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Richard W. Moyer - One of the best experts on this subject based on the ideXlab platform.
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an Amsacta moorei entomopoxvirus ortholog of the poly a polymerase small subunit exhibits methyltransferase activity and is non essential for virus growth
Virology, 2008Co-Authors: Marie N Becker, Tracie M Todd, Richard W. MoyerAbstract:Abstract Unlike the heterodimeric poly(A) polymerase (PAP) of vaccinia virus (VACV), the PAP from the Amsacta moorei entomopoxvirus, AMEV, is potentially derived from three subunits: a single large and two small subunits (AMV060 and AMV115). The VACV small subunit serves as a 2′- O -methyltransferase, a processivity factor for mRNA polyadenylation, and a transcription elongation factor. We wished to determine the structure–function relationships of the three putative AMEV PAP subunits. We show that AMV060 is expressed as an early gene persisting throughout infection, whereas AMV115 is expressed late. We demonstrate that AMV060 exhibits 2′- O -methyltransferase activity but the gene is not essential for virus growth. Absence of the AMV060 protein has no effect on the length of the poly(A) tails present in mRNA. No physical association was found between any of the putative AMEV PAP subunits. We therefore propose that mRNA polyadenylation does not require interactions between these three proteins.
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Amsacta moorei entomopoxvirus inhibitor of apoptosis suppresses cell death by binding grim and hid
Journal of Virology, 2005Co-Authors: Qianjun Li, Peter Liston, Natasha Schokman, Jenny Mei Ho, Richard W. MoyerAbstract:Inhibitor of apoptosis (iap) genes have been identified in the genomes of two independent families of insect viruses, the Baculoviridae and the Entomopoxvirinae. In this report, we examined the functional attributes of the Amsacta moorei entomopoxvirus-encoded IAP protein (AMV-IAP). The binding specificity of the individual baculoviral IAP repeat (BIR) domains of AMV-IAP was investigated by using a random-peptide, phage display library, and sequences similar to the amino termini of proapoptotic Drosophila proteins in the Reaper/Hid/Grim family were identified. Furthermore, the BIR domains of AMV-IAP protein were demonstrated to bind the mammalian IAP inhibitor Smac through the AVPI tetrapeptide sequence, suggesting that the peptide binding pocket and groove found in the insect and mammalian IAPs is conserved in this viral protein. Interaction analysis implicated BIR1 as the high-affinity site for Grim, while BIR2 interacted more strongly with Hid. Both Grim and Hid were demonstrated to interact with AMV-IAP in vivo, and Grim- or Hid-induced cell death was suppressed when AMV-IAP was coexpressed.
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Functional Analysis of the Inhibitor of Apoptosis (iap) Gene Carried by the Entomopoxvirus of Amsacta moorei
Journal of Virology, 2005Co-Authors: Qianjun Li, Peter Liston, Richard W. MoyerAbstract:Apoptosis, or programmed cell death, is a well-conserved and integral process necessary for normal organism development which serves to remove unwanted, damaged, mutated, or infected cells (43, 54, 70). Apoptosis can be initiated by both external and internal stimuli such as UV-induced DNA damage, oncogenic transformation, drugs such as actinomycin D, virus infection, and a variety of extracellular signals (68, 71). These various stimuli lead to the activation of either the intrinsic or extrinsic apoptotic pathway (7). The extrinsic pathway is triggered by the binding of external (death) ligands to their cognate (death) receptors as exemplified by members of the tumor necrosis factor (TNF) superfamily. Receptor-ligand engagement then allows transmission of external signals into the cell. The intrinsic pathway is initiated by signals originating within the cell from a series of death-triggering genes, which in insect systems include the hid, grim, and reaper genes of Drosophila and in mammalian cells the Smac/Diablo, GSPT1, and Omi/HtrA2 genes (39, 68). The apoptotic killing of cells by these death-inducing genes can be blocked and regulated by apoptotic suppressor genes, including members of the inhibitor of apoptosis (iap) gene superfamily (26, 59). Both intrinsic and extrinsic apoptotic pathways result in the formation of a cytosolic protein complex that activates a family of aspartic acid-specific cysteine proteases (caspases). Caspases are a diverse family of proteases, and it is through the action of active caspases that apoptotic death occurs. Caspases are divided into “initiator” (caspases 2, 8, 9, and 10) and “executioner” or “terminal” caspases (caspases 3 and 7) (35, 54). Once activated, the initiator caspases are responsible for activating the executioner caspases by proteolytic cleavage. The activated executioner caspases are then responsible for the proteolytic cleavage and degradation of a broad spectrum of cellular targets, eventually leading to apoptosis that is characterized by changes in the cells such as shrinkage, membrane blebbing, chromatin condensation, formation of the apoptosome, and DNA fragmentation (54, 71). Virus infections frequently lead to the induction of apoptosis in host cells. Apoptosis in infected cells can be considered as a host defense mechanism which ultimately results in reduction of virus production (7, 43). However, viruses have developed strategies to block apoptosis allowing more time for progeny virus to be produced (7, 11, 27, 48). One of the first apoptotic suppressor genes, p35, was discovered in Autographa californica M nucleopolyhedrovirus (AcMNPV). The p35 gene is required for virus growth since deletion of p35 from AcMNPV blocks virus production (13). The P35 protein is believed to function in the terminal stages of apoptosis as a caspase inhibitor that acts to block the lethal effects of terminal caspase activation and prevent cell death. In addition to P35, baculoviruses