The Experts below are selected from a list of 1167 Experts worldwide ranked by ideXlab platform
Javier Benavente - One of the best experts on this subject based on the ideXlab platform.
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Avian Reovirus triggered apoptosis enhances both virus spread and the processing of the viral nonstructural muns protein
Virology, 2014Co-Authors: Javier Rodriguezgrille, Jose Martinezcostas, Lisa K Busch, Javier BenaventeAbstract:Avian Reovirus non-structural protein muNS is partially cleaved in infected chicken embryo fibroblast cells to produce a 55-kDa carboxyterminal protein, termed muNSC, and a 17-kDa aminoterminal polypeptide, designated muNSN. In this study we demonstrate that muNS processing is catalyzed by a caspase 3-like protease activated during the course of Avian Reovirus infection. The cleavage site was mapped by site directed mutagenesis between residues Asp-154 and Ala-155 of the muNS sequence. Although muNS and muNSC, but not muNSN, are able to form inclusions when expressed individually in transfected cells, only muNS is able to recruit specific ARV proteins to these structures. Furthermore, muNSC associates with ARV factories more weakly than muNS, sigmaNS and lambdaA. Finally, the inhibition of caspase activity in ARV-infected cells does not diminish ARV gene expression and replication, but drastically reduces muNS processing and the release and dissemination of progeny viral particles.
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Avian Reovirus μns protein forms homo oligomeric inclusions in a microtubule independent fashion which involves specific regions of its c terminal domain
Journal of Virology, 2010Co-Authors: Alberto Brandariznunez, Javier Benavente, Rebeca Menayavargas, Jose MartinezcostasAbstract:Members of the genus OrthoReovirus replicate in cytoplasmic inclusions termed viral factories. Compelling evidence suggests that the nonstructural protein μNS forms the matrix of the factories and recruits specific viral proteins to these structures. In the first part of this study, we analyzed the properties of Avian Reovirus factories and μNS-derived inclusions and found that they are nonaggresome cytoplasmic globular structures not associated with the cytoskeleton which do not require an intact microtubule network for formation and maturation. We next investigated the capacity of Avian Reovirus μNS to form inclusions in transfected and baculovirus-infected cells. Our results showed that μNS is the main component of the inclusions formed by recombinant baculovirus expression. This, and the fact that μNS is able to self-associate inside the cell, suggests that μNS monomers contain all the interacting domains required for inclusion formation. Examination of the inclusion-forming capacities of truncated μNS versions allowed us to identify the region spanning residues 448 to 635 of μNS as the smallest that was inclusion competent, although residues within the region 140 to 380 seem to be involved in inclusion maturation. Finally, we investigated the roles that four different motifs present in μNS(448-635) play in inclusion formation, and the results suggest that the C-terminal tail domain is a key determinant in dictating the initial orientation of monomer-to-monomer contacts to form basal oligomers that control inclusion shape and inclusion-forming efficiency. Our results contribute to an understanding of the generation of structured protein aggregates that escape the cellular mechanisms of protein recycling.
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Avian Reovirus sigmaa localizes to the nucleolus and enters the nucleus by a nonclassical energy and carrier independent pathway
Journal of Virology, 2009Co-Authors: Lorena Vazqueziglesias, Jose Martinezcostas, Irene Lostaleseijo, Javier BenaventeAbstract:Avian Reovirus sigmaA is a double-stranded RNA (dsRNA)-binding protein that has been shown to stabilize viral core particles and to protect the virus against the antiviral action of interferon. To continue with the characterization of this viral protein, we have investigated its intracellular distribution in Avian cells. Most sigmaA accumulates into cytoplasmic viral factories of infected cells, and yet a significant fraction was detected in the nucleolus. The protein also localizes in the nucleolus of transfected cells, suggesting that nucleolar targeting is not facilitated by the viral infection or by viral factors. Assays performed in both intact cells and digitonin-permeabilized cells demonstrate that sigmaA is able to enter the nucleus via a nucleoporin-dependent nondiffusional mechanism that does not require added cytosolic factors or energy input. These results indicate that sigmaA by itself is able to penetrate into the nucleus using a process that is mechanistically different from the classical nuclear localization signal/importin pathway. On the other hand, two sigmaA arginines that are necessary for dsRNA binding are also required for nucleolar localization, suggesting that dsRNA-binding and nucleolar targeting are intimately linked properties of the viral protein.
