Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha.

Myxoma virus, a Leporipoxvirus and agent of myxomatosis, was shown to possess a gene with the potential to encode an epidermal growth factorlike factor. Its relationship to other members of this family, including the poxvirus growth factors from Shope fibroma virus and vaccinia virus, was analyzed. Alignment of DNA sequences and related open reading frames of myxoma virus and Shope fibroma virus indicated colinearity of genes between these poxviruses.     

Synthesis of a biological active tumor growth factor from the predicted DNA sequence of Shope fibroma virus

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.     

Shope fibroma virus growth factor exhibits epidermal growth factor activities in newborn mice

A synthetic 55-residue peptide consisting of the carboxyl portion of the predicted genomic DNA sequence of Shope fibroma virus growth factor (SFGF residue 26-80) was found to exhibit epidermal growth factor-transforming growth factor activities in newborn mice. The synthetic SFGF accelerated precocious incisor eruption and eyelid opening in newborn mice and also retarded the overall growth rates of hair, body weight and body length when administered in dosages of 4 to 6 micrograms per gram of body weight. The results of whole animal studies indicate that SFGF belongs to the EGF-TGF alpha family and exerts similar biologic effects in newborn animals.     

Studies on the polypeptides of poxvirus. II. Comparison of virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses.

Virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses were examined by SDS-polyacrylamide gel electrophoresis followed by autoradiography. At least 42 vaccinia virus-induced polypeptides were identified among the polypeptides of cells pulse-labeled with [35S]-methionine and/or of fractionated cells labeled with [14C]-leucine for 24 hr. They consisted of 15 polypeptides (early polypeptides) which were synthesized even in the presence of cytosine-1-beta-D-arabinofuranosyl-HCl, and 27 polypeptides (late polypeptides) which were synthesized only in the absence of cytosine-1-beta-D-arabinofuranosyl-HCl. By the same procedure at least 40 cowpox virus-induced polypeptides (14 early polypeptides and 26 late polypeptides) and at least 31 Shope fibroma virus-induced polypeptides (13 early polypeptides and 18 late polypeptides) were identified. Comparative studies of virus-induced polypeptides on the basis of migration in SDS-polyacrylamide gel electrophoresis revealed that 11 polypeptides were early polypeptides common to both vaccinia and cowpox viruses; 21 were late polypeptides common to both vaccinia and cowpox viruses; 4 were early polypeptides common to both vaccinia and Shope fibroma viruses; 7 were late polypeptides common to both vaccinia and Shope fibroma viruses; 5 were early polypeptides common to both cowpox and Shope fibroma viruses; 9 were late polypeptides common to both cowpox and Shope fibroma viruses; 4 were early polypeptides common to all three viruses; and 7 were late polypeptides common to all three viruses.     

Tumorigenic poxviruses: construction of the composite physical map of the Shope fibroma virus genome.

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.     

Studies on the polypeptides of poxvirus. I. Comparison of structural polypeptides in vaccina, cowpox and Shope fibroma viruses.

Radioactively labeled vaccinia, cowpox and Shope fibroma virions free from any detectable contamination with host cell protein, were dissociated into their constituent polypeptides, and these were then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The profiles of constituent polypeptide bands of four strains of vaccinia virus (IHD-W, IHD-J, Lister and DIs) were almost the same, except that a polypeptide of about 41,000 daltons was not detectable in the autoradiogram of strain IHD-W which has no hemagglutinin. The profile of polypeptide bands of cowpox virions was also almost the same as that of vaccinia virions, except for several polypeptides of about 40,000 to 50,000 daltons, but the profile of Shope fibroma virions differed considerably from that of vaccinia or cowpox virions.     

Viral and Host Deoxyribonucleic Acid Synthesis in Shope Fibroma Virus-infected Cells as Studied by Means of High-Resolution Autoradiography

Incorporation of (3)H-thymidine by BSC-1 cells infected with Shope fibroma virus was studied by means of high-resolution electron microscopic radioautography. One-hour pulses with the radioactive precursor were given at various times after infection, during a one-step growth cycle of the virus. In the cytoplasm of infected cells, reacted grains occurred over foci of viroplasm; these foci are believed to represent the true sites of viral deoxyribonucleic acid (DNA) replication. Shope fibroma virus DNA synthesis began before 3 hr postinfection, reached a maximum at 8 to 9 hr, and then declined rapidly. It was demonstrated that the decline in (3)H-thymidine uptake is correlated with the onset of viral morphogenesis. In comparison with the noninfected culture, the nuclear labeling, which reflects host DNA metabolism, was slightly reduced by 4 hr postinfection. Inhibition became more marked as infection progressed, and host DNA synthesis was almost completely suppressed in late stages of viral development.     