also encode at least two other types of apoptotic suppressor proteins, P49 and IAP (12, 17, 41, 73). The P49 protein, produced by the Spodoptera littoralis nucleopolyhedrosis virus, inhibits the activation of initiator caspases, including human caspase-9 and the other P35-sensitive initiator caspases (17, 46, 73). Thus, P49 functions to inhibit apoptosis by blocking activity of the initiator caspases that serves to prevent activation of the downstream effector caspases. Viral iap genes were first described from the Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV), the Op-iap gene, and Cydia pomonella granulosis virus (CpGV), the Cp-iap (8, 15). Structurally, IAPs are characterized by the presence of two signature motifs: the so-called baculovirus IAP repeats (BIRs) and a RING domain (16, 59). RING domains are a specialized form of zinc finger involved in protein-protein interactions. IAP proteins generally act to block apoptosis by interacting through the BIR domain to block the activity of a variety of proapoptotic proteins such as REAPER, HID, and GRIM in insect cells and SMAC/DIABLO in vertebrate cells (34, 63). Op-IAP has been shown to bind to HID, REAPER, and GRIM; to down-regulate Sf-caspase-X; and therefore to inhibit apoptosis induced by various proapoptotic inducers (34, 63). The IAP family of proteins is evolutionarily well conserved, and most IAP proteins appear to be able to function across different species. Unlike p35 and p49 genes, which have only been identified in a few baculoviruses, iap genes have been found widely distributed in most if not all baculoviruses, as well as in eukaryotes, including mammals (16). The Poxviridae comprise a large family of double-stranded DNA-containing viruses, which, unlike baculoviruses, develop in the cytoplasm and include viruses of both vertebrates (Chordopoxvirinae) and invertebrates (Entomopoxvirinae). The vertebrate poxviruses are known to encode a number of proteins that regulate apoptosis, including the serine proteinase inhibitor (serpin), SPI-2/CrmA. The crmA/SPI-2-encoded serpin blocks apoptosis by direct inhibition of initiator caspases, including caspases 1, 8, and 10 (69), as well as granzyme B (40, 47, 58). However, no serpin genes have been identified within the two sequenced insect poxvirus genomes, the entomopoxviruses from Amsacta moorei (AmEPV) (5) and Melanoplus sanguinipes (MsEPV) (1). Furthermore, examination of the genomic sequences suggests none of the many other vertebrate poxvirus apoptosis suppressors exist within AmEPV. AmEPV, a group B (β) entomopoxvirus, has been reported to infect agriculturally important pests, such as Estigmene acrea (24) and Lymantria dispar (5). The 232-kb AmEPV genome was recently sequenced (5) and appears to contain a single iap gene, AMViap (AMV021). As the only candidate apoptotic suppressor gene, it is likely that the single AmEPV iap gene homolog has a role in controlling apoptosis. In this paper, we show that the AMViap gene is active, inhibits apoptosis as expected, and represents one mechanism by which AmEPV can control cell viability.
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NAD+-dependent DNA Ligase Encoded by a Eukaryotic Virus
Journal of Biological Chemistry, 2001Co-Authors: Verl Sriskanda, Richard W. Moyer, Stewart ShumanAbstract:Abstract We report the production, purification, and characterization of an NAD+-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD+ ligase from a source other than eubacteria. AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD+ ligases. Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD+. Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD+. Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD+ ligases. Deletion of domain Ia of AmEPV ligase abolished the sealing of 3′-OH/5′-PO4 nicks and the reaction with NAD+ to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick. Alanine substitutions at residues within domain Ia either reduced (Tyr39, Tyr40, Asp48, and Asp52) or abolished (Tyr51) sealing of a 5′-PO4 nick and adenylyl transfer from NAD+without affecting ligation of DNA-adenylate. We conclude that: (i) NAD+-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD+.
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complete genomic sequence of the Amsacta moorei entomopoxvirus analysis and comparison with other poxviruses
Virology, 2000Co-Authors: Alison L Bawden, Kathryn J Glassberg, James C Diggans, Regina Shaw, William G Farmerie, Richard W. MoyerAbstract:The genome of the genus B entomopoxvirus from Amsacta moorei (AmEPV) was sequenced and found to contain 232,392 bases with 279 unique open reading frames (ORFs) of greater than 60 amino acids. The central core of the viral chromosome is flanked by 9.4-kb inverted terminal repeats (ITRs), each of which contains 13 ORFs, raising the total number of ORFs within the viral chromosome to 292. ORFs with no known homology to other poxvirus genes were shown to constitute 33.6% of the viral genome. Approximately 28.6% of the AmEPV genome encodes homologs of the mammalian poxvirus colinear core genes, which are found dispersed throughout the AmEPV chromosome. There is also no significant gene order conservation between AmEPV and the orthopteran genus B poxvirus of Melanoplus sanguinipes (MsEPV). Novel AmEPV genes include those encoding a putative ABC transporter and a Kunitz-motif protease inhibitor. The most unusual feature of the AmEPV genome relates to the viral encoded poly(A) polymerase. In all other poxviruses this heterodimeric enzyme consists of a single large and a single small subunit. However, AmEPV appears to encode one large and two distinct small poly(A) polymerase subunits. AmEPV is one of the few entomopoxviruses which can be grown and manipulated in cell culture. The complete genomic sequence of AmEPV paves the way for an understanding and comparison of the molecular properties and pathogenesis between the entomopoxviruses of insects and the more intensively studied vertebrate poxviruses.