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crystal structure of the Avian Reovirus inner capsid protein sigmaa
Journal of Virology, 2008Co-Authors: Pablo Guardadocalvo, Jose Martinezcostas, Javier Benavente, Lorena Vazqueziglesias, Antonio L Llamassaiz, Guy Schoehn, G C Fox, Lois X Hermoparrado, Mark J Van RaaijAbstract:Avian Reovirus, an important Avian pathogen, expresses eight structural and four nonstructural proteins. The structural σA protein is a major component of the inner capsid, clamping together λA building blocks. σA has also been implicated in the resistance of Avian Reovirus to the antiviral action of interferon by strongly binding double-stranded RNA in the host cell cytoplasm and thus inhibiting activation of the double-stranded RNA-dependent protein kinase. We have solved the structure of bacterially expressed σA by molecular replacement and refined it using data to 2.3-A resolution. Twelve σA molecules are present in the P1 unit cell, arranged as two short double helical hexamers. A positively charged patch is apparent on the surface of σA on the inside of this helix and mutation of either of two key arginine residues (Arg155 and Arg273) within this patch abolishes double-stranded RNA binding. The structural data, together with gel shift assay, electron microscopy, and sedimentation velocity centrifugation results, provide evidence for cooperative binding of σA to double-stranded RNA. The minimal length of double-stranded RNA required for σA binding was observed to be 14 to 18 bp.
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Avian Reovirus structure and biology
Virus Research, 2007Co-Authors: Javier Benavente, Jose MartinezcostasAbstract:Avian Reoviruses are important pathogens that cause considerable losses to the poultry industry, but they have been poorly characterized at the molecular level in the past, mostly because they have been considered to be very similar to the well-studied mammalian Reoviruses. Studies performed over the last 20 years have revealed that Avian Reoviruses have unique properties and activities, different to those displayed by their mammalian counterparts, and of considerable interest to molecular virologists. Notably, the Avian Reovirus S1 gene is unique, in that it is a functional tricistronic gene that possesses three out-of-phase and partially overlapping open reading frames; the identification of the mechanisms that govern the initiation of translation of the three S1 cistrons, and the study of the properties and activities displayed by their encoded proteins, are particularly interesting areas of research. For instance, Avian Reoviruses are one of the few nonenveloped viruses that cause cell-cell fusion, and their fusogenic phenotype has been associated with a nonstructural 10 kDa transmembrane protein, which is expressed by the second cistron of the S1 gene; the small size of this atypical fusion protein offers an interesting model for studying the mechanisms of cell-cell fusion and for identifying fusogenic domains. Finally, Avian Reoviruses are highly resistant to interferon, and therefore they may be useful for investigating the mechanisms and strategies that viruses utilize to counteract the antiviral actions of interferons.
Jose Martinezcostas - One of the best experts on this subject based on the ideXlab platform.
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Avian Reovirus triggered apoptosis enhances both virus spread and the processing of the viral nonstructural muns protein
Virology, 2014Co-Authors: Javier Rodriguezgrille, Jose Martinezcostas, Lisa K Busch, Javier BenaventeAbstract:Avian Reovirus non-structural protein muNS is partially cleaved in infected chicken embryo fibroblast cells to produce a 55-kDa carboxyterminal protein, termed muNSC, and a 17-kDa aminoterminal polypeptide, designated muNSN. In this study we demonstrate that muNS processing is catalyzed by a caspase 3-like protease activated during the course of Avian Reovirus infection. The cleavage site was mapped by site directed mutagenesis between residues Asp-154 and Ala-155 of the muNS sequence. Although muNS and muNSC, but not muNSN, are able to form inclusions when expressed individually in transfected cells, only muNS is able to recruit specific ARV proteins to these structures. Furthermore, muNSC associates with ARV factories more weakly than muNS, sigmaNS and lambdaA. Finally, the inhibition of caspase activity in ARV-infected cells does not diminish ARV gene expression and replication, but drastically reduces muNS processing and the release and dissemination of progeny viral particles.
