Production and characterization of monoclonal antibodies against shope fibroma virus superoxide dismutase and glutathione-s-transferase.

PURPOSE: The superoxide dismutase (SOD) like proteins encoded by Leporipoxviruses play a role in regulating the redox status of infected cells. The biological function of these proteins is unclear. Why poxviruses encode these proteins are still unknown. Exploiting standard hybridoma techniques, we developed a monoclonal antibody (MAb) against shope fibroma virus superoxide dismutase (sfvSOD) to be used in diagnostics and as tools to understand the role of SOD-like proteins in pathogenesis. METHODS: Hybridoma cell fusion technology was used for production of MAbs. Balb/c mice were immunized with sfvSOD-GST fusion protein. Hybridoma clones were screened using indirect enzyme linked immunosorbent assay (ELISA). Specificity and reactivity of the MAbs were determined by Western blot analysis (WBA) and indirect ELISA. Protein G affinity chromatography was used for the purification of MAbs. RESULTS: Two stable hybridoma clones producing MAbs against the two domains of the fusion protein were obtained. The anti-GST (glutathione-s-transferase) and anti-sfvSOD MAbs were found to react specifically with GST and sfvSOD proteins respectively, in addition to the sfvSOD-GST fusion protein. Isotypes of these MAbs were identified as IgG2b heavy chain and k light chain. CONCLUSION: The anti-sfvSOD MAb (P115.SOD MAb) has been successfully used in studying the enzymatic and biochemical properties of a SOD homolog encoded by sfv. We also developed a strong anti-GST MAb which was also cloned and characterized P115.GST MAb. The anti-GST MAb might be useful in analyzing GST fusion proteins and in immunoaffinity chromatography purification of GST fusion proteins.

Genetic and phylogenetic characterization of the type II cyclobutane pyrimidine dimer photolyases encoded by Leporipoxviruses.

Shope fibroma virus and myxoma virus encode proteins predicted to be Type II photolyases. These are enzymes that catalyze light-dependent repair of cyclobutane pyrimidine dimers (CPDs). When the Shope fibroma virus S127L gene was expressed in an Escherichia coli strain lacking functional CPD repair pathways, the expressed gene protected the bacteria from 70-75% of the ultraviolet (UV) light-induced cytotoxic DNA damage. This proportion suggests that Leporipoxvirus photolyases can only repair CPDs, which typically comprise approximately 70% of the damage caused by short wavelength UV light. To test whether these enzymes can protect virus genomes from UV, we exposed virus suspensions to UV-C light followed by graded exposure to filtered visible light. Viruses encoding a deletion of the putative photolyase gene were unable to photoreactivate UV damage while this treatment again eliminated 70-90% of the lethal photoproducts in wild-type viruses. Western blotting detected photolyase protein in extracts prepared from purified virions and it can be deduced that the poxvirion interior must be fluid enough to permit diffusion of this approximately 50-kDa DNA-binding protein to the sites where it catalyzes photoreactivation. Photolyase promoters are difficult to categorize using bioinformatics methods, as they do not obviously resemble any of the known poxvirus promoter motifs. By fusing the SFV promoter to DNA encoding a luciferase open reading frame, the photolyase promoter was found to exhibit very weak late promoter activity. These data show that the genomes of Leporipoxviruses, similar to that of fowlpox virus, encode catalytically active photolyases. Phylogenetic studies also confirmed the monophyletic origin of poxviruses and suggest an ancient origin for these genes and perhaps poxviruses.

Leporipoxvirus Cu,Zn-superoxide dismutase (SOD) homologs are catalytically inert decoy proteins that bind copper chaperone for SOD.

