- Volume 69, Issue 3, 1988
Volume 69, Issue 3, 1988
- Articles
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ANNOUNCEMENT
JOINT MEETING OF THE VIRUS GROUPS OF THE GERMAN AND BRITISH SOCIETIES FOR GENERAL MICROBIOLOGY
Hamburg 14-17 September 1988
All virologists are cordially invited to attend this meeting
Offers for oral presentation should fall into one of the fields defined in the session titles. Poster contributions may be on any virological topic. Support is available to U.K. participants for accommodation and, to young members of the Society for General Microbiology (U.K.), support for travel and subsistence is also available (details in the February and May issues of the SGM Quarterly). Abstracts (in standard format: title, authors and addresses and text less than 250 words on one A4 page) should be submitted to the U.K. organizers before 31 March 1988 to allow selection for support. Other abstracts must be submitted before 15 May 1988 to any one of the organizers.
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- Bacterial
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An Analysis of Restriction Endonuclease Sites in Cyanophages Infecting the Heterocystous Cyanobacteria Anabaena and Nostoc
More LessSummaryAn analysis of restriction endonuclease cleavage of DNA isolated from cyanophages that infect Anabaena and Nostoc species of cyanobacteria has provided evidence for counter-selection of restriction endonuclease sites. These include sites containing subsequences which are methylated by host (Anabaena PCC 7120) methylase(s) akin to the dam and dcm enzymes of Escherichia coli. Other sites which are counter-selected have no common sequence structure. The latter include those of the endogenous restriction endonucleases of the host, but other absent sequences are not attributable to isoschizomers of any known Anabaena or Nostoc restriction endonuclease. The cyanophages differ in their tolerance to DNA methylation. Isolates A-4L, AN-13 and AN-23 do not tolerate adenosine methylation in the GATC sequence whereas two cyanophages, A-1L and AN-10 (which are related) do tolerate dam-like methylation of this sequence. In addition, A-1L allows cytosine methylation at GGCC sequences, but AN-10 has counter-selected these sequences and the remaining sites are not methylated. Analysis of native and cloned A-4L DNA suggests that counter-selection has occurred against all sequences which would be methylated by the host at either adenosine or cytosine nucleotides.
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- Animal
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The Principal Hydrogen Donor for the Herpes Simplex Virus Type 1-encoded Ribonucleotide Reductase in Infected Cells Is a Cellular Thioredoxin
More LessSummaryIn this study herpes simplex virus type 1-encoded ribonucleotide reductase was shown to be able to utilize thioredoxin purified from the cyanobacterium Anabaena variabilis as a hydrogen donor for the enzyme. An assay has been developed to search for proteins which can function as a hydrogen donor for the viral ribonucleotide reductase. A protein has been identified and purified to homogeneity from infected cell extracts by a combination of fast protein liquid chromatography and gel filtration. This protein, which is also present in mock-infected cells, has been identified as a host cell thioredoxin by similarities in its physical characteristics with other thioredoxins. No evidence for the existence of a major virus-induced thioredoxin was obtained, suggesting that the host cell thioredoxin functions as the hydrogen donor for the herpes simplex virus type 1 ribonucleotide reductase in the infected cell.
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An Antigenic Analysis of the Adenovirus Type 2 Fibre Polypeptide
More LessSummaryTwenty-seven monoclonal antisera were generated against the SDS-denatured fibre of adenovirus type 2. The antisera were characterized using radioimmune assay, fluorescent antibody tests, immune precipitation, Western blotting, haemagglutination and neutralization, and formed six groups as follows: A, type-specific neutralizing antisera which exhibited haemagglutination inhibition (Hi+); B, type-specific non-neutralizing antisera which did not exhibit haemagglutination inhibition (Hi-); C, subgroup-specific neutralizing antisera Hi+; D, a subgroup-specific neutralizing antiserum Hi-; E, subgroup-specific non-neutralizing antisera Hi-; F, a subgroup-specific neutralizing antiserum Hi- which did not react in Western blotting tests. The C-terminal 201 amino acids of the fibre were expressed in Escherichia coli and a total of six antisera from groups A, B and C recognized five epitopes carried on this region which in several models is thought to form the knob of the fibre. At least eight epitopes were expressed by the entire native fibre. The five epitopes of the C-terminal end of the fibre formed three antigenic sites. Two sites each consisted of a neutralizing type-specific and a neutralizing group-specific epitope which overlapped in position. The remaining site consisted of a type-specific, non-neutralizing epitope.
