RESUMO
DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.
Assuntos
Vírus do Fibroma dos Coelhos/genética , Genes Virais , Plasmídeos , Poxviridae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cicloeximida/farmacologia , Genes , Hibridização de Ácido Nucleico , Coelhos , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
Degenerate oligonucleotide probes corresponding to a highly conserved region common to epidermal growth factor, transforming growth factor alpha, and vaccinia growth factor were used to identify a novel growth factor gene in the Shope fibroma virus genome. Sequence analysis indicates that the Shope fibroma growth factor is a distinct new member of this family of growth factors.
Assuntos
Fator de Crescimento Epidérmico/genética , Vírus do Fibroma dos Coelhos/genética , Genes Virais , Genes , Substâncias de Crescimento/genética , Peptídeos/genética , Poxviridae/genética , Fator de Crescimento Transformador alfa , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Homologia de Sequência do Ácido Nucleico , Fatores de Crescimento TransformadoresRESUMO
Malignant rabbit fibroma virus (MV) causes a syndrome that consists of disseminated malignant tumors and immunosuppression complicated by severe Pasteurella multocida infection and death. Tissues from rabbits given MV and rabbit myxoma virus were examined by direct immunofluorescence with the use of antibody against virus antigens. Primary and metastatic tumors caused by MV and rabbit myxoma virus were composed of soft tissue cells containing virus antigens. Skin appendages and epidermis overlying the respective tumors showed scant MV but abundant myxoma virus antigen. Both viruses were present systemically in the reticuloendothelial system. Epithelial cells from the liver, kidney, and lung of myxoma virus-infected rabbits contained virus, whereas in MV tumor-bearing rabbits, these cells were uninvolved. However, nasal mucosal and conjunctival epithelia, the locations of Pasteurella infection, showed squamous metaplasia and contained large amounts of MV and myxoma antigens. By analogy to other respiratory tract pathogens, these epithelial changes were probably etiologically significant for development of pasteurellosis in rabbits bearing virus-induced tumors. Thus by immunopathologic as well as clinical examination, MV produces a syndrome distinct from that seen with rabbit myxoma virus. MV induced severe immunosuppression despite T-lymphocyte hyperplasia in the lymphoid tissues observed. The combination of a systemic virus infection, epithelial alterations that impaired clearance mechanisms, and immunologic dysfunction is likely to contribute to the inability of rabbits given MV to survive their gram-negative infection.
Assuntos
Antígenos Virais/análise , Vírus do Fibroma dos Coelhos/imunologia , Fibroma/etiologia , Myxoma virus/imunologia , Poxviridae/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Antígenos de Neoplasias/análise , Neoplasias da Túnica Conjuntiva/secundário , Feminino , Fibroma/imunologia , Vírus do Fibroma dos Coelhos/genética , Imunofluorescência , Membro Posterior , Histocitoquímica , Metástase Linfática , Sistema Fagocitário Mononuclear/imunologia , Mucosa Nasal/imunologia , Neoplasias Nasais/secundário , CoelhosRESUMO
The clinical, histopathologic, and cultural characteristics of a newly isolated poxvirus, malignant rabbit fibroma virus (MV), were investigated. MV was isolated from tumors induced by an uncloned stock of Shope fibroma virus (SFV). MV, SFV, and rabbit myxoma virus were compared. Similarly to myxoma virus, MV grew to higher titer in vitro than did SFV and produced plaques rather than foci on rabbit kidney cell monolayers. Unlike the local, self-limited fibroblastic proliferations observed in SFV recipients, MV and myxoma caused a fulminant clinical syndrome characterized by malignant histology, metastases, and supervening fatal gram-negative infection with Pasteurella multocida. MV induced a large, protuberant local tumor and discrete metastases histologically resembling myxosarcomas. Draining lymph nodes contained metastases and showed diffuse cortical hyperplasia. Kupffer's cells were prominent in the liver, and macrophages were abundant in the splenic sinusoids. The lungs and trachea were spared, but the conjunctiva and nasal mucosa showed squamous metaplasia and atypia, with overlying Pasteurella infection and underlying tumor. Myxoma virus infection produced similar mucosal changes, but both of these as well as the epidermis overlying the myxomas showed cytoplasmic virus inclusions. Neither the skin nor the epithelial surfaces overlying MV-induced tumors nor the tumors themselves contained virus inclusions. Thus the tumor syndrome caused by MV differed from other known rabbit tumors. Endonuclease restriction digests showed that the MV genome resembled, but was distinct from, rabbit myxoma virus. Opportunistic infection associated with MV-induced disseminated tumor may be an experimental model for the infectious complications that often supervene in host-tumor relationships.
