Expression and characterization of the thymidine kinase gene of African swine fever virus.

The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes. The TK gene was mapped within a 0.72-kbp BglII-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp SalI-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus.     

DNA of Epstein-Barr virus. IV. Linkage map of restriction enzyme fragments of the B95-8 and W91 strains of Epstein-Barr Virus.

The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).     

Evidence for integrated EBV genomes in Raji cellular DNA.

Human lymphoid cell lines cannot be grown in long-term tissue culture, as a rule, unless the cells have been transformed by Epstein-Barr virus (EBV). The latent EBV DNA in established cell lines, is mainly present as free covalently closed circles but viral DNA sequences with properties of integrated DNA also seem to be present. We have extended the studies on the physical state of the EB viral DNA sequences in the cell line Raji which appear at a lower density than that for free EB viral DNA during fractionation on CsCl density gradients. In such material a novel EcoRI EBV DNA fragment is present, which hybridizes to viral sequences homologous to EcoRI A. This fragment is not present in free covalently closed circular EBV DNA. When this EcoRI fragment is further analysed with HindIII a smaller fragment than expected, which contains BamHI W sequences, is detected. The demonstration of this HindIII fragment and its characteristics as a joint, viral-host chromosome fragment will be discussed.     

Phenotypic switching in cells transformed with the herpes simplex virus thymidine kinase gene

Biochemical transformation of Ltk- cells with the herpes simplex virus thymidine kinase (tk) gene resulted in numerous TK+ colonies that survived selection in hypoxanthine-aminopterin-thymidine medium. Many of these TK+ cell lines switched phenotypes and reverted to the TK- state. In this report, we describe the biological and biochemical characteristics of three TK- revertant lines. One (K1B5) transiently expressed TK in the presence of bromodeoxyuridine, which selects for the TK- phenotype. Another TK- sibling (K1B6n) expressed TK only after removal from bromodeoxyuridine-containing medium. The last variant (K1B6me) lost the ability to switch to the TK+ phenotype, although it maintained the herpes simplex virus sequences coding for TK. Loss of the ability of K1B6me cells to express TK was correlated with extensive methylation of the sequence recognized by the restriction endonuclease HpaII (pCpCpGpG). After these cells were treated with 5-azacytidine, they regained the ability to clone in hypoxanthine-aminopterin-thymidine medium and reexpressed virus tk mRNA and enzyme. In addition, the HpaII sites that were previously shown to be refractile to enzyme digestion were converted to a sensitive state, demonstrating that they were no longer methylated.     

Herpes simplex type 1 activation by Epstein-Barr virus nuclear antigen 1

We have investigated the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1), a nuclear protein encoded by EBV, on herpes simplex virus type 1 (HSV-1) infection either in cells constitutively expressing EBNA-1 or in transient expression assays. Rat-1 cells and rat embryo fibroblasts (REF) immortalized by c-myc or E1A were transfected with a specific EBV DNA fragment coding for EBNA-1. Cloned cell lines which constitutively expressed this antigen were infected with HSV-1. Our results indicate that in EBNA-1-expressing cells, virus growth was higher than in control cells for different virus strains or rodent cell lines. This increase was maximal when cells were infected at low multiplicity, as determined by virus growth, and correlated with the stimulation of viral DNA synthesis. REF + c-myc and Vero cells were contransfected by an EBNA-1 expression vector driven by Moloney murine leukaemia virus LTR and HSV-1 immediate-early (α0) or early thymidine kinase upstream promoter regulatory regions linked to chloramphenicol acetyltransferase (CAT) coding sequences as effectors. In both cell lines, stimulation of CAT expression by EBNA-1 was observed only with the immediate-early promoter. These results suggest that EBNA-1 can transactivate immediate-early HSV-1 expression.     

Non-immortalizing P3J-HR-1 Epstein-Barr virus: a deletion mutant of its transforming parent, Jijoye.