Zihni Demirbag - One of the best experts on this subject based on the ideXlab platform.
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Amsacta moorei entomopoxvirus encodes a functional heparin-binding glycosyltransferase (AMV248)
Virus Genes, 2018Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni DemirbagAbstract:Amsacta moorei entomopoxvirus (AMEV) infects certain lepidopteran and orthopteran insects and is the most studied member of the genus Betaentomopoxvirus . It has been considered as a potential vector for gene therapy, a vector to express exogenous proteins and a biological control agent. One of its open reading frames, amv248, encodes a putative glycosyltransferase and is the only known attachment protein conserved in AMEV and chordopoxviruses. The ORF was successfully expressed and the protein was shown to bind soluble heparin, both in silico and in vitro. Our results also showed that, while viral infection was inhibited by soluble glycosaminoglycans (GAGs), GAG-deficient cells were more resistant to the virus. Finally, we revealed that amv248 encodes an active heparin-binding glycosyltransferase which is likely to have a key role in the initiation of infection by AMEV.
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the protein protein interactions between Amsacta moorei entomopoxvirus amev protein kinases pks and all viral proteins
Virus Research, 2018Co-Authors: Hacer Muratoğlu, Remziye Nalçacioğlu, Mehtap Danismazoglu, Zihni DemirbagAbstract:Abstract Entomopoxviruses are an important group of viruses infecting only insects. They belong to Poxviridae which infect both invertebrates and vertebrates, including humans. Protein kinases are known to have roles at virus morphogenesis, host selectivity, the regulation of cell division and apoptosis in some vertebrate poxviruses. In this study, 2 protein kinases (PKs) (AMV153 and AMV197) of Amsacta moorei entomopoxvirus (AMEV) were investigated for the interactions among 230 viral proteins using yeast two-hybrid system (Y2H). For this purpose, two protein kinases and 230 viral genes were cloned into the bait and prey vectors, respectively. Bait vectors were introduced into Saccharomyces cerevisiae AH109. Expression of the bait genes were confirmed by western blot analysis. Both yeast strains of bait were transformed individually with each prey clone and grown on a selective medium (minimal synthetic defined) to determine the protein–protein interactions between bait and prey proteins. Transformations identified totally 16 interactions among AMEV protein kinases and all viral proteins of which 5 belong to AMV153 and 11 belong to AMV197. One of the five interactions detected for AMV153 protein kinase is self-association. Its other four interactions are with two virus entry complex proteins (AMV035 and AMV083), a membrane protein (AMV165) and a subunit of RNA polymerase (AMV230). The other protein kinase, AMV197, interacted with two virus entry complex proteins (AMV035 and AMV083) as AMV153, a caspase-2 enzyme (AMV063), a Holliday junction resolvase (AMV162), a membrane protein (AMV165), a subunit of RNA polymerase (AMV230) and five other hypothetical proteins (AMV026, AMV040, AMV062, AMV069, AMV120) encoded by AMEV genome. Glutathione S-transferase (GST) pull-down assay was used to confirm all interactions described by Y2H analysis. In addition, the theoretical structures of the two of 16 interactions were interpreted by docking analysis. Consistent with Y2H and pull down assays, docking analysis also showed the interactions of AMV063 with AMV153 and AMV197. Detected interactions of the AMEV viral proteins with viral protein kinases could lead to the understanding of the regulation of the viral activities of interacted viral proteins.
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transcriptional analysis of the putative glycosyltransferase gene amv248 of the Amsacta moorei entomopoxvirus
Virus Research, 2018Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni DemirbagAbstract:Abstract Amsacta moorei entomopoxvirus (AMEV), the most studied member of the genus Betaentomopoxvirus , was initially isolated from Red Hairy caterpillar larvae, Amsacta moorei. According to genome sequence and previous studies it was shown that amv248 encodes a putative glycosyltransferase that is the only conserved attachment protein in betaentomopoxviruses. Transcriptional analysis of the amv248 gene by RT-PCR and qPCR showed that transcription starts at 6 h post infection (hpi). Also, transcription was not affected by a DNA replication inhibitor but was severely curtailed by a protein synthesis inhibitor. These results indicate that amv248 belongs to the intermediate class of gene expression. 5′ and 3′ untranslated regions analysis revealed that transcription initiates at position –126 relative to the translational start site, and ends between 50 and 83 bases after the stop codon. To narrow down the size and location of the gene’s promoter, the upstream region as well as several different sized deletions thereof were generated and cloned upstream of a luciferase reporter gene. The constructs were used to measure the Firefly and Renilla luciferase activities in dual assays. The results showed that luciferase activity decreased when bases –198 to –235 of amv248 upstream region were missing. Sequence analysis among the intermediate gene promoters of AMEV showed that TTTAT(T/A)TT(T/A) 2 TTA is possibly a common motif, however, further investigations are needed to confirm this conclusion.