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Operational and Topological Analyses of Antigenic Sites on Influenza C Virus Glycoprotein and Their Dependence on Glycosylation
More LessSummaryIn our previous study, seven monoclonal antibodies specific for influenza C virus glycoprotein (gp88) were prepared and tentatively classified into two groups: group A (J14, J9, Q5, K16) has neutralization activity whereas group B (S16, J6, J15) does not. These antibodies were used to analyse the antigenic structure of gp88 and to examine the effect of glycosylation on the antigenicity of the glycoprotein. Operational analysis with a panel of antigenic variants selected with each of the group A antibodies identified two non-overlapping antigenic sites on the gp88 molecules, site A-1 recognized by J14, J9 and Q5 and site A-2 by K16. Sites A-1 and A-2 were shown, however, to be topographically overlapping by competitive binding assays. Competitive binding analysis with group B antibodies identified two additional non-overlapping antigenic sites, site B-1 recognized by S16 and site B-2 by J6 and J15. It was found in radioimmunoprecipitation experiments that antibodies to sites B-1 and B-2 were reactive not only with gp88 but with its non-glycosylated form (T76) synthesized in the presence of tunicamycin. Antibodies to sites A-1 and A-2, in contrast, immunoprecipitated the T76 polypeptide in only trace amounts or not at all. Additionally, Western blot analysis showed that denatured gp88 blotted on nitrocellulose was reactive with antibodies to sites B-1 and B-2 but not with those to sites A-1 and A-2. These observations suggest that glycosylation of gp88 selectively influences the integrity of antigenic sites A-1 and A-2 which are composed of conformation-dependent epitopes.
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Measles Virus Fusion Protein Presented in an Immune-stimulating Complex (Iscom) Induces Haemolysis-inhibiting and Fusion-inhibiting Antibodies, Virus-specific T Cells and Protection in Mice
SummaryImmune-stimulating complexes (iscoms), which have recently been shown to be highly effective for the antigenic presentation of membrane proteins of viruses, were prepared with affinity-purified fusion (F) protein of measles virus (MV), using an adaptation of the standard method for iscom preparation. Immunization of monkeys with the F iscom preparation induced biologically active anti-F protein antibodies as was shown in haemolysis inhibition and cell-cell fusion inhibition tests. A whole MV iscom preparation, which also contained the haemagglutinin protein, induced not only haemolysis-inhibiting antibodies, but, in contrast to the F iscom preparation, also haemagglutination-inhibiting and virus-neutralizing antibodies. In addition the F iscom preparation was shown to activate measles virus-specific T cells in mice. This was demonstrated by the generation of an MV-specific delayed type hypersensitivity response in F iscom-immunized animals and by the isolation of T cell clones specific for MV F protein with the T helper phenotype. Vaccination of mice with MV iscom or F iscom protected them from MV-induced fatal encephalopathy. The data concerning the immunogenicity of MV proteins presented in iscoms are discussed in relation to their potential for the development of an inactivated measles vaccine.
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W2 Virus Infection of the Crustacean Carcinus mediterraneus: a Reovirus Disease
More LessSummaryMost of the viruses described in marine invertebrates have been related to known virus families only on the basis of ultrastructural properties. Recently a viral agent was isolated and studied in the Mediterranean shore crab Carcinus mediterraneus. This agent, which was 65 to 70 nm in diameter, developed in the cytoplasm of connective tissue cells of C. mediterraneus and produced unusual viral structures, ‘rosettes’, consisting of an empty sphere bounded by arrangements of viral particles. The capsid consisted of two protein shells. After purification, full virions exhibited a density of 1.34 g/ml in CsCl. The nucleic acid composition of virions was estimated at about 22% and was shown to be a dsRNA with at least nine segments in four different size classes. The capsid contained six polypeptides with M r of 120 × 103, 94 × 103, 76 × 103, 44 × 103, 32 × 103 and 24 × 103, as determined by SDS-PAGE. From its biological, ultrastructural and physicochemical properties, we propose that this virus should be classified as a new member of the family Reoviridae.