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.     

Mechanism of pock formation by Shope fibroma virus on monolayers of rabbit cells

Israeli, Ella (Rambam Hospital, Haifa, Israel). Mechanism of pock formation by Shope fibroma virus on monolayers of rabbit cells. J. Bacteriol. 92:727-732. 1966.-The mechanism of pock formation by the Shope fibroma virus (SFV) on rabbit cultures in vitro was studied with the use of p-fluorophenylalanine, 5-bromodeoxyuridine, and 5-iododeoxyuridine. The inhibitors were used to inhibit, and to initiate, virus replication at different times after infection. It was shown that pock formation required virus replication to a threshold value of 25 plaque-forming units per pock area, and that this amount of virus can be accumulated during a period about 3 days less than that required for pock formation. Inhibition of virus growth, and of cell multiplication, after this threshold has been reached, did not prevent pock development. A delay in the onset of virus growth required to reach the threshold virus content, caused an about equivalent delay in the time of pock formation. In the absence of inhibitors, pocks were not formed after infection of 84 rabbit embryo clones, or five mixtures of clones containing five to seven clones each. The results indicate that pock formation by SFV in vitro was the result of cell aggregation, and not of cell multiplication, in special types of cells.     

Comparison of Cytocidal and Noncytocidal Strains of Shope Rabbit Fibroma Virus

Seven strains of Shope fibroma virus were compared for their effect on rabbit cells in vitro. All but one of the naturally occurring strains examined in this study produced a similar response in the infected cultures. This consisted of continued cell multiplication together with changes in cell morphology and growth pattern. In contrast, a recently isolated strain of fibroma virus, the M1 strain, was found to produce a gradual cell destruction under the same cultural conditions. A comparison of the cytocidal M1 strain with a representative noncytocidal strain in vitro showed no differences in the rate of multiplication, plaque type, antigenic composition, or heat lability. Only minor differences were found in the tumors produced in rabbits by these strains.     

Pathogenic poxviruses reveal viral strategies to exploit the ErbB signaling network.

Virulence of poxviruses, the causative agents of smallpox, depends on virus-encoded growth factors related to the mammalian epidermal growth factor (EGF). Here we report that the growth factors of Shope fibroma virus, Myxoma virus and vaccinia virus (SFGF, MGF and VGF) display unique patterns of specificity to ErbB receptor tyrosine kinases; whereas SFGF is a broad-specificity ligand, VGF binds primarily to ErbB-1 homodimers, and the exclusive receptor for MGF is a heterodimer comprised of ErbB-2 and ErbB-3. In spite of 10- to 1000-fold lower binding affinity to their respective receptors, the viral ligands are mitogenically equivalent or even more potent than their mammalian counterparts. This remarkable enhancement of cell growth is due to attenuation of receptor degradation and ubiquitination, which leads to sustained signal transduction. Our results imply that signal potentiation and precise targeting to specific receptor combinations contribute to cell transformation at sites of poxvirus infection, and they underscore the importance of the often ignored low-affinity ligand-receptor interactions.     

Tumorigenic poxviruses up-regulate intracellular superoxide to inhibit apoptosis and promote cell proliferation

Tumorigenic leporipoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD1). The function of the orthologous myxoma virus M131R and Shope fibroma virus S131R gene products is uncertain, but they inhibit SOD1 activity by a process linked to binding its copper chaperone. Using a superoxide-sensitive dye (hydroethidine), we observed that virus infection increased intracellular superoxide levels in an M/S131R-dependent manner. To see whether this effect promotes infection, we deleted the Shope fibroma virus S131R gene and compared the clinical manifestations of wild-type and mutant virus infections in rabbits. S131RDelta virus produced significantly smaller fibroxanthosarcoma-like growths in vivo and, at a point where these growths were already receding, wild-type infections still showed extensive leukocyte infiltration, necrosis, and fibromatous cell proliferation. Coincidentally, whereas Jurkat cells are protected from mitochondria- and Fas-mediated apoptosis by wild-type myxoma virus in vitro, M131RDelta virus could not block Fas-initiated apoptosis as judged by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling, and caspase 3 cleavage assays. These data suggest that tumorigenic poxviruses can modulate intracellular redox status to their advantage to stimulate infected cell growth and inhibit programmed cell death.     