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genome wide analysis of differential mrna expression of Amsacta moorei entomopoxvirus mediated by the gene encoding a viral protein kinase amv197
Virus Research, 2016Co-Authors: Hacer Muratoğlu, Remziye Nalçacioğlu, Basil M Arif, Zihni DemirbagAbstract:Abstract Insect-born entomopoxviruses (Fam: Poxviridae ) are potentially important bio-pesticide against insect pests and expression vectors as well as vectors for transient human gene therapies including recombinant viral vaccines. For these reasons, it is necessary to understand the regulatory genes functions to improve its biotechnological potential. Here, we focused on the characterization of serine/threonine (Ser/Thr; ORF AMV197) protein kinase gene from the Amsacta moorei entomopoxvirus (AMEV), the type species of the genus Betaentomopoxvirus . Transcription of the parental and an amv197-null recombinant AMEV was compared by whole-genome gene expression microarray analysis. Blast2GO analysis reflected a broad diversity of upregulated and downregulated genes. Results showed that expression levels of 102 genes (45%) out of 226 tested genes changed significantly in the recombinant AMEV infected cells. Of these transcripts, 72 (70.58%) were upregulated and 30 (29.41%) were downregulated throughout the infection period. Genes involved in DNA repair, replication and nucleotide metabolism, transcription and RNA modification, and protein modification were mostly upregulated at different times in cells infected with the recombinant virus. Furthermore, transcription of all studied cellular genes including metabolism of apoptosis (Nedd2-like caspase, hemolin and elongation factor-1 alpha ( ef1a ) gene) was downregulated in the absence of amv197 . Quantitative real time reverse transcription-PCR confirmed viral transcriptional changes obtained by microarray. The results of this study indicated that the product of amv197 appears to affect the transcriptional regulation of most viral and many cellular genes. Further investigations are, however, needed to narrow down the role of AMV197 throughout the infection process.
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Amsacta moorei entomopoxvirus encodes a functional esterase amv133 with protease activity
Intervirology, 2015Co-Authors: Emine Ozsahin, Kazım Sezen, Zihni DemirbagAbstract:Objectives: Lipolytic genes have been investigated in several viral genomes, and some of them show enzyme activity which can be used for various functions including the production of DNA replication metabolites, rescue from endosomes, and membrane fusion. Amsacta moorei entomopoxvirus (AMEV) replicates in nearly the entire insect body, especially in the adipose tissue. One of the open reading frames (ORFs) in the AMEV genome, amv133, encodes a putative lipase enzyme. In this study, we therefore investigate the enzyme activity of amv133. Methods:amv133 was aligned with known lipase genes and their homologs in entomopoxviruses. Expressed proteins were partially purified and assayed for lipase, esterase and protease. Results: We found that amv133 contains all the domains required for a functional lipase enzyme and that it shows a significant similarity with homologs in other entomopoxviruses. Since there is a similarity of the catalytic triad between lipases and serine proteases, we also investigated the protease activity of amv133. Lipase, esterase and protease assays showed that amv133 encodes a functional esterase enzyme with protease activity. Conclusion: The current data show that amv133 is a conserved gene in all entomopoxvirus genomes sequenced so far and might contribute greatly to degrading the lipids or proteins and hence improve the virus infection.
Basil M Arif - One of the best experts on this subject based on the ideXlab platform.
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Amsacta moorei entomopoxvirus encodes a functional heparin-binding glycosyltransferase (AMV248)
Virus Genes, 2018Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni DemirbagAbstract:Amsacta moorei entomopoxvirus (AMEV) infects certain lepidopteran and orthopteran insects and is the most studied member of the genus Betaentomopoxvirus . It has been considered as a potential vector for gene therapy, a vector to express exogenous proteins and a biological control agent. One of its open reading frames, amv248, encodes a putative glycosyltransferase and is the only known attachment protein conserved in AMEV and chordopoxviruses. The ORF was successfully expressed and the protein was shown to bind soluble heparin, both in silico and in vitro. Our results also showed that, while viral infection was inhibited by soluble glycosaminoglycans (GAGs), GAG-deficient cells were more resistant to the virus. Finally, we revealed that amv248 encodes an active heparin-binding glycosyltransferase which is likely to have a key role in the initiation of infection by AMEV.
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transcriptional analysis of the putative glycosyltransferase gene amv248 of the Amsacta moorei entomopoxvirus
Virus Research, 2018Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni DemirbagAbstract:Abstract Amsacta moorei entomopoxvirus (AMEV), the most studied member of the genus Betaentomopoxvirus , was initially isolated from Red Hairy caterpillar larvae, Amsacta moorei. According to genome sequence and previous studies it was shown that amv248 encodes a putative glycosyltransferase that is the only conserved attachment protein in betaentomopoxviruses. Transcriptional analysis of the amv248 gene by RT-PCR and qPCR showed that transcription starts at 6 h post infection (hpi). Also, transcription was not affected by a DNA replication inhibitor but was severely curtailed by a protein synthesis inhibitor. These results indicate that amv248 belongs to the intermediate class of gene expression. 5′ and 3′ untranslated regions analysis revealed that transcription initiates at position –126 relative to the translational start site, and ends between 50 and 83 bases after the stop codon. To narrow down the size and location of the gene’s promoter, the upstream region as well as several different sized deletions thereof were generated and cloned upstream of a luciferase reporter gene. The constructs were used to measure the Firefly and Renilla luciferase activities in dual assays. The results showed that luciferase activity decreased when bases –198 to –235 of amv248 upstream region were missing. Sequence analysis among the intermediate gene promoters of AMEV showed that TTTAT(T/A)TT(T/A) 2 TTA is possibly a common motif, however, further investigations are needed to confirm this conclusion.