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Avian Reovirus μns protein forms homo oligomeric inclusions in a microtubule independent fashion which involves specific regions of its c terminal domain
Journal of Virology, 2010Co-Authors: Alberto Brandariznunez, Javier Benavente, Rebeca Menayavargas, Jose MartinezcostasAbstract:Members of the genus OrthoReovirus replicate in cytoplasmic inclusions termed viral factories. Compelling evidence suggests that the nonstructural protein μNS forms the matrix of the factories and recruits specific viral proteins to these structures. In the first part of this study, we analyzed the properties of Avian Reovirus factories and μNS-derived inclusions and found that they are nonaggresome cytoplasmic globular structures not associated with the cytoskeleton which do not require an intact microtubule network for formation and maturation. We next investigated the capacity of Avian Reovirus μNS to form inclusions in transfected and baculovirus-infected cells. Our results showed that μNS is the main component of the inclusions formed by recombinant baculovirus expression. This, and the fact that μNS is able to self-associate inside the cell, suggests that μNS monomers contain all the interacting domains required for inclusion formation. Examination of the inclusion-forming capacities of truncated μNS versions allowed us to identify the region spanning residues 448 to 635 of μNS as the smallest that was inclusion competent, although residues within the region 140 to 380 seem to be involved in inclusion maturation. Finally, we investigated the roles that four different motifs present in μNS(448-635) play in inclusion formation, and the results suggest that the C-terminal tail domain is a key determinant in dictating the initial orientation of monomer-to-monomer contacts to form basal oligomers that control inclusion shape and inclusion-forming efficiency. Our results contribute to an understanding of the generation of structured protein aggregates that escape the cellular mechanisms of protein recycling.
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Avian Reovirus sigmaa localizes to the nucleolus and enters the nucleus by a nonclassical energy and carrier independent pathway
Journal of Virology, 2009Co-Authors: Lorena Vazqueziglesias, Jose Martinezcostas, Irene Lostaleseijo, Javier BenaventeAbstract:Avian Reovirus sigmaA is a double-stranded RNA (dsRNA)-binding protein that has been shown to stabilize viral core particles and to protect the virus against the antiviral action of interferon. To continue with the characterization of this viral protein, we have investigated its intracellular distribution in Avian cells. Most sigmaA accumulates into cytoplasmic viral factories of infected cells, and yet a significant fraction was detected in the nucleolus. The protein also localizes in the nucleolus of transfected cells, suggesting that nucleolar targeting is not facilitated by the viral infection or by viral factors. Assays performed in both intact cells and digitonin-permeabilized cells demonstrate that sigmaA is able to enter the nucleus via a nucleoporin-dependent nondiffusional mechanism that does not require added cytosolic factors or energy input. These results indicate that sigmaA by itself is able to penetrate into the nucleus using a process that is mechanistically different from the classical nuclear localization signal/importin pathway. On the other hand, two sigmaA arginines that are necessary for dsRNA binding are also required for nucleolar localization, suggesting that dsRNA-binding and nucleolar targeting are intimately linked properties of the viral protein.
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crystal structure of the Avian Reovirus inner capsid protein sigmaa
Journal of Virology, 2008Co-Authors: Pablo Guardadocalvo, Jose Martinezcostas, Javier Benavente, Lorena Vazqueziglesias, Antonio L Llamassaiz, Guy Schoehn, G C Fox, Lois X Hermoparrado, Mark J Van RaaijAbstract:Avian Reovirus, an important Avian pathogen, expresses eight structural and four nonstructural proteins. The structural σA protein is a major component of the inner capsid, clamping together λA building blocks. σA has also been implicated in the resistance of Avian Reovirus to the antiviral action of interferon by strongly binding double-stranded RNA in the host cell cytoplasm and thus inhibiting activation of the double-stranded RNA-dependent protein kinase. We have solved the structure of bacterially expressed σA by molecular replacement and refined it using data to 2.3-A resolution. Twelve σA molecules are present in the P1 unit cell, arranged as two short double helical hexamers. A positively charged patch is apparent on the surface of σA on the inside of this helix and mutation of either of two key arginine residues (Arg155 and Arg273) within this patch abolishes double-stranded RNA binding. The structural data, together with gel shift assay, electron microscopy, and sedimentation velocity centrifugation results, provide evidence for cooperative binding of σA to double-stranded RNA. The minimal length of double-stranded RNA required for σA binding was observed to be 14 to 18 bp.
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Avian Reovirus structure and biology
Virus Research, 2007Co-Authors: Javier Benavente, Jose MartinezcostasAbstract:Avian Reoviruses are important pathogens that cause considerable losses to the poultry industry, but they have been poorly characterized at the molecular level in the past, mostly because they have been considered to be very similar to the well-studied mammalian Reoviruses. Studies performed over the last 20 years have revealed that Avian Reoviruses have unique properties and activities, different to those displayed by their mammalian counterparts, and of considerable interest to molecular virologists. Notably, the Avian Reovirus S1 gene is unique, in that it is a functional tricistronic gene that possesses three out-of-phase and partially overlapping open reading frames; the identification of the mechanisms that govern the initiation of translation of the three S1 cistrons, and the study of the properties and activities displayed by their encoded proteins, are particularly interesting areas of research. For instance, Avian Reoviruses are one of the few nonenveloped viruses that cause cell-cell fusion, and their fusogenic phenotype has been associated with a nonstructural 10 kDa transmembrane protein, which is expressed by the second cistron of the S1 gene; the small size of this atypical fusion protein offers an interesting model for studying the mechanisms of cell-cell fusion and for identifying fusogenic domains. Finally, Avian Reoviruses are highly resistant to interferon, and therefore they may be useful for investigating the mechanisms and strategies that viruses utilize to counteract the antiviral actions of interferons.