Assuntos
Vírus do Fibroma dos Coelhos/isolamento & purificação , Fibroma/etiologia , Poxviridae/isolamento & purificação , Infecções Tumorais por Vírus , Animais , Neoplasias da Túnica Conjuntiva/secundário , Enzimas de Restrição do DNA , DNA Viral/isolamento & purificação , Eletroforese em Gel de Ágar , Feminino , Fibroma/patologia , Vírus do Fibroma dos Coelhos/genética , Vírus do Fibroma dos Coelhos/ultraestrutura , Membro Posterior , Myxoma virus/genética , Myxoma virus/ultraestrutura , Mucosa Nasal/patologia , Neoplasias Nasais/secundário , CoelhosRESUMO
The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 (recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation (delta Gf = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high delta Gf value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured delta Gf value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.
Assuntos
DNA Viral , Vírus do Fibroma dos Coelhos/genética , Conformação de Ácido Nucleico , Plasmídeos , Poxviridae/genética , Sequência de Bases , Clonagem Molecular , Replicação do DNA , Eletroforese em Gel de Ágar , Dados de Sequência Molecular , Replicação ViralRESUMO
Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells and recombine at high frequencies. Calcium phosphate precipitates were used to cotransfect Shope fibroma virus-infected cells with different DNA substrates and the recombinant products assayed by genetic and biochemical methods. We have shown previously that bacteriophage lambda DNAs can be used as substrates in these experiments and recombinants assayed on Escherichia coli following DNA recovery and in vitro packaging. Using this assay it was observed that 2-3% of the phage recovered from crosses between point mutants retained heteroduplex at at least one of the mutant sites. The reliability of this genetic analysis was confirmed using DNA substrates that permitted the direct detection of heteroduplex molecules by denaturant gel electrophoresis and Southern blotting. It was further noted that heteroduplex formation coincided with the onset of both replication and recombination suggesting that poxviruses, like certain bacteriophage, make no clear biochemical distinction between these three processes. The fraction of heteroduplex molecules peaked about 12-hr postinfection then declined later in the infection. This decline was probably due to DNA replication rather than mismatch repair because, while high levels of induced DNA polymerase persisted beyond the time of maximal heteroduplex recovery, we were unable to detect any type of mismatch repair activity in cytoplasmic extracts. These results suggest that, although heteroduplex molecules are formed during the progress of poxviral infection, gene conversion through mismatch repair probably does not produce most of the recombinants. The significance of these observations are discussed considering some of the unique properties of poxviral biology.
Assuntos
Replicação do DNA , Vírus do Fibroma dos Coelhos/genética , Ácidos Nucleicos Heteroduplexes/biossíntese , Recombinação Genética , Bacteriófago lambda/genética , Southern Blotting , Células Cultivadas , Reparo do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Eletroforese , Escherichia coli/genética , Células HeLa , Humanos , Cinética , Mutação/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Ácidos Nucleicos Heteroduplexes/fisiologia , Plasmídeos/genética , TransfecçãoRESUMO
A novel member of the serpin family of serine protease inhibitors is presented. A plasmid-like DNA was isolated from rabbit cells by its homology to the genome of Shope fibroma virus (SFV), a tumorigenic poxvirus of rabbits, and was shown elsewhere to encode a serpin-like protein [(1986) Mol. Cell. Biol. 6, 265-276]. Although significant DNA homology exists between the rabbit plasmid serpin open reading frame and the SFV terminal inverted repeat DNA there is no intact serpin counterpart encoded by this region of the SFV genome. The alignment of the novel plasmid-borne polypeptide with the serpin family of proteins confirms its status within this group.
