Although smallpox has been eradicated there are animal poxviruses which are closely related. It is desirable to measure the closeness of this relation to assess whether Variola virus could re-emerge as a complex mutant of an animal poxvirus. The most likely candidate is Monkeypox, which can produce human infection clinically resembling smallpox. The work in this thesis is the beginning of a detailed comparison of the DNA of Variola and Monkeypox. A 15.3kb section of the Variola genome was compared with a corresponding 14.4kb region of Monkeypox. This enabled both a comparison of corresponding sequences and the location of a short sequence present only in variola. Initially restriction enzyme mapping of the two stretches of DNA showed considerable homology and narrowed down the area containing any nonhomologous Variola sequences to within 2.9kb. Sequence comparisons show a level of 96% similarity. When the 2.9kb Variola fragment was compared with the corresponding 2.4kb Monkeypox fragment, a 400bp insert was found in Variola flanked by sequences common to both viruses. Analysis of the insert revealed two overlapping open reading frames present on opposite DNA strands. The DNA and putative polypeptide sequences were compared with known sequences, but no significant homology was detected. The presence or absence of this sequence in other orthopoxviruses is being established, but the expression of these open reading frames in vivo and function of the putative polypeptides is still to be investigated.

Characterization of viruses within the Orthopox genus has, in the past, been done by evaluating a spectrum of biological properties which gave different reactions in the different species. More recently analysis of genomic DNA by restriction endonucleases has proved a valuable additional way of identifying species and revealing intra-species variations among the orthopox viruses. This thesis reports the results of investigations of poxvirus isolates in which both biological characterization and DNA analysis have been used. Specimens of scabs from sick buffaloes in 18 outbreaks of buffalopox in India were extracted and virus was isolated from 13 of them by inoculation on chick embryo chorioallantoic membranes. The lesions resembled those of vaccinia and the isolates were confirmed as orthopox viruses by their susceptibility to neutralization by anti-vaccinial serum. Purified preparations of each isolate were made and the virion DNA extracted from them, was analysed by digestion with endonuclease Hind III, followed by electrophoresis to separate the resulting DNA fragments. The profiles of fragment sizes in the Hind III digest were indistinguishable from those of vaccinia isolates, but differed from other representative orthopoxviruses. This was the first examination of any DNA from the buffalopox virus. Six isolates were characterised in more detail. In biological tests, one of the six showed the ceiling temperature, rabbit virulence, cytopathic effect in cell monolayers and sensitivity to anti-vaccinial serum which is characteristic of vaccinia, and is regarded as an isolate of vaccinia. The other five had a lower ceiling temperature, were avirulent when inoculated in rabbits and were 10 - 50 fold less sensitive than vaccinia to neutralization with anti-vaccinia serum. In these properties they differed from vaccinia and resembled the buffalopox virus described by Baxby and Hill in 1971. Examination of virion DNA, using other restriction endonucleases showed that all five isolates could be grouped together and distinguished from a group of five vaccinia strains in digests with the endonuclease Sac I, Xho I and Eco RI. The sixth isolate which was biologically like vaccinia was also in the vaccinia group in these DNA analyses. Digestion of DNA obtained from all 12 of the non-vaccinia buffalo isolates showed the same profile and this profile differed from that of any of the vaccinia strains and also from the buffalopox isolate made 15 years previously in North India and described by Baxby and Hill. In another series of experiments the viral DNA from a number of isolates of monkeypox was analysed. One of these isolates was the first to be made from a wild caught animal (a squirrel) and the question was whether this isolate could be identified with isolates made from humans who had become infected in the same area of Zaire. All the isolates had previously been characterized biologically as being monkeypox virus. DNA was prepared from the squirrel isolate and from two human isolates from about the same time and district. No difference could be detected in the profiles of fragment sizes generated by digestion with the endonucleases Hind III or Pst I. To substantiate this apparent identity, DNA was prepared from another 9 monkeypox isolates from human infections with monkeypox in Liberia, Sierra Leone, Nigeria and Zaire as well as from five outbreaks in captive monkeys in Europe or America. The isolates covered the period from 1958 to 1986. The fragment size profiles established by electrophoresis of Pst I digests of all these DNA preparations were compared and found to fall into one of three distinct patterns. All the animal isolates from outside Africa had the same pattern as the isolates from Liberia and Sierra Leone. A second pattern was given by the two isolates from Nigeria and all the seven isolates from Zaire showed a third pattern. These observations supported the idea that the squirrel had been infected with the same variant of monkeypox virus that was causing the sporadic human infections in that country. The significance of the difference between the three patterns was investigated by constructing maps showing the location of Pst I cleavage sites on the genome of one isolate from each of the three main areas. A map had been published for Hind III cleavage sites on the genome of the Denmark strain of monkeypox virus. DNA from the Denmark strain was digested with Hind III, separated by electrophoresis in agarose gels and the fragments transferred by blotting onto nylon membranes. Fragments of DNA excised from gels after electrophoresis of Pst I digests were labelled by nick translation with 32 P-nucleotide and hybridized to the Hind III blots in order to determine the part of the genome from which each fragment came. By repetitions of this technique it was possible to construct Pst I maps of the genomes of representative strains of each of the three geographical variants of the virus. The sites which were different in the various isolates were found to be located in the centre and towards the left-hand end of the genome maps.