Effect of marker distance and orientation on recombinant formation in poxvirus-infected cells

Little is known about the mechanism of poxvirus recombination even though construction of recombinant viruses by recombination-dependent methods is a widely adopted technique. We have shown previously that transfected DNAs are efficiently recombined while replicating in cells infected with Shope fibroma virus. Because recombinant DNA can be recovered from infected cells as a high-molecular-weight head-to-tail concatemer, it was possible to transfect genetically marked lambda DNAs into infected cells and assay recombinants as bacteriophage particles following in vitro packaging. This approach was used in this study to examine how marker distance and marker orientation influence recombination in Shope fibroma virus-infected cells. Simple two-factor crosses were readily modelled by using a mapping function derived from classical phage studies and showed low negative interference (I = -2.8 +/- 0.5) in crosses involving markers greater than 100 bp apart. More complex four- and five-factor crosses showed that the recombination frequency per unit distance was not constant (rising as the marker separation was reduced from 100 to 1 bp) and that crosses performed in poxvirus-infected cells are subject to high negative interference. One consequence is that marker orientation does not dramatically influence the outcome of most Shope fibroma virus-catalyzed crosses in clear contrast to what is observed in adenovirus or simian virus 40-infected cells. These results can be interpreted to indicate that similar statistical and physical constraints influence both viral and phage recombination and suggest that heteroduplexes may be important intermediates in the poxvirus recombination process.     

Effect of Persistent Fibroma Virus Infection on Susceptibility of Cells to Other Viruses

Shope fibroma virus establishes a persistent cytoplasmic infection in primary (RK) and serially cultivated (DRK(3)) rabbit kidney cells which is accompanied by a morphological alteration of the cells. The response of such cells to superinfection by other viruses was compared with that of control cells by determining plaque production and virus yield of superinfecting viruses. It was found that the growth of other poxviruses, myxoma and vaccinia, was greatly inhibited in the fibroma virus-infected cells, but that of pseudorabies and herpes simplex viruses, which are unrelated deoxyribonucleic acid viruses, was virtually unaffected. The ribonucleic acid (RNA) viruses, poliovirus 1 and coxsackievirus B1, did not produce plaques on either RK or fibroma virus-infected (F-RK) monolayers. However, the growth of several other RNA viruses, vesicular stomatitis virus, encephalomyocarditis virus, Sindbis virus, and Newcastle disease virus, was enhanced in F-RK cells. None of these latter RNA viruses produced any infectious progeny in DRK(3) cells, but they all plaqued on and produced good yields in DRK(3) cells persistently infected with fibroma virus. This phenomenon is termed facilitation. Facilitation results from the infection of DRK(3) cells by fibroma virus. Neither interference nor facilitation were due to changes in the adsorption or eclipse of the superinfecting virus.     

Deletion analysis of two tandemly arranged virulence genes in myxoma virus, M11L and myxoma growth factor.