Assuntos
DNA/análise , Plasmídeos , Proteínas/genética , Animais , Sequência de Bases , Vírus do Fibroma dos Coelhos/genética , Técnicas In Vitro , Conformação Proteica , Coelhos , Homologia de Sequência do Ácido Nucleico , Inibidores de Serina ProteinaseRESUMO
We have examined the effects of transcription on recombination frequencies in poxvirus-infected cells. A synthetic poxviral promoter was shown to function as a hybrid early/late transcription element when fused to a luciferase reporter gene, and then cloned into genetically-marked recombination substrates. These lambda DNA substrates were transfected into cells infected with Shope fibroma virus (SFV) and the recombinants detected by recovering the transfected DNA, packaging it in vitro into infectious particles, and then assaying the yield of recombinants on Escherichia coli. Controls showed that the poxviral promoter conferred no replicative advantage, or disadvantage, on molecules encoding the promoter. Furthermore, the promoter had no detectable effect on the recombination frequency when recombination was measured in the interval immediately adjacent to the promoter-insertion site. However, the promoter did appear to stimulate recombination at a distance, in a manner that appeared to be dependent on the level of transcription, and the effect was observed regardless of whether or not the promoter was present on one or both of the recombinational substrates. The peak of recombinational enhancement was centered about 500 bp away from the promoter element, where the frequency of recombination was 30-50% higher than that seen when the recombinational substrates lacked the promoter. Possible explanations for these observations are discussed.
Assuntos
Vírus do Fibroma dos Coelhos/genética , Regiões Promotoras Genéticas , Recombinação Genética , Transcrição Gênica , Sequência de Bases , Técnicas de Cultura , Replicação do DNA , Escherichia coli/genética , Dados de Sequência MolecularRESUMO
Differences observed in the virulence of two related leporipoxviruses are closely tied to a particular region of their genomes. For the virulent poxvirus of this pair, malignant rabbit fibroma virus (MV), this region is the BamHI "C" fragment, which is 10.7 kb. For the avirulent poxvirus, Shope fibroma virus, SFV, this region is the corresponding BamHI "D" fragment, which is 13.1 kb. As part of our attempt to understand the virulence of these two viruses, we sequenced these two DNA fragments. The sequence for the BamHI "C" fragment of MV is reported elsewhere (Strayer et al., 1991). We report here the sequence for SFV's BamHI "D" fragment and resultant open reading frames, and compare both DNA and open reading frame structures to those of MV and other known poxviruses. The BamHI "D" fragment of SFV contains 12 open reading frames of 100 amino acids or more, arranged similarly to orf's in MV and vaccinia. Striking similarities between SFV and MV are seen in certain parts of this restriction fragment, including substantial stretches of DNA in which the two viruses are identical. Clear homologies exist between these leporipox virus genomes and those of other related poxviruses. To understand the pathogenesis of virus infection, one must appreciate the structure of those viral genes that play important roles in infection.
Assuntos
DNA Viral/genética , Vírus do Fibroma dos Coelhos/genética , Poxviridae/genética , Sequência de Bases , Desoxirribonuclease BamHI , Vírus do Fibroma dos Coelhos/patogenicidade , Dados de Sequência Molecular , Fases de Leitura Aberta , Poxviridae/patogenicidade , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Virulência/genéticaRESUMO
To investigate if Shope fibroma virus (SFV), a leporipoxvirus that induces benign tumors in adult rabbits, can trigger the second step of carcinogenesis in vitro or malignant transformation, an already immortalized rabbit cell line (SIRC) was inoculated with ultraviolet-irradiated virus. The resulting cell transformants displayed the characteristic properties of the malignant phenotype: lack of infectious particles, low serum requirement, high efficiency of cloning, resistance to superinfection, presence of viral DNA sequences in the nucleus, expression of viral proteins and induction of tumors in rabbits. However, this transformation was not stable since in all cell lines studied, a loss of the malignant phenotype was recorded close to the 50th passage. To assess the oncogenic potential of SFV, NIH 3T3 cells were transfected with SFV DNA. The results of these experiments indicate that SFV DNA can induce the formation of foci in certain NIH 3T3 cell lines. Taken together these results support the notion that SFV can elicit the transformation of cells in vitro.