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In vitro Transcription and Translation of Bluetongue Virus mRNA
More LessSummaryFractionation of in vitro transcribed bluetongue virus (BTV) mRNA by agarose gel electrophoresis resulted in the separation of eight of the 10 species. The relative molar ratio of the mRNAs confirmed that mRNA 5 was transcribed more frequently than would be predicted from the size of the S5 genome segment, while mRNA 10 was transcribed less frequently. In vitro translation of unfractionated BTV mRNAs resulted in the synthesis of the seven known structural proteins (P1 to P7) and two known non-structural proteins (NS1 and NS2). Two additional non-structural proteins (NS3 and NS3A) with M r of 28K and 25K respectively were identified. The protein coding assignments for the medium- and small-sized double-stranded RNA genome segments of BTV serotype 10 were found to correspond to those reported for BTV-1 and BTV-17. The peptide maps of NS1, NS2, NS3 and NS3A synthesized in vitro corresponded to those of their counterparts synthesized in infected cells. Protein NS3A appeared to be a truncated form of NS3, since its peptide map completely overlapped that of NS3. Proteins NS3 and NS3A were present in very small amounts in the soluble fraction of the cytoplasm of infected cells, and were synthesized in variable amounts in vitro, whereas the other nine viral proteins were synthesized in constant molar ratios. A difference in the relative molar ratios in which some of the BTV proteins were synthesized in vitro and in vivo was observed. In vivo, protein NS1 was translated in the largest amount but in vitro, NS2 was the most efficiently translated protein. Conversely, protein P6 was translated much more efficiently in vitro than in vivo.
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The P2 and P3 Regions of the Poliovirus Genome are Preferentially Translated at Alkaline pH in Infected HeLa Cells
More LessSummaryHeLa cells infected with poliovirus exclusively synthesized proteins coded by the P2 and P3 regions of the viral genome when they were placed in an alkaline medium lacking sodium ions. The amount of viral protein synthesized was augmented by increasing the concentration of KCl. Also in the presence of KCl, the cells continued synthesizing this altered pattern of proteins for longer times. This effect occurred in the presence of guanidine, it was reversible, and the normal pattern of poliovirus proteins reappeared when control medium was added, even when guanidine was present. These findings suggest that truncated viral RNA is not made under these conditions. Pactamycin and sodium fluoride, two known inhibitors of the initiation of translation, blocked protein synthesis in cells placed in alkaline medium, indicating the possibility that initiation at internal sites of the viral mRNA may take place under these conditions. Finally, the proteins synthesized were analysed by two-dimensional gel electrophoresis. The proteins migrated as authentic viral proteins indicating that if internal initiation takes place, at least some of these initiation events occur in phase with the initiation codon present in the poliovirus genome.
