Amsacta moorei entomopoxvirus encodes a protein kinase with dual activity and a broad substrate spectrum including two putative cellular substrates.

Amsacta moorei entomopoxvirus (AMEV) is a poxvirus that can only infect insects. This virus is an attractive research material because it is similar to smallpox virus. AMEV is one of many viruses that encode protein kinases that drive the host's cellular mechanisms, modifying immune responses to it, and regulating viral protein activity. We report here the functional characterization of a serine/threonine (Ser/Thr) protein kinase (PK) gene (ORF AMV197) of AMEV. Expression of the AMV197 gene in baculovirus expression system yielded a ~ 35.5 kDa protein. PK activity of expressed AMV197 was shown by standard PK assay. Substrate profiling of AMV197 protein by peptide microarray indicated that the expressed protein phosphorylated 81 of 624 substrates which belong to 28 families of PK substrates. While the hypothetical AMV197 protein phosphorylates Ser/Thr only, we demonstrated that the expressed PK also phosphorylates probes with tyrosine residues on the array which is a rare property among PKs. Pull-down assay of the AMV197 protein with the subcellular protein fractionations of Ld652 cells showed that it is using two cellular proteins (18 and 42 kDa) as novel putative substrates. Our results suggest that AMEV can regulate cellular mechanisms by phosphorylating cellular proteins through AMV197 PK. However, further experiments are needed to identify the exact role of this PK in the replication of AMEV.

The Melolontha melolontha entomopoxvirus fusolin protein is a chitin-active lytic polysaccharide monooxygenase that displays extreme stability.

Spindles are intracellular crystals of the fusolin protein that enhances the oral virulence of insect poxviruses by disruption of the larval chitinous peritrophic matrix. The enigmatic fusolin protein is classified as a lytic polysaccharide monooxygenase (LPMO) by sequence and structure. Although circumstantial evidence points towards a role for fusolin in chitin degradation, no biochemical data exist to verify this claim. In the present study, we demonstrate that fusolin released from over 40-year-old spindles, stored for 10 years at 4 °C, are chitin-degrading LPMOs. Not only was fusolin active after long-term storage, but it also withstood high temperature and oxidative stress in its crystalline form, highlighting extreme stability that is beneficial to viral persistence and desirable for potential biotechnology applications.

Amsacta moorei entomopoxvirus encodes a functional esterase (amv133) with protease activity.

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.

Further research on the biological function of inclusion bodies of Anomala cuprea entomopoxvirus, with special reference to the effect on the insecticidal activity of a Bacillus thuringiensis formulation.

BACKGROUND: Entomopoxviruses (EVs) form two types of inclusion body: spheroids, which contain virions, and spindles, which do not. The authors tested whether the spindles from a coleopteran EV, Anomala cuprea EV (ACEV), enhanced the insecticidal activity of a commercial Bacillus thuringiensis (Bt) formulation and the susceptibility of scarabaeid pest species in Japan to the virus's spheroids, to assess whether ACEV inclusion bodies are potential biological control agents for pest insects. RESULTS: Peroral inoculation with both ACEV spindles and the Bt toxin only or the complete Bt formulation shortened the survival and increased the mortality of treated insects compared with those of insects inoculated with Bt without the spindles (8-38 h of decrease in LT50 values among assays). ACEV showed high infectivity to a major scarabaeid pest species in Japanese sugar cane fields. CONCLUSION: The results suggest that spindles or the constituent protein fusolin can be used as a coagent with Bt formulations, and that fusolin coexpression with a Bt toxin in crops might improve the insecticidal efficacy. In addition, the spheroids are potential biocontrol agents for some scarabaeid pests that are not easy to control because of their underground habitation.

The N-terminal region of an entomopoxvirus fusolin is essential for the enhancement of peroral infection, whereas the C-terminal region is eliminated in digestive juice.