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genome wide analysis of differential mrna expression of Amsacta moorei entomopoxvirus mediated by the gene encoding a viral protein kinase amv197
Virus Research, 2016Co-Authors: Hacer Muratoğlu, Remziye Nalçacioğlu, Basil M Arif, Zihni DemirbagAbstract:Abstract Insect-born entomopoxviruses (Fam: Poxviridae ) are potentially important bio-pesticide against insect pests and expression vectors as well as vectors for transient human gene therapies including recombinant viral vaccines. For these reasons, it is necessary to understand the regulatory genes functions to improve its biotechnological potential. Here, we focused on the characterization of serine/threonine (Ser/Thr; ORF AMV197) protein kinase gene from the Amsacta moorei entomopoxvirus (AMEV), the type species of the genus Betaentomopoxvirus . Transcription of the parental and an amv197-null recombinant AMEV was compared by whole-genome gene expression microarray analysis. Blast2GO analysis reflected a broad diversity of upregulated and downregulated genes. Results showed that expression levels of 102 genes (45%) out of 226 tested genes changed significantly in the recombinant AMEV infected cells. Of these transcripts, 72 (70.58%) were upregulated and 30 (29.41%) were downregulated throughout the infection period. Genes involved in DNA repair, replication and nucleotide metabolism, transcription and RNA modification, and protein modification were mostly upregulated at different times in cells infected with the recombinant virus. Furthermore, transcription of all studied cellular genes including metabolism of apoptosis (Nedd2-like caspase, hemolin and elongation factor-1 alpha ( ef1a ) gene) was downregulated in the absence of amv197 . Quantitative real time reverse transcription-PCR confirmed viral transcriptional changes obtained by microarray. The results of this study indicated that the product of amv197 appears to affect the transcriptional regulation of most viral and many cellular genes. Further investigations are, however, needed to narrow down the role of AMV197 throughout the infection process.
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induction of apoptosis by the Amsacta moorei entomopoxvirus
Journal of General Virology, 2013Co-Authors: Srini C Perera, Remziye Nalçacioğlu, Zihni Demirbag, Peter J Krell, Basil M ArifAbstract:CF-70-B2 cells derived from the spruce budworm (Choristoneura fumiferana) undergo apoptosis when infected with Amsacta moorei entomopoxvirus (AMEV), as characterized by membrane blebbing, formation of apoptotic bodies, TdT-mediated dUTP nick-end labelling (TUNEL) staining, condensed chromatin and induction of caspase-3/7 activity. The apoptotic response was reduced when cells were infected with UV-inactivated AMEV, but not when infected in the presence of the DNA synthesis inhibitor, cytosine β-d-arabinofuranoside. Hence, only pre-DNA replication events were involved in inducing the antiviral response in CF-70-B2 cells. The virus eventually overcame the host’s antiviral response and replicated to high progeny virus titres accompanied by high levels of caspase-3/7 activity. The CF-70-B2 cells were less productive of progeny virus in comparison to LD-652, a Lymantria dispar cell line routinely used for propagation of AMEV. At late stages of infection, LD-652 cells also showed characteristics of apoptosis such as oligosomal DNA fragmentation, TUNEL staining, condensed chromatin and increased caspase-3/7 activity. Induction of apoptosis in LD-652 cells was dependent on viral DNA replication and/or late gene expression. A significantly reduced rate of infection was observed in the presence of general caspase inhibitors Q-VD-OPH and Z-VAD-FMK, indicating caspases may be involved in productive virus infection.
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expression of heterologous genes in the Amsacta moorei entomopoxvirus
Journal of Virological Methods, 2010Co-Authors: Srini C Perera, Peter J Krell, Philip Wong, Basil M ArifAbstract:Abstract Spheroidin (SPH) is the most abundant late protein in cells infected with the Amsacta moorei entomopoxvirus (AMEV). This locus can be used for expression of exogenous genes because it is not essential for virus replication. The sph promoter contains a conserved TAAATG motif, which serves as the site of initiation for both transcription and translation. Additional sequences downstream of the conserved motif have been shown to be involved in high-level expression of the sph gene. As a first step towards developing a protein expression vector based on the sph locus, four recombinant AMEV viruses expressing either gfp or lacZ were constructed. Both reporter genes were expressed under the control of the sph promoter containing the TAAATG motif. An additional 6 bp or 21 bp of sph coding region was included in three of the recombinants, to be expressed as an N-terminal fusion protein of GFP or LacZ. GFP and β-galactosidase expression was observed at 2 days post-infection and continued throughout the observation period. The highest level of reporter gene expression was observed in the recombinant containing 21 bp from the sph coding region. These results indicate that sph locus of AMEV can be used successfully to express exogenous genes.