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Poxviruses are an extensive family of large DNA viruses, including the human pathogen variola virus, which causes smallpox. Vertebrate poxviruses encode numerous proteins of unknown function that contain an ankyrin repeat (ANK) domain. ANK domains have been shown to mediate protein-protein interactions and are present in a large number of cellular proteins involved in pathways such as transcription, cell cycle regulation and development. Recently, an F-box-like motif, which conserved key residues of the cellular F-box motif, was identified at the C-terminus of these proteins. Most cellular F-box proteins are specificity factors of SCF1 ubiquitin ligases, recruiting substrate proteins to the SCF1 complex where they are poly-ubiquitinated. F-box proteins use their N-terminal F-box to bind the SCF1 complex, and a second interaction motif, such as a leucine-rich-repeat domain, but never an ANK domain, to recruit the substrate. Poxviral ANK/F-box-like proteins exhibit a reversed domain order compared to cellular F-box proteins, and a novel domain combination of ANK domain and putative F-box. The aim of this study was to examine the functionality of the poxviral F-box-like motif and to assess whether poxviral ANK/F-box-like proteins could function as specificity factors of cellular ubiquitin ligases. Using immunoprecipitation of transiently expressed recombinant proteins, several poxvirus F-box-like motifs that represented the sequence variation and differences in motif length observed among poxvirus ANK/F-box-like proteins, were tested and found to be functional F-box domains. The poxviral ANK/F-box proteins interacted with all three subunits of the SCF1 complex, Skp1 (directly), Cul1 and Rbx1 (indirectly) regardless of the manner of SCF1 expression (transient over-expression or endogenous). A representative poxviral F-box was found to be both necessary and sufficient for binding SCF1, while the ANK domain of the same protein was expendable. SCF1 ubiquitin ligases bound by a poxviral ANK/F-box protein remained competent in forming poly-ubiquitin chains, indicating that these poxviral proteins are not inhibitors of SCF1. This evidence strongly suggests that poxvirus ANK/F-box proteins function as specificity factors of SCF1 ubiquitin ligases. Individual chordopoxviruses each encode several discrete ANK/F-box proteins. The five ANK/F-box proteins of one poxvirus, orf virus, were competent in binding SCF1, and when transiently expressed, were found to exhibit different sub-cellular localization patterns that were consistent in two cell lines and in the presence and absence of orf virus infection. Furthermore, the ANK domains of the five orf virus ANK/F-box proteins were distinct in primary amino acid sequence and in predicted tertiary structure. Together theses findings indicate that the five orf virus ANK/F-box proteins interact with different binding partners. In summary, this study identifies a novel class of F-box proteins encoded by chordopoxviruses that exhibits a reversed domain order and a novel domain combination of ANK domain with a truncated, but functional, F-box motif. Bioinformatic analysis, structure modeling, and microscopy findings suggest that chordopoxvirus ANK/F-box proteins function as specificity factors of SCF1 ubiquitin ligases. Several viruses use specificity factors to target cellular anti-viral factors to the ubiquitination system, a virulence mechanism that now seems likely to also exist in poxviruses.

Shope fibroma virus (SFV) N1R is a member of a family of poxvirus proteins that is associated with virulence and largely defined by the presence of a C-terminal RING finger motif and localization to virus factories within the cytoplasm of infected cells. Altered proteins, with deletions and site-specific mutations, were transiently expressed in vaccinia virus infected cells to discern regions of the protein that are required for localization. Deletion mutagenesis implicated a requirement of a small central region of the RING for localization, but the RING motif alone was not sufficient. A chimeric protein, however, in which the RING motif of the herpes simplex virus-1 ICP0 protein replaced the SFV N1R RING motif did localize to virus factories, indicating that the specificity for factory localization resided outside the RING motif of N1R. Critical evaluation of an alignment of poxviral N1R homologs identified a short, highly conserved N-terminal sequence 24-YINIT-28. When this sequence was deleted from N1R localization was abolished. Recombinant N1R protein isolated from vaccinia virus (VV) infected cells bound to calf-thymus DNA cellulose. Elution from this matrix required 0.5–0.75M NaCl, suggesting N1R localizes to the factory through an inherent DNA binding activity. Structural prediction analysis inferred that the conserved N-terminal region required for N1Rs factory localization forms a short β strand and subsequent alignment analysis with β sheet DNA binding proteins uncovered significant homology with the ribbon-helix-helix motif family which utilize a short β sheet for specific DNA interaction. Characterization of the factory localization of five N1R mutants, each having a single potential β strand residue replaced with Ala revealed that Asn 26 was the most important residue for factory localization. In contrast to N1R, which strongly binds DNA and rapidly sediments with the virus factories, SFV-N1RAsn26ΔAla mutant protein was found in the soluble fraction of infected cell lysates and failed to bind DNA cellulose. These results indicate that the N1R RING finger motif may not be central to DNA interactions and that N1R β strand residues particularly Asn 26 are involved in DNA binding and targeting N1R to the virus factories. Overexpression of N1R in vaccinia virus (VV) infected cells was found to inhibit virus induced apoptosis. To clarify the role of N1R protein with respect to apoptosis and to examine whether the related ectromelia virus virulence factor p28 (EVp28) might also play a role in apoptosis protection, a p28-mutant EV virus and the VV-N1R virus were tested for their ability to interfere with apoptosis induced by different signals. VV and EV infection were found to protect cells from Ultra Violet (UV) light, Tumor necrosis factor alpha (TNFα) and anti-Fas induced apoptosis. Expression of SFV N1R and EVp28 however, only protected infected HeLa cells from apoptosis induced by UV light, and did not protect from apoptosis induced by TNFα or anti-Fas antibody. Immunoblot analysis indicated EVp28 blocks processing of procaspase-3 suggesting EVp28 acts upstream of this protease in response to UV induced apoptotic signals. The requirement of EVp28 to promote replication and virulence in vivo may be related to apoptosis suppression because the number of progeny virus harvested from p28-mutant EV virus infected cells compared to wild type EV was similar following mock UV induced apoptosis, but significantly reduced following apoptosis induction by UV. / Graduate

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