The spindles of Anomala cuprea entomopoxvirus (AncuEPV), which are composed of glycoprotein fusolin, are known to enhance the peroral infectivity of AncuEPV itself and of nucleopolyhedroviruses. This has been demonstrated to involve the disruption of intestinal peritrophic membrane (PM), composed of chitin matrix, glycosaminoglycans, and proteins. To identify essential and nonessential regions for this enhancement activity, AncuEPV fusolin and its deletion mutants were expressed in Sf21 cells using a baculovirus system, and their enhancement abilities were analyzed. The recombinant fusolin enhanced the peroral infectivity of Bombyx mori nucleopolyhedrovirus up to 320-fold and facilitated the infection of host insect with AncuEPV. Deletion mutagenesis revealed that the N-terminal region (amino acids 1 to 253), a possible chitin-binding domain, is essential for the enhancement of infection, whereas the C-terminal region is entirely dispensable. The glycosylation-defective mutants N191Q, whose Asn(191) is replaced with Gln, and DeltaSIG, whose signal peptide is deleted, showed considerably reduced and abolished enhancing activities, respectively, indicating that the carbohydrate chain is important in the enhancing activity. Interestingly, the C-terminal dispensable region was digested by a serine protease(s) in insect digestive juice. Moreover, both the N-terminal conserved region and the carbohydrate chain were necessary not only for chitin binding but also for stability in digestive juice. A triple amino acid replacement mutant, IHE (Ile-His-Glu(161) to Ala-Ala-Ala), was stable in digestive juice and had chitin-binding ability but did not retain its enhancing activity. These results suggest that the enhancement of infectivity involves more than the tolerance to digestive juice and chitin-binding ability.

Characterization of mimivirus NAD+-dependent DNA ligase.

Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication, modification, and repair. Here we produced, purified, and characterized mimivirus DNA ligase (MimiLIG), an NAD+-dependent nick joining enzyme homologous to bacterial LigA and entomopoxvirus DNA ligase. MimiLIG is a 636-aa polypeptide composed of an N-terminal NAD+ specificity module (domain Ia), linked to nucleotidyltransferase, OB-fold, helix-hairpin-helix, and BRCT domains, but it lacks the tetracysteine Zn-binding module found in all bacterial LigA enzymes. MimiLIG requires conserved domain Ia residues Tyr36, Asp46, Tyr49, and Asp50 for its initial reaction with NAD+ to form the ligase-AMP intermediate, but not for the third step of phosphodiester formation at a preadenylylated nick. MimiLIG differs from bacterial LigA enzymes in that its activity is strongly dependent on the C-terminal BRCT domain, deletion of which reduced its specific activity in nick joining by 75-fold without affecting the ligase adenylylation step. The DeltaBRCT mutant of MimiLIG was impaired in sealing at a preadenylylated nick. We propose that eukaryal DNA viruses acquired the NAD+-dependent ligases by horizontal transfer from a bacterium and that MimiLIG predates entomopoxvirus ligase, which lacks both the tetracysteine and BRCT domains. We speculate that the dissemination of NAD+-dependent ligase from bacterium to eukaryotic virus might have occurred within an amoebal host.

Unique ligation properties of eukaryotic NAD+-dependent DNA ligase from Melanoplus sanguinipes entomopoxvirus.

The eukaryotic Melanoplus sanguinipes entomopoxvirus (MsEPV) genome reveals a homologous sequence to eubacterial nicotinamide adenine dinucleotide (NAD(+))-dependent DNA ligases [J. Virol. 73 (1999) 533]. This 522-amino acid open reading frame (ORF) contains all conserved nucleotidyl transferase motifs but lacks the zinc finger motif and BRCT domain found in conventional eubacterial NAD(+) ligases. Nevertheless, cloned MsEPV ligase seals DNA nicks in a NAD(+)-dependent fashion, while adenosine 5'-monophosphate (ATP) cannot serve as an adenylation cofactor. The ligation activity of MsEPV ligase requires Mg(2+) or Mn(2+). MsEPV ligase seals sticky ends efficiently, but has little activity on 1-nucleotide gap or blunt-ended DNA substrates even in the presence of polyethylene glycol. In comparison, bacterial NAD(+)-dependent ligases seal blunt-ended DNA substrates in the presence of polyethylene glycol. MsEPV DNA ligase readily joins DNA nicks with mismatches at either side of the nick junction, except for mismatches at the nick junction containing an A base in the template strand (A/A, G/A, and C/A). MsEPV NAD(+)-dependent DNA ligase can join DNA probes on RNA templates, a unique property that distinguishes this enzyme from other conventional bacterial NAD(+) DNA ligases. T4 ATP-dependent DNA ligase shows no detectable mismatch ligation at the 3' side of the nick but substantial 5' T/G mismatch ligation on an RNA template. In contrast, MsEPV ligase joins mismatches at the 3' side of the nick more frequently than at the 5' side of the nick on an RNA template. The complementary specificities of these two enzymes suggest alternative primer design for genomic profiling approaches that use allele-specific detection directly from RNA transcripts.

In vivo enhancement of nucleopolyhedrovirus of oriental Armyworm, Mythimna separata using spindles from Helicoverpa armigera entomopoxvirus.