Richard L. Hall - One of the best experts on this subject based on the ideXlab platform.
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Transient, nonlethal expression of genes in vertebrate cells by recombinant entomopoxviruses.
Journal of Virology, 1997Co-Authors: Yi Li, Richard L. Hall, Richard W. MoyerAbstract:The group B entomopoxvirus (EPV) from Amsacta moorei (AmEPV) productively infects only insect cells. A series of AmEPV-lacZ recombinants was constructed in which the lacZ gene was regulated by either late (the AmEPV spheroidin or the cowpox virus A-type inclusion [ATI]) or early (the AmEPV esp [early strong promoter; derived from a 42-kDa AmEPV protein] or the Melolontha melolontha EPV fusolin, fus) virus promoters. When the AmEPV recombinants were used to infect vertebrate cells, beta-galactosidase expression occurred (in >30% of the cells) when lacZ was regulated by either the fus or esp early promoters but not when lacZ was regulated by the late promoters (spheroidin or ATI). Therefore, AmEPV enters vertebrate cells and undergoes at least a partial uncoating and early, but not late, viral genes are expressed. Neither viral DNA synthesis nor cytopathic effects were observed under any infection conditions. When an AmEPV recombinant virus containing the Aequorea victoria green fluorescent protein gene (gfp) under the control of the esp promoter was used to infect vertebrate cells at a low multiplicity of infection, single fluorescent cells resulted, which continued to divide over a period of several days, ultimately forming fluorescent cell clusters, suggesting that vertebrate cells survive the infection and continue to grow. Therefore, AmEPV may prove to be a highly efficient, nontoxic method of gene delivery into vertebrate cells for transient gene expression.
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Characterization of a DNA Topoisomerase Encoded byAmsacta mooreiEntomopoxvirus
Virology, 1997Co-Authors: Birgitte Ø. Petersen, Richard W. Moyer, Richard L. Hall, Stewart ShumanAbstract:Abstract We have identified an Amsacta moorei entomopoxvirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxviruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant enzyme was partially purified. AmEPV topoisomerase is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma virus, and Orf virus enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT↓. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase ( k obs = 0.08 sec −1 ; K cl = 0.22) are similar to those of the vaccinia virus enzyme.
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TheAmsacta mooreiEntomopoxvirus Spheroidin Gene Is Improperly Transcribed in Vertebrate Poxviruses
Virology, 1996Co-Authors: Richard L. Hall, Yi Li, Joyce A. Feller, Richard W. MoyerAbstract:Abstract The Amsacta moorei entomopoxvirus (AmEPV) spheroidin is the most highly expressed late viral gene product in infected insect cells. However, when a cassette containing the spheroidin gene and putative promoter was inserted into cowpox (CPV) or vaccinia viruses, only very low levels of spheroidin gene expression were observed. Primer extension analysis suggests much lower spheroidin gene transcript levels than seen either for the highly expressed CPV A-type inclusion gene or for the spheroidin gene within infected insect cells, indicating that in vertebrate cells, the spheroidin promoter functions poorly if at all. Examination of the spheroidin mRNA synthesized in recombinant CPV shows that the 5′ start site of the spheroidin transcript was also unexpectedly imprecise and upstream (approximately 31 bp) of the well-defined start site normally observed in AmEPV-infected insect cells. Sequencing of the 5′ terminus of the CPV recombinant spheroidin mRNA suggested that 5′ poly(A), a characteristic feature of late poxvirus mRNAs and spheroidin mRNA derived from insect cells, was absent, despite the presence of the typical vertebrate poxvirus late promoter consensus sequence, TAAATG. Our results indicate that insect and vertebrate poxvirus promoters may not be universally interchangeable and imply that there are regulatory features of gene expression unique to the infected insect cell environment.
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the effect of inhibitors on the growth of the entomopoxvirus from Amsacta moorei in lymantria dispar gypsy moth cells
Virology, 1995Co-Authors: J Winter, Richard L. Hall, Richard W. MoyerAbstract:Abstract Within the family of Poxviridae, the entomopoxviruses are the most distant relatives of the more well-known and intensively studied orthopoxviruses (vaccinia and variola). The growth of the entomopoxvirus from Amsacta moorei (AmEPV) has been characterized in cell culture and compared to that of vaccinia virus (VV), the prototypic orthopoxvirus. The overall characteristics of infected cell cultures were generally similar between the two viruses. One striking difference noted was the apparent absence of proteolytic processing of late AmEPV viral proteins, a hallmark of vertebrate poxvirus infections associated with viral morphogenesis. AmEPV, like VV, was found to be sensitive to all the inhibitors of viral infection tested including phosphonoacetic acid, 1-β- d -arabinofuranosylcytosine, and isatin-β-thiosemicarbazone (IBT), a compound associated with the rather specific inhibition of vertebrate poxviruses. While both VV and AmEPV are inhibited by IBT, the inhibition of AmEPV, unlike that of VV, is not accompanied by either a breakdown of ribosomal RNA or a global inhibition of late viral protein synthesis. Instead, in the presence of IBT, AmEPV enveloped, immature virions form devoid of a well-differentiated core, which unlike mature virions fail to insert into occlusion bodies.
