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. 2004 Oct;78(19):10265-75.
doi: 10.1128/JVI.78.19.10265-10275.2004.

Amsacta moorei entomopoxvirus expresses an active superoxide dismutase

M N Becker  1 , W B Greenleaf, D A Ostrov, R W Moyer
Affiliations

Amsacta moorei entomopoxvirus expresses an active superoxide dismutase

M N Becker et al. J Virol. 2004 Oct.

Abstract

The entomopoxvirus from Amsacta moorei serves as the prototype of the group B entomopoxviruses. One of the interesting genes found in Amsacta moorei entomopoxvirus (AmEPV) is a superoxide dismutase (sod) (open reading frame AMV255). Superoxide dismutases (SODs) catalyze the conversion of superoxide radicals to hydrogen peroxide and oxygen. Many vertebrate poxviruses contain a sod gene, but to date, none have been demonstrated to be active. There are three families of SODs, characterized by their metal ion-binding partners, Fe, Mn, or Cu and Zn. Poxvirus enzymes belong to the Cu-Zn SOD family. Unlike inactive vertebrate poxvirus SODs, AMVSOD contains all the amino acids necessary for function. We expressed and purified a 6X-His-tagged version of the AMVSOD in Escherichia coli. The recombinant AMVSOD demonstrates superoxide dismutase activity both in an in situ gel assay and by stopped flow spectrophotometry. The k(cat)/K(m) for AMVSOD is 4 x 10(7) M(-1)s(-1). In infected cells, the AMVSOD protein behaves as a dimer and is catalytically active; however, disruption of the gene in AMEPV has little or no effect on growth of the virus in cell culture. An analysis of mRNA expression indicates that AMVsod is expressed late during infection of Lymantria dispar (Ld652) cells and produces a discrete nonpolydisperse transcript. Characterization of protein expression with a monoclonal antibody generated against AMVSOD confirms that the AMVSOD protein can be classified as a late, postreplicative gene. Therefore, AMVSOD is the first example of an active poxvirus SOD.

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Figures

FIG. 1.
FIG. 1.
Alignment of sod genes from viral and cellular sources. The sequences were aligned with AlignX software (Invitrogen-Informax). The sequences used were AmEPV AMV255 (AMV255, GenBank accession number NP_065037), bovine SOD (P00442), human SOD (P00441), vaccinia virus SOD (strain Wyeth, AAK76404), myxoma virus SOD (MYX, NP_051845), Shope fibroma virus SOD (SFV, strain Kasza, NP_052020). The key metal binding residues are indicated by ∧, intrastrand disulfide linkages by * and the catalytic arginine by #. The ∼ represents residues at the dimer interface for bovine SOD. AMV255 contains all of the key elements necessary for function compared with bovine SOD; however, the vaccinia virus, myxoma virus, and Shope fibroma virus sod genes have alterations to the gene which would render them inactive (4, 12, 13).
FIG. 2.
FIG. 2.
Purification and activity assay for His-SOD. His-SOD protein isolated from E. coli was electrophoresed on 10% polyacrylamide-SDS gels (A, B, and C) and a native polyacrylamide gel (D). The size standards refer to panels A, B, and C. (A) A Coomassie-stained gel of denatured purified protein. (B) Immunoblot with the His tag antibody, showing that all of the visible bands on the Coomassie-stained gel are also immunoreactive and therefore represent multiple forms of the His-SOD protein. (C and D) The activity of the recombinant protein was assayed following separation on either SDS-PAGE (panel C) or native polyacrylamide gels (panel D) in the absence of reducing agent, with the inhibition of NBT reduction technique of Beauchamp and Fridovich (10). The clear zones on the gels indicate SOD activity.
FIG. 3.
FIG. 3.
Activity of His-SOD determined with stopped-flow spectrophotometry. The initial velocities of the catalyzed disproportionation of superoxide anion at 20°C are shown. Final solutions contained 100 mM CHES, 1 mM CAPS, 1 mM EDTA, and 0.25 μM His-SOD at pH 9.5. The total enzyme concentration is based on the total Cu occupancy. The solid line is a least squares fit of the data to the Michaelis-Menten equation with kcat/Km = 4.1 ± 0.1 × 107 M−1 s−1.
FIG. 4.
FIG. 4.
Time course of mRNA expression in wild-type AmEPV as determined by Northern blotting. Each blot contains 3 μg of total RNA per lane. The time points are 3, 6, 9, 12, 15, 18, 24, and 48 h postinfection (hpi). The rightmost three lanes of each blot contain RNA isolated from mock-infected cells at 12, 18, and 24 h, respectively. Each blot was hybridized with a single oligonucleotide to one AmEPV gene. The sequences of the oligonucleotide probes are given in Table 2. (A) AMV255 sod gene. The oligonucleotide probe is near the 5′ end of the gene. (B) AMV139 p4a. (C) AMV147 p4b. (D) AMV054 RAP94. (E) AMV016 thymidine kinase. (F) AMV199 DNA ligase.
FIG. 5.
FIG. 5.
AmEPV SOD activity during infection. (A) Coomassie-stained gel of infected and uninfected cellular protein extracts. Extraction with chloroform-ethanol is indicated by + (lanes 3 and 4). Extracts were from Ld652 cells infected with wild-type AmEPV at a multiplicity of infection of 0.15 (lanes 2 and 4) or mock infected (lanes 1 and 3), and proteins were harvested at 48 h postinfection. Lane 5 contains purified His-SOD protein. (B) In situ gel activity assay. Protein extracts were separated by SDS-PAGE and then processed for activity as described in Materials and Methods. The arrow indicates the band of activity in the wild-type AmEPV extract (lane 2).
FIG. 6.
FIG. 6.
Size determination of AMVSOD from infected cells. (A) Chromatogram from a Superdex 75 column of a mixture of proteins from wild-type AmEPV-infected cells. Arrows at the top indicate the locations of peaks for each size standard. Elution volume is indicated on the x axis. (B) Chromatogram of bovine SOD for comparison. The calculated size is 34.6 kDa. (C) The standard curve for the column is derived from proteins of known size: albumin, 67 kDa; ovalbumin, 43 kDa; chymotrypsinogen A, 25 kDa; and RNase A, 13.7 kDa. B indicates the position of the blue dextran peak used to determine the void volume of the column. (D) Western blot of 0.5-ml fractions from the column with monoclonal antibody 2B8-1C9 to detect AMVSOD. Lane 1, total protein; lane 2, 0.25 μg of purified His-SOD, lanes 3 to 10 are labeled according to the starting elution volume of the fraction and correspond to fractions 19 to 26, respectively. AMVSOD elutes predominantly at 10 to 11 ml.
FIG. 7.
FIG. 7.
Inactivation of the sod gene in AmEPV. Immunoblot analysis with the SOD monoclonal antibody 2B8-1C9. Each lane contains 10 μg of denatured, unreduced protein. Lane 1 contains protein from mock-infected cells; lane 2 contains protein from wild-type AmEPV-infected cells. Δsodgfp refers to the two different recombinant viruses, each containing the GFP marker but in different orientations with respect to the transcription of the intact sod gene. The monoclonal antibody was used at a 1:1,000 dilution.
FIG. 8.
FIG. 8.
Model of the AMVSOD dimer. The model shows an AMVSOD dimer (in red) superimposed on a bovine SOD dimer (PDB 1E9O) in blue. Disulfide bonds are shown in yellow. The model shows a high level of similarity between the two SOD proteins. The figure was generated with SETOR.
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