Long Huw Lee - One of the best experts on this subject based on the ideXlab platform.
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Identification and characterization of RNA-binding activities of Avian Reovirus non-structural protein rNS
2014Co-Authors: Hsiensheng Yin, Long Huw LeeAbstract:Cytoplasmic extracts prepared from Avian Reovirus (ARV) strain S1133-infected chicken embryo fibro-blasts were examined for the presence of RNA-binding proteins in order to identify and charac-terize ARV RNA-binding proteins. Analysis of bind-ing activity to poly(A)–Sepharose indicated that infected cells contained significant amounts of a protein that co-migrated with ARV protein rNS present in total virus-infected cell extracts. De-termination of the N-terminal amino acid sequence of several peptide fragments generated by V8 protease digestion of the poly(A)–Sepharose-purified protein confirmed that this viral protein was rNS. Competition assays showed that single-stranded RNA from the unrelated Avian pathoge
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cytokine mrna expression in chicken experimentally infected with different Avian Reovirus strains
Taiwan Veterinary Journal, 2014Co-Authors: Pinchun Shen, Jialing Yang, Long Huw LeeAbstract:This study was undertaken to elucidate the cytokine response in chicken infected with strains of Avian Reovirus (ARV) S1133 and 2408. The expression levels of cytokine mRNA in the spleen and viral S1 RNA in various tissues at 1.5 and 2.5 days post inoculation (dpi) were examined using real-time quantitative PCR. Among the cytokines examined, the mRNA expression levels of IL-6, IFN-γ, IL-10 and iNOS at 2.5 dpi were significantly upregulated and higher in chickens infected with strain 2408 than in chickens infected with strain S1133, particularly IL-6 and IFN-γ. A significantly higher levels of viral S1 RNA were detected in the examined tissues from chickens infected with strain 2408 than with strain S1133 over the experimental course, among which the foot pad and spleen were more predominant. The highest levels of IL-6 and IFN-γ mRNA expression correlated with the viral S1 RNA levels in the spleen and the marked clinical diseases and gross lesions, suggesting that IL-6 and IFN-γ may play a role in the pathogenesis of ARV infection.
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characterization of interleukin 1β mrna expression in chicken macrophages in response to Avian Reovirus
Journal of General Virology, 2008Co-Authors: Hungjen Liu, Jui Huang Shien, Shiowher Chiou, Long Huw LeeAbstract:Inhibitors of viral disassembly or RNA and protein synthesis, viral disassembly intermediates (infectious subviral particles, ISVP), binary ethylenimine-inactivated virions, and viral particles lacking genomic double-stranded (ds) RNA (empty particles) were used to assess the expression of interleukin-1β (IL-1β) mRNA in chicken (chIL-1β) macrophages in response to Avian Reovirus. The results demonstrate that two distinct expression patterns of chIL-1β mRNA mediated by different steps in viral replication were found. Viral disassembly was required for the induction of a rapid, transient expression pattern of chIL-1β mRNA that was rapidly induced at 30 min, with maximal levels reached by 2 h, and fell to a low level within 6 h post-inoculation, while viral RNA synthesis rather than protein translation, which was subsequent to membrane penetration, was required to induce a stable, sustained expression pattern of chIL-1β mRNA that occurred at and after 6 h post-inoculation. In addition, the induction of chIL-1β mRNA expression by the empty particles and ISVP was extremely weak, compared with the active dsRNA+ virions or binary ethylenimine-inactivated virions, suggesting that the presence of dsRNA, even if transcriptionally inactive, may be an important factor in this response.