The P3J-HR-1 strain of Epstein-Barr virus (EBV) fails to immortalize human lymphocytes. We wished to understand the nature of the genomic alterations which correlated with the loss of this ability. As a first step, the heterogeneity of DNA molecules in the P3J-HR-1 line was eliminated by cell cloning. Then a physical map was prepared of virion DNA from one cell clone, designated FF452-3. By comparison with the genomes of two EBVs, B95-8 and FF41, which are competent to immortalize lymphocytes, we identified a total of eight modifications of BamHI and EcoRI restriction endonuclease fragments of EBV (FF452-3) DNA consisting of insertions, deletions, or loss of a restriction endonuclease recognition site. To determine which of these alterations might be responsible for the loss of transforming phenotype, we examined homologous DNA fragments of the Jijoye strain of EBV, the progenitor of the HR-1 strain which still retains the ability to immortalize lymphocytes. We also studied viral DNA in lymphocytes transformed in vitro by Jijoye virus. Six of the eight alterations were found both in Jijoye and in clonal HR-1 DNA and were presumably genomic traits characteristic of this lineage of EBV. A small deletion in the BamHI-K fragment of HR-1 DNA was not found in Jijoye virion DNA, but this deletion was present in intracellular Jijoye DNA. Thus only one major genomic lesion in HR-1 DNA, a deletion of at least 2.4 x 10(6) molecular weight of DNA from a fused BamHI-H-Y fragment, consistently distinguished Jijoye DNA from its non-immortalizing P3J-HR-1 derivative. This deletion is likely to affect EBV genes which are directly or indirectly involved in immortalizing lymphocytes.     

Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus.

We have constructed a hybrid plasmid by insertion of the thymidine kinase (TK) gene of Herpes simplex virus (HSV) type I at the BamHI site on Escherichia coli plasmid pBR322. The restriction endonuclease cleavage site map for the viral DNA fragment was determined for ten nucleases, and the insert in the recombinant plasmid has the same restriction nuclease digestion pattern as bona fide viral DNA. This result indicates that the plasmid contains an accurate copy of the viral DNA. The viral TK gene carried on the plasmid can be introduced into mammalian cells where it is expressed. This source of DNA with a selectable marker should be of considerable practical use in gene-transfer experiments in mammalian cells.     

Two families of sequences in the small RNA-encoding region of Epstein-Barr virus (EBV) correlate with EBV types A and B

DNA sequence analysis was carried out on the 1-kilobase SacI-EcoRI region of the EcoRI J fragment of four strains of Epstein-Barr virus (EBV) (MABA, P3HR-1, FF41, and NPC-5), and the sequences were compared with the prototype sequence from strain B95-8. Ten single-base changes which grouped the strains into two families (1 and 2) were found. Restriction endonuclease polymorphisms predicted from the sequences were used to classify the EBV DNA from a further 26 EBV-positive cell lines into these two families. The EBNA-2 types (A or B) of the strains were found to correlate with the J region type; EBNA-2 type A DNA regularly contained J region sequence type 1, while EBNA-2 type B DNA generally carried J region sequence type 2. These data are consistent with the notion of there being two distinct families of EBV with discrete, conserved differences in DNA sequence.     

Epstein-Barr virus with heterogeneous DNA disrupts latency.

By cloning the HR-1 Burkitt lymphoma line, we previously uncovered two distinct biological variants of nontransforming Epstein-Barr virus (EBV). The most commonly cloned variant has a low rate of spontaneous viral synthesis and is unable to induce early antigen in Raji cells (EAI-). A rare variant spontaneously releases virus which is capable of inducing early antigen in Raji cells (EAI+). Since EAI- virus lacks heterogeneous DNA (het-) and EAI+ virus contains heterogeneous DNA (het+), we suggested that spontaneous viral synthesis and induction of early antigen are biological properties which correlate with the presence of het sequences. The present experiments provide three new lines of experimental evidence in favor of this hypothesis. (i) Revertant subclones of the EAI+ het+ variant which have lost the het DNA concomitantly lost EAI ability. Thus, het DNA is not stably associated with the cells as are the episomes. (ii) het DNA was acquired by two het- subclones of the HR-1 line after superinfection with EAI+ virus. After superinfection, these clones synthesized EAI+ het+ virus. Thus, het DNA may be maintained in the HR-1 line by cell-to-cell spread. (iii) Virus with het DNA activated full expression of endogenous latent EBV of the transforming phenotype in a line of immortalized neonatal lymphocytes designated X50-7. By use of restriction endonuclease polymorphisms unique to both the superinfecting and endogenous genomes, we show that the genome of the activated virus resembles that of the virus which was endogenous to X50-7 cells. This result suggests that het sequences result in transactivation of the latent EBV. het DNA had homology with EBV sequences which are not normally contiguous on the physical map of the genome. het DNA was always accompanied by the presence of DNA of nonheterogenous HR-1. Thus, het DNA is a form of "defective" EBV DNA. However, the biological effect of this defective DNA is to enhance rather than to interfere with EBV replication. This is a novel property of defective virus.     