Many Chordopoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD). The biological function of these proteins is unknown, although the proteins encoded by Leporipoxviruses have been shown to promote a slow decline in the level of superoxide dismutase activity in virus-infected cells. To gain more insights into their function, we have further characterized the enzymatic and biochemical properties of a SOD homolog encoded by Shope fibroma virus. Shope fibroma virus SOD has retained the zinc binding properties of its cellular homolog, but cannot bind copper. Site-directed mutagenesis showed that it requires at least four amino acid substitutions to partially restore copper binding activity, but even these changes still did not restore catalytic activity. Reciprocal co-immunoprecipitation experiments showed that recombinant Shope fibroma virus SOD forms very stable complexes with cellular copper chaperones for SOD and these observations were confirmed using glutathione-S-transferase tagged proteins. Similar viral SOD/chaperone complexes were formed in cells infected with a closely related myxoma virus, where we also noted that some of the SOD antigen co-localizes with mitochondrial markers using confocal fluorescence microscopy. About 2% of the viral SOD was subsequently detected in gradient-purified mitochondria extracted from virus-infected cells. These poxviral SOD homologs do not form stable complexes with cellular Cu,Zn-SOD or affect its concentration. We suggest that Leporipoxvirus SOD homologs are catalytically inert decoy proteins that are designed to interfere in the proper metallation and activation of cellular Cu,Zn-SOD. This reaction might be advantageous for tumorigenic poxviruses, since higher levels of superoxide have been proposed to have anti-apoptotic and tumorigenic activity.

Shope fibroma virus DNA topoisomerase catalyses holliday junction resolution and hairpin formation in vitro.

The telomeres of poxviral chromosomes comprise covalently closed hairpin structures bearing mismatched bases. These hairpins are formed as concatemeric replication intermediates and are processed into mature, unit-length genomes. The structural transitions and enzymes involved in telomere resolution are poorly understood. Here we show that the type I topoisomerase of Shope fibroma virus (SFV) can promote a recombination reaction which converts cloned SFV replication intermediates into hairpin-ended molecules resembling mature poxviral telomeres. Recombinant SFV topoisomerase linearised a palindromic plasmid bearing 1.5 kb of DNA encoding the SFV concatemer junction, at a site near the centre of inverted-repeat symmetry. Most of these linear reaction products bore hairpin tips as judged by denaturing gel electrophoresis. The resolution reaction required palindromic SFV DNA sequences and was inhibited by compounds which block branch migration (MgCl2) or poxviral topoisomerases. The resolution reaction was also slow, needed substantial quantities of topoisomerase, and required that the palindrome be extruded in a cruciform configuration. DNA cleavage experiments identified a pair of suitably oriented topoisomerase recognition sites, 90 bases from the centre of the cloned SFV terminal inverted repeat, which may mark the resolution site. These data suggest a resolution scheme in which branch migration of a Holliday junction through a site occupied by covalently bound topoisomerase molecules, could lead to telomere resolution.

DNA ligase gene disruptions can depress viral growth and replication in poxvirus-infected cells.

Poxvirus-encoded DNA ligases are assumed to play a role in viral DNA replication; however mutational inactivation of vaccinia ligase has not been reported to affect viral growth rates in culture. This communication re-examines this surprising aspect of poxviral biology using both Shope fibroma virus (SFV) and vaccinia virus. SFV and vaccinia ligase deficiencies create essentially identical phenotypes. In particular, ligase-deficient SFV strains are mildly UV sensitive and etoposide resistant, phenotypes previously shown to characterize ligase-deficient vaccinia strains. Moreover, we find that ligase mutations can inhibit the growth of both SFV and vaccinia virus in vitro. The poor growth observed in the absence of a viral ligase is correlated with a two- to tenfold reduction in viral and extragenomic DNA synthesis. This phenotype is host dependent. No differences in viral growth or DNA yield were seen when vaccinia strains were cultured on rabbit (SIRC) cells, but ligase deficiencies reduced growth and DNA yields when vaccinia was plated on BSC-40 cells or SFV on SIRC cells. Despite these replicative defects, mutational inactivation of SFV ligase produced no detectable increase in the number of viral DNA breaks and had no effect on virus-catalyzed extragenomic DNA recombination or UV repair. We conclude that poxviral ligases do play a role in viral DNA replication, but the replicative defect is obscured in some cell lines.

SFV topoisomerase: sequence specificity in a genetically mapped interval.