Assuntos
Transformação Celular Neoplásica/etiologia , Transformação Celular Viral , Vírus do Fibroma dos Coelhos/patogenicidade , Poxviridae/patogenicidade , Animais , Linhagem Celular , Transformação Celular Viral/efeitos dos fármacos , Transformação Celular Viral/efeitos da radiação , Cocarcinogênese , DNA Viral/genética , Fibroblastos , Vírus do Fibroma dos Coelhos/genética , Vírus do Fibroma dos Coelhos/efeitos da radiação , Camundongos , Neoplasias Experimentais/etiologia , Coelhos , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Raios UltravioletaAssuntos
Transformação Celular Neoplásica , Vírus do Fibroma dos Coelhos/genética , Poxviridae/genética , Infecções Tumorais por Vírus/microbiologia , Animais , Linhagem Celular , Células Cultivadas , Córnea , DNA Viral/genética , Modelos Animais de Doenças , Fibroblastos , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Coelhos , TransfecçãoRESUMO
The properties of NIH 3T3 cells transfected with Shope fibroma virus (SFV) DNA were investigated. Six focus-derived cell lines were established that display the following properties: a) They all contained SFV DNA sequences at early passages, b) five of them also expressed SFV RNA at early passages and induced tumors in nude mice, and c) four lines were anchorage independent. Transfection with cloned DNA fragments containing terminal sequences did not induce foci, except with fragment C, which contains the growth-factor gene. However, these transfected cells failed to cause tumors, suggesting that the growth factor alone may not be responsible for tumorigenesis.
Assuntos
Transformação Celular Viral , DNA Viral/genética , Vírus do Fibroma dos Coelhos/genética , Poxviridae/genética , Transfecção , Animais , Linhagem Celular Transformada , Camundongos , Camundongos NusRESUMO
The sites for the restriction enzymes BamHI, Bg/I, HindIII, PstI, PvuII, and SstI on the linear DNA genome of Shope fibroma virus, a tumorigenic poxvirus of rabbits, have been determined by digestions of the cloned BamHI and HindIII restriction fragments and by hybridization of 32P-labeled cloned fragments to Southern blots of Shope fibroma virus DNA cleaved partially or completely with the various enzymes. The linear genome is shown to be 160 kilobases in length and to possess terminal inverted repeat sequences of between 12.2 and 12.5 kilobases extending inwards from the cross-linked DNA telomeres. The fine map of the Shope fibroma virus terminal inverted repeats has been constructed and shown to be distinctly different from that of members of the orthopoxvirus group, such as vaccinia, by the absence of detectable tandemly repeated sequences near the termini and by the lack of detectable sequence homology with vaccinia termini.
Assuntos
Clonagem Molecular , Vírus do Fibroma dos Coelhos/genética , Genes Virais , Poxviridae/genética , Animais , Sequência de Bases , Linhagem Celular , Enzimas de Restrição do DNA , Peso Molecular , Hibridização de Ácido NucleicoRESUMO
A 55-residue peptide comprising the carboxyl portion (residues 26-80) of the Shope fibroma virus growth factor (SFGF), a predicted 80-residue DNA virus gene product that encoded a homologous sequence with the epidermal growth factor transforming growth factor alpha family, was synthesized by a stepwise solid-phase method. The synthetic SFGF (26-80) purified to homogeneity by reverse-phase HPLC was characterized by fission ionization mass spectrometry and amino acid analysis. The disulfide pairings were established by enzymatic digestion and mass spectrometry and were found to be similar to those of EGF and TGF alpha. Synthetic SFGF (26-80) was found to share about 10% of the activities as EGF in the radioreceptor binding to A431 cells, stimulation of [3H]thymidine uptake in NRK cells, and induction of colony formation in soft-agar assay. Our results therefore confirmed that SFGF contained the putative biological activities of the EGF-TGF alpha family and that production of SFGF by Shope fibroma virus infected cells may account for the proliferative diseases associated with this particular virus.