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Variation in Dengue Type 2 Viruses Isolated in Bangkok during 1980
SummaryDengue type-2 viruses isolated in metropolitan Bangkok during 1980 (Bangkok/80) were characterized by oligonucleotide fingerprinting, restriction enzyme (RE) mapping and antigenic analysis using monoclonal antibody probes. Of 10 isolates analysed by oligonucleotide fingerprinting, nine were very closely related, showing 72.5% to 91.4% oligonucleotide homology. One isolate (D80-141) produced a distinctly different fingerprint (55.7% to 58.0% homology) and was less related to other Bangkok/80 dengue-2 virus isolates than to a 1964 Bangkok isolate (16681). RE mapping conducted on complementary dsDNA prepared from three Bangkok/80 isolates, strain 16681 and the prototype New Guinea C strain confirmed that D80-141 was genetically distinct. On antigenic analysis, only one of 22 monoclonal antibody probes produced against representative 1980 Bangkok dengue-2 isolates, D80-100 and D80-141, was able to distinguish between these virus strains. Monoclonal antibody 47-10/10, prepared using D80-100 virus and directed at the NS1 non-structural glycoprotein, had a significantly lower (100-fold) solid phase radioimmune assay endpoint titre for D80-141 antigen than for D80-100 antigen. By the indirect immunofluorescence assay, 47-10/10 had lower antibody endpoint titres against D80-141, the NGC strain and 13 (12%) of 110 Bangkok/80 isolates than to a control antibody preparation. These results suggest that strain D80-141 represents a second minor topotype of dengue-2 which was circulating concurrently with the major endemic topotype in Bangkok in early 1980.
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Nucleotide Sequence of the Rubella Virus Capsid Protein Gene Reveals an Unusually High G/C Content
SummaryThe nucleotide sequence of the rubella virus capsid protein (C) gene has been determined from a cDNA clone derived from the 40S genomic RNA. The sequence covers the coding region of the C protein (831 nucleotides), 70 nucleotides of the 5′ untranslated region, and the 5′ end of the downstream E2 membrane protein gene. The capsid gene is unusually rich in C (41.6%) and G (31.2%) residues (G + C 72.8%), and poor in A (15.4%) and U residues (11.8%). There are regions with long runs of up to 45% C or 35% G residues. The codon usage is non-random, with a strong preference for C and G residues in the third position. Starting from two in-frame AUG codons (seven amino acid residues apart) an open reading frame (ORF) was identified that extended in frame into the ORF coding for the downstream E2 membrane protein gene. Since the amino terminus of the capsid protein is blocked, we could not determine which of the AUGs serve as the initiating codon. To verify that the deduced ORF was correct, we have determined the amino acid sequence of 13 tryptic peptides corresponding to one-third of the C protein. Our data show that the C protein is about 277 residues in length (M r about 30750). It is very hydrophilic and rich in prolines (14.1%) and arginines (14.4%). Clusters of these amino acids are concentrated in the aminoterminal third of the C protein. No sequence homology to the capsid protein of several alphaviruses was observed. Together with our previous sequence data we have now completed the sequence of the genes coding for the structural proteins C, E2 and E1 of rubella virus.
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Nucleotide Sequence of the Fusion and Haemagglutinin-Neuraminidase Glycoprotein Genes of Newcastle Disease Virus, Strain Ulster: Molecular Basis for Variations in Pathogenicity between Strains
More LessSummaryThe nucleotide sequences of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoprotein genes of the extremely avirulent Newcastle disease virus (NDV) strain Ulster have been determined by sequencing cDNA clones derived from viral genomic RNA. Open reading frames, assumed to encode the F0 and HN0 glycoprotein precursors, were 553 and 616 amino acids long, respectively. Comparisons of the two glycoprotein sequences with those of more virulent NDV strains suggested an explanation for the molecular basis of the wide-ranging differences in virulence observed between strains of NDV. The open reading frame corresponding to the Ulster HN glycoprotein extended beyond the C terminus of more virulent strains. This C-terminal extension was assumed to be responsible for the origin of the HN precursor (HN0) found in strain Ulster and other extremely avirulent strains of NDV. There were fewer basic amino acids at the cleavage site of F0 in strain Ulster than are present in more virulent strains, which may be responsible for the absence of cleavage and activation of F0 from this strain in many host cells. In more virulent strains of NDV, as well as in other paramyxoviruses, a phenylalanine residue occurs at the N terminus of the F1 cleavage fragment. The occurrence of a leucine residue at this position in strain Ulster may be partly responsible for the lack of virulence of this strain.