Poxviral DNA topoisomerases are sequence-specific enzymes whose activities are thought to influence such diverse processes as transcription, DNA replication, and genetic recombination. To obtain further insights into the relatedness of these enzymes, and their influence on virus-mediated recombination, we have determined the target-specificity and other catalytic properties of the Shope fibroma virus (SFV) topoisomerase. SFV topoisomerase was expressed in Escherichia coli and purified as a glutathione S-transferase (GST) or (his)6-tagged fusion protein. The recombinant Leporipox-virus (SFV) enzyme displayed catalytic properties very similar to vaccinia topoisomerase. In particular SFV topoisomerase recognizes the same pentanucleotide motif [5'-(C/T)CCTT-3'] and promotes the same DNA relaxation, strand transfer, and strand cleavage reactions catalyzed by the Orthopoxviral (vaccinia) enzyme. The SFV enzyme can also efficiently cleave DNA 3' of the variant site 5'-CCCTG-3' in certain sequence contexts. These studies identified several sites where SFV topoisomerases interact with a recombinational substrate and permitted a comparison of recombination frequencies across intervals which did, or did not, span these sites. We failed to detect any effect of topoisomerase-recognition sites on recombination frequencies, except for a small (< 2-fold) stimulation seen when the substrates encoded a nearby poxviral promoter. This and other work shows that poxviral topoisomerases from several genera share common target specificities, but other enzymatic systems probably catalyze the high-frequency recombination seen in poxvirus-infected cells.

Myxoma virus and Shope fibroma virus encode dual-specificity tyrosine/serine phosphatases which are essential for virus viability.

Sequence analysis of the genomes of the Leporipoxviruses myxoma virus and Shope fibroma virus (SFV) led to the discovery of open reading frames homologous to the vaccinia H1L gene encoding a soluble protein phosphatase with dual tyrosine/serine specificity. These viral phosphatase genes were subsequently localized to the myxoma BamHI-I fragment and the SFV BamHI-M fragment, and the resulting encoded proteins were designated I1L and M1L, respectively. The localization and orientation of the myxoma I1L and SFV M1L open reading frames within the well conserved central core of the viral genomes closely mirror that of the Orthopoxviruses vaccinia virus and variola virus. The myxoma I1L and SFV M1L phosphatases each contain the conserved tyrosine phosphatase signature sequence motif, (I/V)HCXAGXXR(S/T)G, including the active site cysteine, found previously to be essential for phosphotyrosine dephosphorylation. The vaccinia H1L phosphatase was originally shown to have the ability to dephosphorylate phosphotyrosyl and phosphoseryl residues in vitro. To assess whether this is a common feature of poxvirus phosphatases, myxoma I1L was expressed as a GST-fusion protein, purified, and shown to dephosphorylate substrates containing tyrosine and serine phosphorylated residues, in a similar fashion to vaccinia H1L. A myxoma I1L variant, in which the active site cysteine 110 was mutated to serine, was expressed in a parallel fashion to the wild-type I1L protein and found to be completely deficient in its ability to dephosphorylate both phosphotyrosine and phosphoserine amino acids. In an attempt to ascertain the biological requirement for the myxoma I1L phosphatase, we constructed a recombinant myxoma virus containing a disrupted I1L open reading frame. This I1L mutant virus was able to successfully propagate in tissue culture only in the presence of a wild-type complementing gene, and pure virus clones containing only the disrupted allele were not viable. Thus, we conclude that the myxoma I1L dual specificity phosphatase is an essential factor for virus viability.

Identification of a poxvirus gene encoding a uracil DNA glycosylase.

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.

Identification and DNA sequence of the large subunit of the capping enzyme from Shope fibroma virus.

A 3.6-kb region of the Shope fibroma virus (SFV) BamHI D fragment located in the central region of the viral genome was sequenced. Three open reading frames (ORFs) were identified, D3R, D4L, and D5R. Each of these ORFs have a counterpart organized identically within the HindIII fragment D of the vaccinia virus genome (D1R, D2L, and D3R). Homology scores and assays of viral cores indicate that SFV D3R encodes the large subunit of the SFV mRNA capping enzyme.

Myxoma virus and malignant rabbit fibroma virus encode a serpin-like protein important for virus virulence.