Myxoma virus (MYX) is a leporipoxvirus of rabbits that induces a lethal syndrome characterized by disseminated tumorlike lesions, generalized immunosuppression, and secondary gram-negative bacterial infection. A MYX deletion mutant (vMYX-GF- delta M11L) was constructed to remove the entire myxoma growth factor (MGF) coding sequence and that for the C-terminal five amino acids of the partially overlapping upstream gene, M11L. Unexpectedly, this deletion completely abrogates the capacity of MYX to cause the characteristic disease symptoms of myxomatosis. Upon inoculation of rabbits with vMYX-GF- delta M11L, recipient animals developed only a benign, localized nodule reminiscent of a Shope fibroma virus-induced tumor in which a single primary lesion appeared at the site of injection and then completely regressed within 14 days, leaving the animals resistant to challenge with wild-type MYX. No evidence of the purulent conjunctivitis and rhinitis that always accompany wild-type MYX infection was observed. To ascertain whether the attenuation observed in vMYX-GF- delta M11L was due to a combined effect of the MGF deletion and alteration of the upstream M11L gene, two additional MYX recombinants were constructed: an MGF- virus (vMYX-GF-) containing an intact M11L gene and an M11L- virus (vMYX-M11L-) containing an intact MGF gene. Infection with vMYX-GF- resulted in moderated symptoms of myxomatosis, but all clinical stages of the disease were still detectable. In contrast, disruption of M11L alone dramatically reduced the virus virulence, resulting in a nonlethal syndrome whose clinical course was nevertheless distinct from that of vMYX-GF- delta M11L. Upon inoculation with vMYX-M11L-, rabbits developed primary and secondary tumors which were larger and more circumscribed than those of wild-type MYX recipients. Whereas wild-type MYX infection always includes severe, purulent conjunctivitis and rhinitis, vMYX-M11L- recipients remained healthy and displayed only minimal signs of respiratory distress. By about 30 days after infection, the tumors induced by vMYX-M11L- had completely regressed and these animals were immune to challenge with wild-type MYX. Histological analysis indicated that tumors induced by vMYX-M11L- are much more heavily infiltrated with macrophages and heterophils and that the sites of viral replication are more edematous and necrotic than those of wild-type infection, suggesting that the host was able to mount a more vigorous inflammatory response to vMYX-M11L- infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells

In attempts to determine the mechanism of proliferation of arterial smooth muscle cells (SMC) in intimal atheromatous lesions, autocrine secretion of growth factors by SMC has recently received much attention. Here we report a new growth factor named smooth muscle cell derived growth factor (SDGF). Cultured rabbit medial SMC secreted SDGF for 1 week during their incubation in serum-free media only after at least 4 passages. SDGF differed from platelet derived growth factor (PDGF) physicochemically, immunologically, and biologically. The properties of SDGF also seemed different from those of other known growth factors that stimulate the proliferation of mesenchymal cells.     

Nerve growth factor, a differentiating agent, and epidermal growth factor, a mitogen, increase the activities of different S6 kinases in PC12 cells

Treatment of PC12 cells with either nerve growth factor, a differentiating agent, or epidermal growth factor, a mitogen, leads to an increase in the phosphorylation of the ribosomal protein S6. The soluble fraction of PC12 cells contains two S6 kinases, separable on heparin-Sepharose. Treatment of the cells with nerve growth factor results in an increase in the activity of one of these kinases; treatment of the cells with epidermal growth factor results in an increase in the activity of the other one. The data suggest that the patterns of phosphorylation and, in turn, the functional properties of S6 are different in cells instructed to differentiate from those in cells instructed to divide.     

A novel member of the serpin superfamily is encoded on a circular plasmid-like DNA species isolated from rabbit cells

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.     

High levels of genetic recombination among cotransfected plasmid DNAs in poxvirus-infected mammalian cells.

The frequency of recombination between transfected plasmid DNAs was measured by using cultured cells infected with a variety of poxviruses. Plasmid derivatives of pBR322 containing XhoI linker insertion mutations in the tetracycline gene were used to assess recombination frequencies in rabbit cells infected with the leporipoxviruses Shope fibroma virus and myxoma virus and the orthopoxvirus vaccinia virus. Recombination frequencies were calculated by Southern blotting, which detects novel plasmid restriction fragments generated by genetic recombination, and by a plasmid rescue procedure in which the reconstruction of an intact tetracycline gene in the transfected rabbit cell was monitored by transformation back into Escherichia coli. The highest recombination frequencies were measured in cells infected with Shope fibroma virus and myxoma virus, and a minimum recombination frequency of at least one recombination event per 7 kilobases was calculated within 24 h posttransfection under these conditions. The deduced recombination frequency in vaccinia virus-infected cells was at least fivefold lower and was not detectable in mock-infected cells, suggesting that the induced recombination activity detected by these methods was under viral control. The results of kinetic studies, analysis with methylation-sensitive restriction enzymes, and the use of phosphonoacetic acid, a specific inhibitor of poxvirus DNA polymerase, indicated that recombination between transfecting DNAs occurred concomitantly with DNA replication but that the two processes could be partially uncoupled. We conclude that the dramatic expansion of recombination activities in the cytoplasm of poxvirus-infected cells is virus specific and offers a good model system with which to analyze the mechanism of recombination in a eucaryotic environment.