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Evolution of Avian Coronavirus IBV: Sequence of the Matrix Glycoprotein Gene and Intergenic Region of Several Serotypes
More LessSummaryWe have sequenced 200 to 240 bases of the matrix (M) glycoprotein gene of 23 strains of infectious bronchitis virus (IBV) representing the A (D207), B (D3896), C (D3128), D (D212), Massachusetts (Mass), UK11 and UK12 serotypes. The bases examined code for the external, hydrophilic region and the first membrane-embedded hydrophobic region of M, both regions comprising approximately 20 amino acids. As predicted from protein M r studies the A/D and B/C serotypes had two and one potential glycosylation sites respectively. This variation appeared to derive from a combination of base substitutions and deletions/insertions. The glycosylation sequence Asn-Cys-Thr was highly conserved. Overall, the exposed part of M exhibited a fourfold greater extent of amino acid variation than did the membrane-embedded sequence. The transcription-associated homology region sequence (CUUAACAA) in the 5′ intergenic region was identical in all strains but there was considerable variation as to its location. The M gene of UK12 appeared to have evolved from a group A-like M gene by a two stage process involving a base substitution in the intergenic region which generated a new AUG translation start codon followed by deletion of the original AUG. Isolate UK11 closely resembled Mass strains in the intergenic region but was dissimilar from all strains in the protein coding region. The M sequences of serotypes B and C were identical and those of the A and D serotypes very similar. These results are discussed in relation to recent sequencing of part of the spike glycoprotein gene of some of these strains and the discovery of in vitro recombination of murine hepatitis coronavirus.
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Comparative Studies on Structural and Antigenic Properties of Two Serotypes of Infectious Bursal Disease Virus
More LessSummaryThe electrophoretic mobilities of the two genome segments and the structural polypeptides of the chicken strain Cu-1 (serotype I) and the turkey isolate 23/82 (serotype II) of infectious bursal disease virus were compared. There is a close antigenic relationship between the smaller of the two major structural proteins (32K) of both strains. Neutralizing monoclonal antibodies are induced by the larger protein (40K in Cu-1) which differentiates between the two serotypes. The 40K structural protein also has epitopes which do not induce neutralizing antibodies and which are common to both strains. There is evidence that the antigenic region responsible for the production of neutralizing antibodies is highly conformation-dependent. Passively administered neutralizing antibodies directed against the 40K structural polypeptide of Cu-1 confer protective immunity to susceptible chickens, whereas antibodies directed against the 32K structural protein do not have any protective effect.
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Isolation of a Variant Endogenous Avian Leukosis Virus: Non-productive Exogenous Infection with Endogenous Viruses Containing p27 and p27°
More LessSummaryTen replication-competent endogenous avian leukosis viruses (ALVs) with subgroup E specificity were isolated from six commercial chicken lines of mixed breed. All the ALVs isolated belonged to a variant class of endogenous viruses with a major group-specific antigen, p27, identical in size to that of exogenous viruses and different from the p27° of endogenous viruses isolated from White Leghorn chickens. It was possible to infect chickens exogenously with endogenous viruses (containing p27) and with Rous-associated virus type 0 (containing p27°) but infection was non-productive. Congenital transmission and shedding did not occur in chickens infected with endogenous viruses containing p27° or in chickens infected with endogenous viruses containing p27. Restriction of endogenous virus replication in some tissues (heart, spleen and meconia) of viraemic chickens was observed suggesting that exogenous infection of chickens with endogenous viruses differs from an infection with exogenous ALVs.
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Epigenetic Control and Reintegration of Extrachromosomal Proviral DNA in HL60 Cells Chronically Infected with Human T Cell Leukaemia Virus Type 1
More LessSummaryExtrachromosomal human T cell leukaemia virus type 1 (HTLV-I) proviruses are persistently maintained in HTLV-I-infected human promyelocytic leukaemia (HL60) cells even 24 months after viral infection. By successive recloning of these HTLV-I-infected clones, and by Southern blot analysis of their HTLV-I proviruses, we concluded the following. The copy number of extrachromosomal proviruses fluctuated, and this fluctuation was probably dependent on the epigenetic conditions in the host cell, HL60. The transient appearance of a high copy number of extrachromosomal proviruses was followed by an increase in the copy number of integrated proviruses. Persistence of extrachromosomal proviruses appeared to be caused by an intracellular rather than an intercellular mechanism.