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Avian Reovirus core protein μa expressed in escherichia coli possesses both ntpase and rtpase activities
Journal of General Virology, 2007Co-Authors: Jui Huang Shien, Hungjen Liu, Hsiensheng Yin, Long Huw LeeAbstract:Analysis of the amino acid sequence of core protein μA of Avian Reovirus has indicated that it may share similar functions to protein μ2 of mammalian Reovirus. Since μ2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant μA ( μA) was designed and used to test these activities. μA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that μA possessed NTPase activity that enabled the protein to hydrolyse the β–γ phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k cat values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of μA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein μA. The activity of μA is dependent on the divalent cations Mg2+ or Mn2+, but not Ca2+ or Zn2+. Optimal NTPase activity of μA was achieved between pH 5.5 and 6.0. In addition, μA enzymic activity increased with temperature up to 40 °C and was almost totally inhibited at temperatures higher than 55 °C. Tests of phosphate release from RNA substrates with μA or K408A/K412A μA indicated that μA, but not K408A/K412A μA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of μA might be carried out at the same active site, and that protein μA could play important roles during viral RNA synthesis.
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the sequence and phylogenetic analysis of Avian Reovirus genome segments m1 m2 and m3 encoding the minor core protein μa the major outer capsid protein μb and the nonstructural protein μns
Journal of Virological Methods, 2006Co-Authors: Jui Huang Shien, Hungjen Liu, Long Huw LeeAbstract:Abstract The sequences and phylogenetic analyses of the M-class genome segments of 12 Avian Reovirus strains are described. The S1133 M1 genome segment is 2283 base pairs long, encoding a protein μA consisted of 732 amino acids. Each M2 or M3 genome segment of 12 Avian Reovirus strains is 2158 or 1996 base pairs long, respectively, encoding a protein μB or μNS consisted of 676 and 635 amino acids, respectively. The S1133 genome segment has the 5′ GCUUUU terminal motif, but each M2 and M3 genome segment displays the 5′ GCUUUUU terminal motif which is common to other known Avian Reovirus genome segments. The UCAUC 3′-terminal sequences of the M-class genome segments are shared by both Avian and mammalian Reoviruses. Noncoding regions of both 5′- and 3′-termini of the S1133 M1 genome segment consist of 12 and 72 nucleotides, respectively, those of each M2 genome segment consist of 29 and 98 nucleotides, respectively, and those of each M3 genome segment are 24 and 64 nucleotides, respectively. Analysis of the average degree of the M-class gene and the deduced μ-class protein sequence identities indicated that the M2 genes and the μB proteins have the greatest level of sequence divergence. Computer searches revealed that the μA possesses a sequence motif (NH2-Leu-Ala-Leu-Asp-Pro-Pro-Phe-COOH) (residues 458–464) indicative of N-6 adenine-specific DNA methylase. Examination of the μB amino acid sequences indicated that the cleavage site of μB into μBN and μBC is between positions 42 and 43 near the N-terminus of the protein, and this site is conserved for each protein. During in vitro treatment of virions with trypsin to yield infectious subviral particles, both the N-terminal fragment δ and the C-terminal fragment φ were shown to be generated. The site of trypsin cleavage was identified in the deduced amino acid sequence of μB by determining the amino-terminal sequences of φ proteins: between arginine 582 and glycine 583. The predicted length of δ generated from μBC is very similar to that of δ generated from mammalian Reovirus μ1C. Taken together, protein μB is structurally, and probably functionally, similar to its mammalian homolog, μ1. In addition, two regions near the C-terminal and with a propensity to form α-helical coiled-coil structures as previously indicated are observed for each protein μB. Phylogenetic analysis of the M-class genes revealed that the predicted phylograms delineated 3 M1, 5 M2, and 2 M3 lineages, no correlation with serotype or pathotype of the viruses. The results also showed that M2 lineages I–V consist of a mixture of viruses from the M1 and M3 genes of lineages I–III, reflecting frequent reassortment of these genes among virus strains.
Hung J Liu - One of the best experts on this subject based on the ideXlab platform.
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Avian Reovirus influences phosphorylation of several factors involved in host protein translation including eukaryotic translation elongation factor 2 eef2 in vero cells
Biochemical and Biophysical Research Communications, 2009Co-Authors: Lai Wang, Ru C Lin, Wei R Huang, Hung J LiuAbstract:Viral infection usually influences cellular protein synthesis either actively or passively via modification of various translation initiation factors. Here we demonstrated that infection with Avian Reovirus (ARV) interfered with cellular protein synthesis. This study demonstrated for the first time that ARV influenced the phosphorylation of translation initiation factors including eIF4E and eIF-4G. Interestingly, ARV also induced phosphorylation of eukaryotic translation elongation factor (eEF2) in a time- and dose-dependent manner. Inhibition of mTOR by rapamycin notably increased the level of phosphorylated eEF2 in infected cells. However, rapamycin did not show any negative effects on ARV replication, suggesting that phosphorylation of eEF2 in infected cells did not reduce ARV propagation. These results demonstrated for the first time that ARV promotes phosphorylation of eEF2 which in turn influenced host protein production not simply by modulating the function of translation initiation factors but also by regulating elongation factor eEF2.