When the third instar larvae of M. separata were exposed to eight varying concentrations of polyhedral occlusion bodies (POB's) of nucleopolyhedrovirus of M. separata (MsNPV) ranging from 2.6 x 10(-1) to 2.6 x 10(8) POB's/ml, the percent mortality and incubation period ranged from 16-100% and 14 to 9 days respectively. On the other hand when the same third instar larvae of M. separata were exposed to only five varying concentration of POB's of MsNPV ranging from 2.6 x 10(2) to 2.6 x 10(6), POB's/ml along with a constant dose of entomopox viral spindles from Helicoverpa armigera, the per cent mortality ranged from 63 to 100% with reduction in incubation period from 7 to 4 days respectively. The enhancement index (log10) of the virus was 2.76 or reduction of more than 500 times in LC50. The ability and the mechanism of the spindles from H. armigera entomopoxvirus to enhance the infectivity of MsNPV has been discussed.

Assessment of foreign protein production by recombinant Heliothis (Helicoverpa) armigera entomopoxviruses in Spodoptera frugiperda cells.

This report describes the first production of recombinant forms of Heliothis (Helicoverpa) armigera entomopoxvirus (HaEPV). These HaEPVs are engineered at either the spheroidin or fusolin locus, to produce the green fluorescent marker protein (GFP). The growth properties of these recombinant HaEPVs, in comparison to the parental HaEPV, were assessed in cultured Spodoptera frugiperda Sf9 cells. Additionally, GFP production by these recombinant HaEPVs was compared to that of a GFP-expressing recombinant of the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) in the same in vitro system, at various multiplicities of infection. Expression of GFP from the HaEPV spheroidin locus produced up to 60% of that generated from the AcNPV polyhedrin locus, albeit over a longer period of infection. A considerably lower yield was recorded from the HaEPV fusolin locus, a result that contrasted markedly with the apparent activity of this promoter in caterpillar infections in vivo. The potential applications for further development of HaEPV expression systems are discussed.

A bacterially produced virus enhancing factor from an entomopoxvirus enhances nucleopolyhedrovirus infection in armyworm larvae.

Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have expressed a virus enhancing factor (EF) from Pseudaletia separata entomopoxvirus, which enhances P. unipuncta multi nucleopolyhedrovirus (PsunMNPV) infection in larvae of the armyworm, P. separata. The lysates of transformed E. coli cells, which were not active in enhancing PsunMNPV infection, became active when treated with either trypsin or thrombin. The GST-EF fusion protein in a lysate was purified with a bulk GST purification module and cleaved into the EF and GST moieties with thrombin. Removal of the GST moiety with glutathione-Sepharose 4B resulted in a highly purified EF preparation, which enhanced PsunMNPV infection in armyworm larvae and PsunMNPV fusion with an armyworm cell line, SIE-MSH-805-F.

Spindle bodies of Heliothis armigera entomopoxvirus develop in structures associated with host cell endoplasmic reticulum.

Immunoelectron microscopy has shown that morphogenesis of spindle bodies (SB) of Heliothis armigera entomopoxvirus involves an iterative process of condensation, aggregation, and crystallization of the major constituent protein (fusolin) within the perinuclear space and endoplasmic reticulum (ER) of infected cells and in vesicles derived from ER constituents. The ER-specific chaperone BiP has been observed to be associated with developing SBs at all stages of this process, and it is postulated that its sequestration within these bodies may have consequences for host cell metabolism.

The Amsacta moorei entomopoxvirus spheroidin gene is improperly transcribed in vertebrate poxviruses.

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 spheroldin 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.

Identification and characterization of a filament-associated protein encoded by Amsacta moorei entomopoxvirus.

A novel protein which is expressed at high levels in insect cells infected with Amsacta moorei entomopoxvirus was identified by our laboratory. This viral gene product migrates as a 25/27-kDa doublet when subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. It is expressed at late times of infection and is present in infected cells but is absent in purified extracellular virions and occlusion bodies. The gene encoding this polypeptide was mapped on the viral genome, and cDNA clones were generated and sequenced. The predicted protein was shown to be phosphorylated and contained an unusual 10-unit proline-glutamic acid repeat element. A polyclonal antiserum was produced against a recombinant form of the protein expressed in Escherichia coli, and a monoclonal antibody which reacted with the proline-glutamic acid motif was also identified. Immunofluorescence and immunoelectron microscopy techniques revealed that this protein is associated with large cytoplasmic fibrils which accumulate in the cytoplasm between 96 and 120 h postinfection. We subsequently called this viral polypeptide filament-associated late protein of entomopoxvirus. The fibrils containing this polypeptide are closely associated with occlusion bodies and may play a role in their morphogenesis and maturation.