Assuntos
Vírus do Fibroma dos Coelhos/genética , Substâncias de Crescimento/síntese química , Poxviridae/genética , Sequência de Aminoácidos , Bioensaio , DNA Viral/genética , Genes Virais , Substâncias de Crescimento/isolamento & purificação , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Five strains of Shope fibroma virus (SFV), a strain of rabbit myxoma virus, and a strain of vaccinia virus were compared by restriction endonuclease digestion of their viral DNAs. Restriction digest patterns revealed that SFV and rabbit myxoma, both members of the Leporipoxvirus genus, were distinct from vaccinia, an Orthopoxvirus. All strains of SFV examined had a high degree of nucleotide sequence homology as shown by conservation of restriction sites within their genomes. However, restriction patterns of SFV and myxoma were quite different from one another suggesting that the genomes from these two viruses of the Leporipoxvirus genus do not share a large, highly conserved region of homology as do the viruses belonging to the Orthopoxvirus genus. Restriction mapping identified inverted terminal repeats of approximately 12 kb in length. Restriction fragments representing all but 400 bp of the termini were cloned in plasmid vectors.
Assuntos
Clonagem Molecular , Vírus do Fibroma dos Coelhos/genética , Genes Virais , Poxviridae/genética , Animais , Enzimas de Restrição do DNA , Plasmídeos , Coelhos , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Shope fibroma virus (SFV), a tumorigenic poxvirus, has a 160-kb linear double-stranded DNA genome and possesses terminal inverted repeats (TIRs) of 12.4 kb. The DNA sequence of the terminal 5.5 kb of the viral genome is presented and together with previously published sequences completes the entire sequence of the SFV TIR. The terminal 400-bp region contains no major open reading frames (ORFs) but does possess five related imperfect palindromes. The remaining 5.1 kb of the sequence contains seven tightly clustered and tandemly oriented ORFs, four larger than 100 amino acids in length (T1, T2, T4, and T5) and three smaller ORFs (T3A, T3B, and T3C). All are transcribed toward the viral hairpin and almost all possess the consensus sequence TTTTTNT near their 3' ends which has been implicated for the transcription termination of vaccinia virus early genes. Searches of the published DNA database revealed no sequences with significant homology with this region of the SFV genome but when the protein database was searched with the translation products of ORFs T1-T5 it was found that the N-terminus of the putative T4 polypeptide is closely related to the signal sequence of the hemagglutinin precursor from influenza A virus, suggesting that the T4 polypeptide may be secreted from SFV-infected cells. Examination of other SFV ORFs shows that T1 and T2 also possess signal-like hydrophobic amino acid stretches close to their N-termini. The protein database search also revealed that the putative T2 protein has significant homology to the insulin family of polypeptides. In terms of sequence repetitions, seven tandemly repeated copies of the hexanucleotide ATTGTT and three flanking regions of dyad symmetry were detected, all in ORF T3C. A search for palindromic sequences also revealed two clusters, one in ORF T3A/B and a second in ORF T2. ORF T2 harbors five short sequence domains, each of which consists of a 6-bp short palindrome and a 10- to 18-bp larger palindrome. The significance of these palindromic domains in this ORF is unclear but the coincidence of the end of one larger palindrome with the end of the translated protein sequence that has homology with the B chain of insulin suggests that the palindromes may divide the T2 protein into several functional units. The salient organizational features of the complete SFV TIR are also discussed in light of what is known about other poxviral TIRs.
