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Richard W. Moyer – 1st expert on this subject based on the ideXlab platform

  • an Amsacta moorei entomopoxvirus ortholog of the poly a polymerase small subunit exhibits methyltransferase activity and is non essential for virus growth
    Virology, 2008
    Co-Authors: Marie N Becker, Tracie M Todd, Richard W. Moyer

    Abstract:

    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.

  • Amsacta moorei entomopoxvirus inhibitor of apoptosis suppresses cell death by binding grim and hid
    Journal of Virology, 2005
    Co-Authors: Qianjun Li, Peter Liston, Natasha Schokman, Jenny Mei Ho, Richard W. Moyer

    Abstract:

    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.

  • Functional Analysis of the Inhibitor of Apoptosis (iap) Gene Carried by the Entomopoxvirus of Amsacta moorei
    Journal of Virology, 2005
    Co-Authors: Qianjun Li, Peter Liston, Richard W. Moyer

    Abstract:

    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.

Zihni Demirbag – 2nd expert on this subject based on the ideXlab platform

  • Amsacta moorei entomopoxvirus encodes a functional heparin-binding glycosyltransferase (AMV248)
    Virus Genes, 2018
    Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni Demirbag

    Abstract:

    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.

  • the protein protein interactions between Amsacta moorei entomopoxvirus amev protein kinases pks and all viral proteins
    Virus Research, 2018
    Co-Authors: Hacer Muratoğlu, Remziye Nalçacioğlu, Mehtap Danismazoglu, Zihni Demirbag

    Abstract:

    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.

  • transcriptional analysis of the putative glycosyltransferase gene amv248 of the Amsacta moorei entomopoxvirus
    Virus Research, 2018
    Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni Demirbag

    Abstract:

    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.

Basil M Arif – 3rd expert on this subject based on the ideXlab platform

  • Amsacta moorei entomopoxvirus encodes a functional heparin-binding glycosyltransferase (AMV248)
    Virus Genes, 2018
    Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni Demirbag

    Abstract:

    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.

  • transcriptional analysis of the putative glycosyltransferase gene amv248 of the Amsacta moorei entomopoxvirus
    Virus Research, 2018
    Co-Authors: Cihan Inan, Hacer Muratoğlu, Basil M Arif, Zihni Demirbag

    Abstract:

    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.

  • genome wide analysis of differential mrna expression of Amsacta moorei entomopoxvirus mediated by the gene encoding a viral protein kinase amv197
    Virus Research, 2016
    Co-Authors: Hacer Muratoğlu, Remziye Nalçacioğlu, Basil M Arif, Zihni Demirbag

    Abstract:

    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.