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proteasome inhibition reduces Avian Reovirus replication and apoptosis induction in cultured cells
Journal of Virological Methods, 2008Co-Authors: Yu T Chen, Chi H Lin, Hung J LiuAbstract:The interplay between Avian Reovirus (ARV) replication and apoptosis and proteasome pathway was studied in cultured cells. It is shown that inhibition of the proteasome did not affect viral entry and host cell translation but had influence on ARV replication and ARV-induced apoptosis. Evidence is provided to demonstrate that ubiquitin-proteasome blocked ARV replication at an early step in viral life cycle. However, viral transcription and protein translation were also reduced markedly after addition of proteasome inhibitor MG132. Treatment of BHK-21 cells with the MG132 markedly decreased virus titer as well as prevented virus-induced apoptosis. The expression of ARV proteins σC, σA, and σNS was also reduced markedly, suggesting that suppression of virus replication is due to down-regulation of these ARV proteins by ubiquitin-proteasome system. MG132 was also shown to suppress ARV σC-induced phosphrylation of p53 on serine 46, caspase 3 activities, and DNA fragmentation leading to complete inhibition of ARV-induced apoptosis.
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Suppression of protein expression of three Avian Reovirus S-class genome segments by RNA interference.
Veterinary microbiology, 2007Co-Authors: Julius L C Chulu, Long H Lee, Feng L Lin, Hung J LiuAbstract:RNA interference was used to suppress protein expression of three S-class genome segments of Avian Reovirus (ARV). Viral progeny titer was successfully down-regulated by RNA interference. Suppression of S1 genome segment, which has three open reading frames, not only decreased the expression level of the structural protein sigmaC but also reduced cell fusion and the level of Ser(15)-phosphorylated p53 protein caused by the nonstructural proteins p10 and p17, respectively. Suppression of S2 or S4 genome segment by RNA interference could also reduce the expression level of sigmaA or sigmaNS. Interestingly, suppression of sigmaNS resulted in down regulation of the expression of other viral products. In terms of variability of different genes among viral strains and of the impact after their suppression, it seems that the viral products involved in construction of viroplasm or core particles, like sigmaNS, are considerable choices to efficiently inhibit ARV multiplication by RNA interference. Using a GFP reporter system, it was discovered that ARV could not inhibit activated RNA interference, suggesting that RNA interference may be used in the suppression of ARV infection.
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apoptosis induction by Avian Reovirus through p53 and mitochondria mediated pathway
Biochemical and Biophysical Research Communications, 2007Co-Authors: Julius L C Chulu, Wen L Shih, Long H Lee, Ya C Lee, Shu H Liao, Feng L Lin, Hung J LiuAbstract:Although induction of apoptosis by Avian Reovirus has been demonstrated in primary chicken embryonic fibroblast and several cell lines, to date, the potential significance of Avian Reovirus (ARV)-induced apoptosis and its pathways in cultured cells are still largely unknown. We now provide the first evidence of upregulation of p53 and Bax and specifically for Bax translocation from cytosol to mitochondria following infection with a cytoplasmically replicating RNA virus. Bax translocation to the mitochondria led to the release of mitochondrial proapoptic factors cytochrome c and Smac/DIABLO from mitochondria to the cytosol, but not the release of apoptosis-inducting factor. Activation of caspases-9 and -3 which cleaves the enzyme poly(ADP-ribose) polymerase in ARV-infected BHK-21 cells was also detected. Internucleosomal DNA cleavage was prevented by caspase inhibitors, further demonstrating that ARV-induced apoptosis was executed through caspase-dependent mechanisms. Stable expression of human bcl-2 in BHK-21 cells not only blocked ARV-induced apoptosis and DNA fragmentation but also reduced the level of infectious virus production and its spread in BHK-21 cells infected with ARV at a low multiplicity of infection. All our data suggest that p53 and the mitochondria-mediated pathway played an important regulatory role in ARV-induced apoptosis in BHK-21 cells. To further study the pathogenesis of ARV infection, a dual-labeling assay was used for the simultaneous detection of cells containing viral antigen and apoptotic cells. Dual-labeling assay revealed that the majority of antigen-expressing cells were not apoptotic. Remarkably, some apoptotic but non-antigen-expressing cells were frequently located in the vicinity of antigen-expressing cells. Syncytium formation in ARV-infected BHK-21 cells undergoing apoptosis, was apparent in large syncytia at late infection times, indicating a correlation between virus replication and apoptosis in cultured cells.