Assuntos
DNA Viral/genética , Vírus do Fibroma dos Coelhos/genética , Genes Virais , Poxviridae/genética , Sequência de Aminoácidos , Sequência de Bases , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
DNA Topoisomerases Tipo I/genética , DNA Viral/genética , Vírus do Fibroma dos Coelhos/genética , Poxviridae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Vírus do Fibroma dos Coelhos/enzimologia , Dados de Sequência Molecular , Plasmídeos , Mapeamento por RestriçãoRESUMO
Malignant rabbit fibroma virus (MRV) has been shown to be a lethal tumorigenic poxvirus of rabbits derived from a recombination event between Shope fibroma virus (SFV), which induces benign fibromas in rabbits, and myxoma virus, the agent of myxomatosis. We have cloned and sequenced all of the MRV recombination junctions, which are located near the left and right terminal inverted repeat (TIR) regions, and present a composite map of the MRV genome with respect to the relevant gene products. The two junctions closet to the MRV termini, at identical positions at the left and right ends, are at nucleotide 5272 and result in an in-frame fusion protein (ORF T-5) in which the N-terminal 232 aa are derived from an SFV sequence linked to a C-terminus derived from myxoma. At the left MRV TIR the recombination junction distal from the terminus maps to nucleotide 9946 but leaves the adjacent gene virtually unchanged from its SFV homolog. At the right terminus, the relevant junction sequences from MRV and myxoma could not be cloned in wild-type Escherichia coli but were maintained stably in a recA recBC sbcB host. The SFV/myxoma junction at this location maps 5' to a growth factor gene (SFGF) which is related to those encoding epidermal growth factor and transforming growth factor-alpha. As a result, the myxoma growth factor gene has been deleted in MRV and replaced in toto by the SFV gene. The recombination junction upstream from the SFGF gene creates an in-frame fusion in ORF T11-R in which the N-terminal amino acids are derived from myxoma and the remainder from SFV. In summary, MRV has received the following ORFs from SFV: at the left terminus T5 (fusion), T6, T7, and T8; at the right terminus, T5 (fusion), T6, T7, T8, T9-R, SFGF, and T11-R (fusion).
Assuntos
DNA Viral/genética , Vírus do Fibroma dos Coelhos/genética , Genes Virais , Myxoma virus/genética , Neoplasias Experimentais/microbiologia , Poxviridae/genética , Recombinação Genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Dados de Sequência Molecular , Neoplasias Experimentais/genética , Coelhos/microbiologiaRESUMO
The thymidine kinase (TK) gene of Shope fibroma virus (SFV), a tumorigenic leporipoxvirus, was localized within the viral genome with degenerate oligonucleotide probes. These probes were constructed to two regions of high sequence conservation between the vaccinia virus TK gene and those of several known eucaryotic cellular TK genes, including human, mouse, hamster, and chicken TK genes. The oligonucleotide probes initially localized the SFV TK gene 50 kilobases (kb) from the right terminus of the 160-kb SFV genome within the 9.5-kb BamHI-HindIII fragment E. Fine-mapping analysis indicated that the TK gene was within a 1.2-kb AvaI-HaeIII fragment, and DNA sequencing of this region revealed an open reading frame capable of encoding a polypeptide of 176 amino acids possessing considerable homology to the TK genes of the vaccinia, variola, and monkeypox orthopoxviruses and also to a variety of cellular TK genes. Homology matrix analysis and homology scores suggest that the SFV TK gene has diverged significantly from its counterpart members in the orthopoxvirus genus. Nevertheless, the presence of conserved upstream open reading frames on the 5' side of all of the poxvirus TK genes indicates a similarity of functional organization between the orthopoxviruses and leporipoxviruses. These data suggest a common ancestral origin for at least some of the unique internal regions of the leporipoxviruses and orthopoxviruses as exemplified by SFV and vaccinia virus, respectively.
Assuntos
Vírus do Fibroma dos Coelhos/genética , Poxviridae/genética , Timidina Quinase/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Enzimas de Restrição do DNA , DNA Viral/genética , Regulação da Expressão Gênica , Genes Virais , Oligodesoxirribonucleotídeos/síntese química , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
An isolate of Shope fibroma virus (SFV), designated Indiana (SFV-I), was previously described to be tumorigenic in vivo as SFV, cytocidal in vitro as the orthopoxviruses (vaccinia, rabbitpox, etc.), and to share antigenic determinants with SFV and vaccinia. The genetic relatedness of SFV-I to SFV and vaccinia was studied by means of Southern blotting and hybridization. The results indicated that SFV-I shares extensive DNA homology with vaccinia, but few common sequences with SFV. By contrast, SFV and vaccinia show no sequence homology. These findings suggest that SFV-I is an orthopoxvirus which carries some genetic information from the leporipoxviruses. Recombinants between two genera of poxviruses have not been reported before.