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Alterations in Humoral Immune Response to Bovine Leukaemia Virus Antigens in Cattle with Lymphoma
More LessSummarySera collected from cattle with enzootic bovine lymphoma (EBL) were compared to sera from clinically normal bovine leukaemia virus (BLV)-infected cattle for immunoglobulin concentration and for antibodies detecting BLV proteins tested using three different viral isolates. Monoclonal antibodies to bovine immunoglobulin isotypes were used in Western blot analysis to identify isotype reactivity to specific viral antigens. IgG titres to BLV were determined by ELISA. Serum immunoglobulin (G1, G2 and M) concentrations were assessed by radial immunodiffusion. Although EBL was associated with reduced total immunoglobulin production, sera from cattle with EBL had significantly (P < 0.001) higher specific IgG titres and produced antibodies against a greater and more varied number of BLV proteins than did sera from clinically normal BLV-infected cattle. Variations were consistent within groups of cattle and did not depend on the viral isolate used.
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Antiviral, Anti-glycoprotein and Neutralizing Antibodies in Foals with Equine Infectious Anaemia Virus
More LessSummaryEquine infectious anaemia virus is related by genome sequence homology to human immunodeficiency virus, caprine arthritis-encephalitis virus and visna virus. Failure of the host to mount a strong neutralizing response detectable in vitro or to eliminate persistent infection in vivo characterizes lentivirus infections in the natural host. In this study the specificities and neutralizing activity of antibodies induced during experimental infection with equine infectious anaemia virus were investigated using antiviral ELISA, radioimmunoprecipitation and neutralization assays. ELISA antibody titres of 105 to 106 were demonstrated in samples collected 30 and 60 days after infection. Immunoprecipitation titrations demonstrated that antibody titres to the glycoproteins gp90 and gp45 were 10 to 100 times higher than titres to the internal structural protein, p24. Low levels of neutralizing antibody appeared at 23 to 46 days post-infection. The presence of low levels of neutralizing activity in the presence of high levels of anti-glycoprotein activity suggests that the major immunogenic sites on the viral surface are not sensitive to neutralization.
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Precursor Polypeptides of Caprine Arthritis-Encephalitis Lentivirus Structural Proteins
More LessSummaryThe synthesis of caprine arthritis-encephalitis virus structural proteins was analysed in infected cells labelled with [35S]methionine and [3H]glucosamine and by translation of virion RNA in vitro. Viral polypeptides were isolated from infected cell lysates or from in vitro translation products by immunoprecipitation with specific antisera and resolved by SDS-PAGE. Results indicated that the gag gene-encoded p28, p19 and p16 virion core proteins were formed by cleavage processing of a 55K M r precursor with several intermediate polypeptides. The gp135 virion surface glycoprotein, encoded by the env gene, was formed by post-translational modification of a glycosylated precursor of 150K apparent M r. This precursor was formed by glycosylation of a 90K primary env gene product.
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Isolation of a Simian Virus 40-Simian Agent 12 Recombinant Papovavirus That Synthesizes a Hybrid Large Tumour Antigen
More LessSummaryThe simian virus 40 (SV40) mutant dl1055 carries a 55 base pair deletion in the viral early region coding sequences that causes premature termination of large tumour (T) antigen translation resulting in a protein fragment consisting of the amino-terminal 399 residues. The mutation renders the virus defective. We report the characterization of hyb4001, which was isolated as a single plaque following cotransfection of permissive cells with dl1055 DNA and the DNA of a related papovavirus, simian agent 12 (SA12). Hyb4001 arose by two homologous recombination events involving crossovers in regions of 7 and 12 base pairs of perfect homology between the two viruses. Hyb4001 synthesizes a hybrid T antigen with the amino-terminal 382 residues encoded by SV40, residues 383 to 449 encoded by SA12, and the carboxy-terminal 259 residues encoded by SV40.
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