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Avian Reovirus induced apoptosis related to tissue injury
Avian Pathology, 2007Co-Authors: Hsin Y Lin, Sue T Chuang, Yu T Chen, Wen L Shih, Ching D Chang, Hung J LiuAbstract:Apoptosis plays an important role in pathogenesis of many viral infections. Infection of chicken with Avian Reovirus S1133 causes tissue injury related to virus-induced apoptosis. To determine whether Avian Reovirus (ARV) induced apoptosis in chicken tissues, six 3-week-old specific pathogen free White Leghorn chicks were inoculated with ARV S1133. Tissues were dual-labelled for the simultaneous detection of viral antigen containing and apoptotic cells. DNA laddering was detected in ARV-infected but not mock-infected chicken tissues. Dual-labelling assay revealed that the majority of antigen-expressing cells were not apoptotic. Surprisingly, some apoptotic but non-antigen-expressing cells were frequently located in the vicinity of antigen-expressing cells. Syncytium formation in ARV-infected chicken tissues undergoing apoptosis was apparent, suggesting a correlation between virus replication and apoptosis in chicken tissues.
Hungjen Liu - One of the best experts on this subject based on the ideXlab platform.
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Avian Reovirus s1133 induced apoptosis is associated with bip grp79 mediated bim translocation to the endoplasmic reticulum
Apoptosis, 2015Co-Authors: Pingyuan Lin, Chi I Chang, Hungjen Liu, Chingdong Chang, Yochia Chen, Wenling ShihAbstract:In this study the mechanism of Avian Reovirus (ARV) S1133-induced pathogenesis was investigated, with a focus on the contribution of ER stress to apoptosis. Our results showed that upregulation of the ER stress response protein, as well as caspase-3 activation, occurred in ARV S1133-infected cultured cells and in SPF White Leghorn chicks organs. Upon infection, Bim was translocated specifically to the ER, but not mitochondria, in the middle to late infectious stages. In addition, ARV S1133 induced JNK phosphorylation and promoted JNK-Bim complex formation, which correlated with the Bim translocation and apoptosis induction that was observed at the same time point. Knockdown of BiP/GRP78 by siRNA and inhibition of BiP/GRP78 using EGCG both abolished the formation of the JNK-Bim complex, caspase-3 activation, and subsequent apoptosis induction by ARV S1133 efficiently. These results suggest that BiP/GRP78 played critical roles and works upstream of JNK-Bim in response to the ARV S1133-mediated apoptosis process.
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characterization of interleukin 1β mrna expression in chicken macrophages in response to Avian Reovirus
Journal of General Virology, 2008Co-Authors: Hungjen Liu, Jui Huang Shien, Shiowher Chiou, Long Huw LeeAbstract:Inhibitors of viral disassembly or RNA and protein synthesis, viral disassembly intermediates (infectious subviral particles, ISVP), binary ethylenimine-inactivated virions, and viral particles lacking genomic double-stranded (ds) RNA (empty particles) were used to assess the expression of interleukin-1β (IL-1β) mRNA in chicken (chIL-1β) macrophages in response to Avian Reovirus. The results demonstrate that two distinct expression patterns of chIL-1β mRNA mediated by different steps in viral replication were found. Viral disassembly was required for the induction of a rapid, transient expression pattern of chIL-1β mRNA that was rapidly induced at 30 min, with maximal levels reached by 2 h, and fell to a low level within 6 h post-inoculation, while viral RNA synthesis rather than protein translation, which was subsequent to membrane penetration, was required to induce a stable, sustained expression pattern of chIL-1β mRNA that occurred at and after 6 h post-inoculation. In addition, the induction of chIL-1β mRNA expression by the empty particles and ISVP was extremely weak, compared with the active dsRNA+ virions or binary ethylenimine-inactivated virions, suggesting that the presence of dsRNA, even if transcriptionally inactive, may be an important factor in this response.