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identification of an Amsacta spheroidin like protein within the occlusion bodies of choristoneura entomopoxviruses
Virology, 1993Co-Authors: Richard L. Hall, Richard W. MoyerAbstract:Abstract Like baculoviruses, a characteristic feature of entomopoxviruses is the amalgamation of virions within environmentally stable occlusion bodies. It is this occluded form of the virus that is primarily responsible for dissemination to other insects. While the major protein (polyhedrin) of baculovirus occlusions is quite similar between viruses, it has been reported that the major occlusion body protein (spheroidin) of two group B entomopoxviruses, Amsacta moorei (AmEPV) and Choristoneura biennis (CbEPV) is quite different both in terms of amine acid sequence and coding capacity of the corresponding spheroidin genes (115 and 47 kDa for AmEPV and CbEPV, respectively). We report the discovery of a AmEPV spheroidin gene homolog in both CbEPV and a second Choristoneura virus, Choristoneura fumiferana (CfEPV). Antibodies directed against the AmEPV 115-kDa spheroidin reacted with the major protein of ∼115 kDa found within the occlusion body preparation from both Choristoneura viruses. Direct protein microsequencing of small portions of the 115-kDa protein from CbEPV has resulted in peptide sequences identical to those of corresponding regions of the AmEPV spheroidin gene. We suggest that it is this Choristoneura gene which encodes spheroidin. All attempts, however, to find a homolog of the previously reported CbEPV spheroidin gene within AmEPV have been unsuccessful. We also show this newly identified Choristoneura homolog of the AmEPV spheroidin gene as well as the AmEPV spheroidin gene itself are both located at the 3′ end of an NPH I gene and are highly homologous in all three viruses, indicating that this region of the genome in the three viruses is co-linear. These results and others suggest that while the insect viruses lack the traditional central core of conserved genes observed for the vertebrate poxviruses, the insect poxviruses may have also evolved an alternative central core of conserved genes, unique to the invertebrate poxviruses.
Hacer Muratoğlu - One of the best experts on this subject based on the ideXlab platform.
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Amsacta moorei entomopoxvirus encodes a functional heparin-binding glycosyltransferase (AMV248)
Virus Genes, 2018Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni DemirbagAbstract:Amsacta moorei entomopoxvirus (AMEV) infects certain lepidopteran and orthopteran insects and is the most studied member of the genus Betaentomopoxvirus . It has been considered as a potential vector for gene therapy, a vector to express exogenous proteins and a biological control agent. One of its open reading frames, amv248, encodes a putative glycosyltransferase and is the only known attachment protein conserved in AMEV and chordopoxviruses. The ORF was successfully expressed and the protein was shown to bind soluble heparin, both in silico and in vitro. Our results also showed that, while viral infection was inhibited by soluble glycosaminoglycans (GAGs), GAG-deficient cells were more resistant to the virus. Finally, we revealed that amv248 encodes an active heparin-binding glycosyltransferase which is likely to have a key role in the initiation of infection by AMEV.
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the protein protein interactions between Amsacta moorei entomopoxvirus amev protein kinases pks and all viral proteins
Virus Research, 2018Co-Authors: Hacer Muratoğlu, Remziye Nalçacioğlu, Mehtap Danismazoglu, Zihni DemirbagAbstract:Abstract Entomopoxviruses are an important group of viruses infecting only insects. They belong to Poxviridae which infect both invertebrates and vertebrates, including humans. Protein kinases are known to have roles at virus morphogenesis, host selectivity, the regulation of cell division and apoptosis in some vertebrate poxviruses. In this study, 2 protein kinases (PKs) (AMV153 and AMV197) of Amsacta moorei entomopoxvirus (AMEV) were investigated for the interactions among 230 viral proteins using yeast two-hybrid system (Y2H). For this purpose, two protein kinases and 230 viral genes were cloned into the bait and prey vectors, respectively. Bait vectors were introduced into Saccharomyces cerevisiae AH109. Expression of the bait genes were confirmed by western blot analysis. Both yeast strains of bait were transformed individually with each prey clone and grown on a selective medium (minimal synthetic defined) to determine the protein–protein interactions between bait and prey proteins. Transformations identified totally 16 interactions among AMEV protein kinases and all viral proteins of which 5 belong to AMV153 and 11 belong to AMV197. One of the five interactions detected for AMV153 protein kinase is self-association. Its other four interactions are with two virus entry complex proteins (AMV035 and AMV083), a membrane protein (AMV165) and a subunit of RNA polymerase (AMV230). The other protein kinase, AMV197, interacted with two virus entry complex proteins (AMV035 and AMV083) as AMV153, a caspase-2 enzyme (AMV063), a Holliday junction resolvase (AMV162), a membrane protein (AMV165), a subunit of RNA polymerase (AMV230) and five other hypothetical proteins (AMV026, AMV040, AMV062, AMV069, AMV120) encoded by AMEV genome. Glutathione S-transferase (GST) pull-down assay was used to confirm all interactions described by Y2H analysis. In addition, the theoretical structures of the two of 16 interactions were interpreted by docking analysis. Consistent with Y2H and pull down assays, docking analysis also showed the interactions of AMV063 with AMV153 and AMV197. Detected interactions of the AMEV viral proteins with viral protein kinases could lead to the understanding of the regulation of the viral activities of interacted viral proteins.