The leporipoxviruses Shope fibroma virus (SFV), the myxoma virus (MYX), and the SFV/MYX recombinant malignant rabbit fibroma virus (MRV) are closely related yet induce profoundly different diseases in the European rabbit. SFV, which produces a benign tumor at the site of inoculation, is cleared by the immune system after approximately 2 weeks whereas MYX and MRV induce a rapidly lethal systemic infection characterized by generalized suppression of host immune functions. DNA sequencing studies reveal that MRV and MYX possess homologous gene members of the T6/T8/T9 family originally described in the terminal inverted repeat (TIR) of SFV. We also describe a gene present in both MYX and MRV genomes, but which has apparently evolved in the SFV genome into a fragmented pseudogene that appears to contribute to the aggressive nature of MYX and MRV infections. Translation of this open reading frame, designated MYXOMA SERPIN 1 (SERP1), reveals a protein sequence with highly significant homology to the super-family of serine protease inhibitors (serpins) which also includes a number of other poxviral proteins. In the MYX genome the SERP1 gene lies entirely within the TIR sequences and is thus present as two copies, while in the MRV genome SERP1 is present in the unique sequences adjacent to the TIR boundary and hence is a single copy. The amino acid homology between the putative active site of SERP1 and those of other serpins predicts that the target enzyme will be different from the known catalog of serine antiprotease substrates. Deletion of this gene from MRV significantly attenuates the disease spectrum induced by the normally lethal virus. Although the MRV-S1 deletion construct (MRV with SERP1 gene deleted) grows in all tissue culture cells tested in a fashion identical to the MRV parent, the majority of rabbits infected with MRV-S1 are able to mount an effective immune response and totally recover from the virus infection to become resistant to subsequent challenge by MRV or MYX.

Identification and DNA sequence of the Shope fibroma virus DNA topoisomerase gene.

The Shope fibroma virus (SFV) DNA topoisomerase gene has been identified and mapped to the BamHI D fragment near the midpoint of the genome. The DNA sequence of the SFV BamHI S fragment together with the contiguous BamHI-ClaI subfragment of BamHI D which encompasses the topoisomerase gene and two flanking genes has been determined and analyzed. Both the SFV DNA topoisomerase and the two flanking genes are closely related in terms of sequence and spatial organization to the homologous sequences from the midpoint of the vaccinia virus genome, indicating that these proteins are conserved not only in their sequence but also by position within the poxvirus genome. To confirm the assignment of the SFV gene, the putative SFV DNA topoisomerase has been expressed as an active fusion protein in Escherichia coli and this system should be useful in the analysis of topoisomerase function following the introduction of targeted mutations into the topoisomerase gene. The results of this work shed further light on the evolutionary relationship of the different poxvirus genera and indicate that central unique regions of the poxvirus genomes contain many of the essential viral genes and are thus highly conserved.

Shope fibroma virus. II. Role of the virion-associated nucleases.

The effect of Shope fibroma virus (SFV) infection on host DNA synthesis was investigated. The cytocidal strain, SFV-I, inhibited the incorporation of [3H]thymidine into nuclear DNA very shortly (2 h) after infection, whereas the noncytocidal strain, SFV-W, did so later (10 h postinfection) and to a lesser extent. Furthermore, a two- to threefold stimulation of host DNA synthesis was recorded in SFV-W-infected cells 3 to 4 h after infection. Since virion-associated nucleases have been implicated in the shutoff of host synthesis, these and other enzymatic activities were measured in purified virion preparations. The SFV strains and vaccinia virus contained equivalent amounts of DNA-dependent RNA polymerase, ATPase, and protein kinase activities. However, in SFV-W the pH 4.5 exonuclease activity was lower than in SFV-I and vaccinia virus, and the level of pH 7.8 endonuclease was almost undetectable. To test whether the lack of endonucleolytic activity had some effect on the removal of the cross-links in the parental DNA that occurs after viral penetration, the fate of the virion SFV DNA was followed. The majority (80%) of the SFV-I and SFV-W DNA molecules extracted after viral adsorption sedimented in alkaline sucrose gradients as cross-linked. After 3 h of infection, 75% of the SFV-I DNA molecules lacked cross-links, whereas 78% of the SFV-W DNA still remained cross-linked. The same results were obtained when the presence of cross-links was tested in restriction fragments. Taken together, these results indicate that virion-associated nucleases are involved in the early shutoff of host DNA synthesis and in the elimination of cross-links from the parental viral DNA.