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Avian Reovirus core protein μa expressed in escherichia coli possesses both ntpase and rtpase activities
Journal of General Virology, 2007Co-Authors: Jui Huang Shien, Hungjen Liu, Hsiensheng Yin, Long Huw LeeAbstract:Analysis of the amino acid sequence of core protein μA of Avian Reovirus has indicated that it may share similar functions to protein μ2 of mammalian Reovirus. Since μ2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant μA ( μA) was designed and used to test these activities. μA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that μA possessed NTPase activity that enabled the protein to hydrolyse the β–γ phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k cat values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of μA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein μA. The activity of μA is dependent on the divalent cations Mg2+ or Mn2+, but not Ca2+ or Zn2+. Optimal NTPase activity of μA was achieved between pH 5.5 and 6.0. In addition, μA enzymic activity increased with temperature up to 40 °C and was almost totally inhibited at temperatures higher than 55 °C. Tests of phosphate release from RNA substrates with μA or K408A/K412A μA indicated that μA, but not K408A/K412A μA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of μA might be carried out at the same active site, and that protein μA could play important roles during viral RNA synthesis.
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expression of Avian Reovirus σc protein in transgenic plants
Journal of Virological Methods, 2006Co-Authors: Liang Kai Huang, Sin Chung Liao, Ching Chun Chang, Hungjen LiuAbstract:Abstract Avian Reovirus (ARV) structural protein, σC encoded by S1 genome segment, is the prime candidate to become a vaccine against ARV infection. Two plant nuclear expression vectors with expression of σC-encoding gene driven by CaMV 35S promoter and rice actin promoter were constructed, respectively. Agrobacterium containing the S1 expression constructs were used to transform alfalfa, and transformants were selected using hygromysin. The integration of S1 transgene in alfalfa chromosome was confirmed by PCR and histochemical GUS staining. Western blot analysis using antiserum against σC was carried out to determine the expression of σC protein in transgenic alfalfa cells. The highest expression levels of σC protein in the cellular extracts of selected p35S-S1 and pAct1-S1 transgenic alfalfa lines were 0.008% and 0.007% of the total soluble protein, respectively. The transgenic alfalfa cells with expression of σC protein pave the way for the development of edible vaccine.
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the sequence and phylogenetic analysis of Avian Reovirus genome segments m1 m2 and m3 encoding the minor core protein μa the major outer capsid protein μb and the nonstructural protein μns
Journal of Virological Methods, 2006Co-Authors: Jui Huang Shien, Hungjen Liu, Long Huw LeeAbstract:Abstract The sequences and phylogenetic analyses of the M-class genome segments of 12 Avian Reovirus strains are described. The S1133 M1 genome segment is 2283 base pairs long, encoding a protein μA consisted of 732 amino acids. Each M2 or M3 genome segment of 12 Avian Reovirus strains is 2158 or 1996 base pairs long, respectively, encoding a protein μB or μNS consisted of 676 and 635 amino acids, respectively. The S1133 genome segment has the 5′ GCUUUU terminal motif, but each M2 and M3 genome segment displays the 5′ GCUUUUU terminal motif which is common to other known Avian Reovirus genome segments. The UCAUC 3′-terminal sequences of the M-class genome segments are shared by both Avian and mammalian Reoviruses. Noncoding regions of both 5′- and 3′-termini of the S1133 M1 genome segment consist of 12 and 72 nucleotides, respectively, those of each M2 genome segment consist of 29 and 98 nucleotides, respectively, and those of each M3 genome segment are 24 and 64 nucleotides, respectively. Analysis of the average degree of the M-class gene and the deduced μ-class protein sequence identities indicated that the M2 genes and the μB proteins have the greatest level of sequence divergence. Computer searches revealed that the μA possesses a sequence motif (NH2-Leu-Ala-Leu-Asp-Pro-Pro-Phe-COOH) (residues 458–464) indicative of N-6 adenine-specific DNA methylase. Examination of the μB amino acid sequences indicated that the cleavage site of μB into μBN and μBC is between positions 42 and 43 near the N-terminus of the protein, and this site is conserved for each protein. During in vitro treatment of virions with trypsin to yield infectious subviral particles, both the N-terminal fragment δ and the C-terminal fragment φ were shown to be generated. The site of trypsin cleavage was identified in the deduced amino acid sequence of μB by determining the amino-terminal sequences of φ proteins: between arginine 582 and glycine 583. The predicted length of δ generated from μBC is very similar to that of δ generated from mammalian Reovirus μ1C. Taken together, protein μB is structurally, and probably functionally, similar to its mammalian homolog, μ1. In addition, two regions near the C-terminal and with a propensity to form α-helical coiled-coil structures as previously indicated are observed for each protein μB. Phylogenetic analysis of the M-class genes revealed that the predicted phylograms delineated 3 M1, 5 M2, and 2 M3 lineages, no correlation with serotype or pathotype of the viruses. The results also showed that M2 lineages I–V consist of a mixture of viruses from the M1 and M3 genes of lineages I–III, reflecting frequent reassortment of these genes among virus strains.