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transcriptional analysis of the putative glycosyltransferase gene amv248 of the Amsacta moorei entomopoxvirus
Virus Research, 2018Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni DemirbagAbstract:Abstract Amsacta moorei entomopoxvirus (AMEV), the most studied member of the genus Betaentomopoxvirus , was initially isolated from Red Hairy caterpillar larvae, Amsacta moorei. According to genome sequence and previous studies it was shown that amv248 encodes a putative glycosyltransferase that is the only conserved attachment protein in betaentomopoxviruses. Transcriptional analysis of the amv248 gene by RT-PCR and qPCR showed that transcription starts at 6 h post infection (hpi). Also, transcription was not affected by a DNA replication inhibitor but was severely curtailed by a protein synthesis inhibitor. These results indicate that amv248 belongs to the intermediate class of gene expression. 5′ and 3′ untranslated regions analysis revealed that transcription initiates at position –126 relative to the translational start site, and ends between 50 and 83 bases after the stop codon. To narrow down the size and location of the gene’s promoter, the upstream region as well as several different sized deletions thereof were generated and cloned upstream of a luciferase reporter gene. The constructs were used to measure the Firefly and Renilla luciferase activities in dual assays. The results showed that luciferase activity decreased when bases –198 to –235 of amv248 upstream region were missing. Sequence analysis among the intermediate gene promoters of AMEV showed that TTTAT(T/A)TT(T/A) 2 TTA is possibly a common motif, however, further investigations are needed to confirm this conclusion.
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genome wide analysis of differential mrna expression of Amsacta moorei entomopoxvirus mediated by the gene encoding a viral protein kinase amv197
Virus Research, 2016Co-Authors: Hacer Muratoğlu, Remziye Nalçacioğlu, Basil M Arif, Zihni DemirbagAbstract:Abstract Insect-born entomopoxviruses (Fam: Poxviridae ) are potentially important bio-pesticide against insect pests and expression vectors as well as vectors for transient human gene therapies including recombinant viral vaccines. For these reasons, it is necessary to understand the regulatory genes functions to improve its biotechnological potential. Here, we focused on the characterization of serine/threonine (Ser/Thr; ORF AMV197) protein kinase gene from the Amsacta moorei entomopoxvirus (AMEV), the type species of the genus Betaentomopoxvirus . Transcription of the parental and an amv197-null recombinant AMEV was compared by whole-genome gene expression microarray analysis. Blast2GO analysis reflected a broad diversity of upregulated and downregulated genes. Results showed that expression levels of 102 genes (45%) out of 226 tested genes changed significantly in the recombinant AMEV infected cells. Of these transcripts, 72 (70.58%) were upregulated and 30 (29.41%) were downregulated throughout the infection period. Genes involved in DNA repair, replication and nucleotide metabolism, transcription and RNA modification, and protein modification were mostly upregulated at different times in cells infected with the recombinant virus. Furthermore, transcription of all studied cellular genes including metabolism of apoptosis (Nedd2-like caspase, hemolin and elongation factor-1 alpha ( ef1a ) gene) was downregulated in the absence of amv197 . Quantitative real time reverse transcription-PCR confirmed viral transcriptional changes obtained by microarray. The results of this study indicated that the product of amv197 appears to affect the transcriptional regulation of most viral and many cellular genes. Further investigations are, however, needed to narrow down the role of AMV197 throughout the infection process.
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Transcriptional and structural analyses of Amsacta moorei entomopoxvirus protein kinase gene (AMV197, pk)
Annals of Microbiology, 2010Co-Authors: Hacer Muratoğlu, Remziye Nalçacioğlu, Zihni DemirbagAbstract:The Amsacta moorei entomopoxvirus (AMEV) genome has 279 open reading frames (ORFs) among which is the AMV197, composed of 900 nt and potentially encoding a protein of 299 amino acids. Sequence-derived amino acid analysis suggested it to be a serine/threonine protein kinase (PK) having conserved PK and serine/threonine PK domains. For transcriptional analysis of the AMV197 pk gene, Ld652 cells were infected with AMEV and mRNA was isolated at different times thereafter. RT–PCR analysis indicated that the transcription of the AMV197 pk gene started at 4 h post infection (h p.i.) and continued to be expressed through 24 h p.i. Infection of Ld cells in the presence of Ara-C (inhibits DNA replication), followed by RT–PCR showed that AMV197 pk is transcribed as an early gene. Transcription was initiated at 54 nt upstream of the translation start site. The vaccinia virus early promoter element G was also found at the correct position (−21) in the AMV197 pk gene. Rapid amplification of the 3′ ends of the AMV197 pk transcript showed that there are two polyadenylation start points. They are located at 22 and 32 nucleotides downstream of translation stop site. Also, the translational stop site and poly (A) signal of AMV197 pk are overlapped. The termination signal TTTTTGT sequence of vaccinia virus early genes was found just upstream of the 3′ end of AMV197 pk gene. Conserved amino acid subdomains of the AMV197 PK were found by sequence comparisons with PK’s from other organisms. Analysis of the protein sequence of AMV197 pk gene reveals close identity with PK genes of other organisms.
