without showing any effect on JEV RNA despite the fact that both when biochemically purified showed significant enrichment in ER membrane-enriched fractions. Additionally, inhibition of the IRE1 RNase activity by STF083010, a specific drug, diminished viral protein levels and reduced the titre of the virus produced from infected Neuro2a cells. The results present evidence for the first report of a beneficial effect of RIDD activation on the viral life cycle.

scholarly journals N-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cells

2013 ◽  
Vol 94 (10) ◽  
pp. 2249-2258 ◽  
Author(s):  
Kentaro Yoshii ◽  
Natsumi Yanagihara ◽  
Mariko Ishizuka ◽  
Mizuki Sakai ◽  
Hiroaki Kariwa
Keyword(s):  
Envelope Protein ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Consensus Sequence ◽  
Encephalitis Virus ◽  
Secretory Process ◽  
Virus Envelope ◽  
Tick Borne Encephalitis ◽  
Virus Envelope Protein ◽  
Tick Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a zoonotic disease agent that causes severe encephalitis in humans. The envelope protein E of TBEV has one N-linked glycosylation consensus sequence, but little is known about the biological function of the N-linked glycan. In this study, the function of protein E glycosylation was investigated using recombinant TBEV with or without the protein E N-linked glycan. Virion infectivity was not affected after removing the N-linked glycans using N-glycosidase F. In mammalian cells, loss of glycosylation affected the conformation of protein E during secretion, reducing the infectivity of secreted virions. Mice subcutaneously infected with TBEV lacking protein E glycosylation showed no signs of disease, and viral multiplication in peripheral organs was reduced relative to that with the parental virus. In contrast, loss of glycosylation did not affect the secretory process of infectious virions in tick cells. Furthermore, inhibition of transport to the Golgi apparatus affected TBEV secretion in mammalian cells, but not in tick cells, indicating that TBEV was secreted through an unidentified pathway after synthesis in endoplasmic reticulum in tick cells. These results increase our understanding of the molecular mechanisms of TBEV maturation.

scholarly journals Evaluation of Extracellular Subviral Particles of Dengue Virus Type 2 and Japanese Encephalitis Virus Produced by Spodoptera frugiperda Cells for Use as Vaccine and Diagnostic Antigens

2010 ◽  
Vol 17 (10) ◽  
pp. 1560-1566 ◽  
Author(s):  
Miwa Kuwahara ◽  
Eiji Konishi
Keyword(s):  
Dengue Virus ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Spodoptera Frugiperda ◽  
Mammalian Cells ◽  
Encephalitis Virus ◽  
Commercial Application ◽  
Sf9 Cells ◽  
High Level ◽  
Level Production

ABSTRACT New or improved vaccines against dengue virus types 1 to 4 (DENV1 to DENV4) and Japanese encephalitis virus (JEV), the causative agents of dengue fever and Japanese encephalitis (JE), respectively, are urgently required. The use of noninfectious subviral extracellular particles (EPs) is an inexpensive and safe strategy for the production of protein-based flavivirus vaccines. Although coexpression of premembrane (prM) and envelope (E) proteins has been demonstrated to produce EPs in mammalian cells, low yields have hindered their commercial application. Therefore, we used an insect cell expression system with Spodoptera frugiperda-derived Sf9 cells to investigate high-level production of DENV2 and JEV EPs. Sf9 cells transfected with the prM and E genes of DENV2 or JEV secreted corresponding viral antigens in a particulate form that were biochemically and biophysically equivalent to the authentic antigens obtained from infected C6/36 mosquito cells. Additionally, equivalent neutralizing antibody titers were induced in mice immunized either with EPs produced by transfected Sf9 cells or with EPs produced by transfected mammalian cells, in the context of coimmunization with a DNA vaccine that expresses EPs. Furthermore, the results of an enzyme-linked immunosorbent assay (ELISA) using an EP antigen derived from Sf9 cells correlated significantly with the results obtained by a neutralization test and an ELISA using an EP antigen derived from mammalian cells. Finally, Sf9 cells could produce 10- to 100-fold larger amounts of E antigen than mammalian cells. These results indicate the potential of Sf9 cells for high-level production of flavivirus protein vaccines and diagnostic antigens.

scholarly journals Nuclear Import and Export of Venezuelan Equine Encephalitis Virus Nonstructural Protein 2

2007 ◽  
Vol 81 (19) ◽  
pp. 10268-10279 ◽  
Author(s):  
Stephanie A. Montgomery ◽  
Robert E. Johnston
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nonstructural Protein ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Nonstructural Protein 2

ABSTRACT Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-α1 but not with karyopherin-α2, -3, or -4, suggesting that karyopherin-α1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.

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