Transformation of rodent cells by a cloned DNA fragment of herpes simplex virus type 2.

Transformation of rodent cells with isolated restriction endonuclease fragments of herpes simplex virus type 2 DNA identified a region of the genome located between map positions 0.58 and 0.62. These sequences were cloned into pBR322, and the recombinant plasmid was used to transform primary rat embryo cells and NIH 3T3 cells. The transformants were selected for their ability to form dense foci on a monolayer or to form colonies in semisolid medium. In contrast to the parental rat or mouse cells, cell lines transformed with the cloned herpes simplex virus type 2 fragment grow to high saturation densities, replicate in medium containing 1% serum, form colonies in dilute methylcellulose, show reduced levels of fibronectin, and are tumorigenic in nude mice and in their syngeneic hosts. Southern blot hybridizations have detected sequences homologous to the viral fragment in high-molecular-weight DNA from the transformed cell lines that are not present in DNA from normal rodents. In all cases, the plasmid DNA was present in less than one copy per cell, and the patterns of viral sequences changed with passage of the cell line in vivo.     

Organization of the Epstein-Barr virus DNA molecule. II. Fine mapping of the boundaries of the internal repeat cluster of B95-8 and identification of additional small tandem repeats adjacent to the HR-1 deletion.

We used cloned BamHI fragments from Epstein-Barr virus strain B95-8 [EBV(B95-8)]DNA to obtain detailed restriction maps of the region of the genome adjacent to the large internal repeat cluster. These maps together with the results of hybridization experiments using a 3.1-kilobase repeat probe defined more precisely the location of the injection between the internal repeat cluster and the flanking unique-sequence DNA. On one side (UL), the repeat sequences extended 600 +/- 80 base pairs (bp) into BamHI-Y; on the other side (US), they extended 1,300 +/- 200 bp into BamHI-C. Therefore, EBV(B95-8) DNA contained a nonintegral number of 3.1-kilobase repeat units, namely, 12.6 copies. The mapping studies also revealed a second series of internal tandem repetitions in EBV(B95-8) DNA located within the BamHI-H fragment. This cluster comprised 11 copies of a 135-bp repeat unit which contained a single site for the NotI restriction endonuclease. Hybridization to these cloned EBV(B95-8) fragments using total EBV(HR-1) DNA as probe indicated that the deletion in EBV(HR-1) removed all 3,000 bp of unique-sequence DNA which lay between the large 3.1-kilobase and the small 135-bp repeat clusters. Thus, the deletion which destroyed the transforming ability in the EBV(HR-1) virus was bounded on either side by tandem repetitions.     

Molecular cloning of the complete Epstein-Barr virus genome as a set of overlapping restriction endonuclease fragments.

A complete collection of fragments of Epstein-Barr virus DNA, obtained by cleavage with restriction endonuclease Eco RI, has been cloned. Fourteen different internal fragments of the virus genome, derived from linear virion DNA of the B95-8 strain, and sequences corresponding to the terminal regions of virion DNA, derived from intracellular circular EBV DNA isolated from 895-8 cells, were cloned. Sizes of fragments were determined by agarose gel electrophoresis and their sum leads to an estimated molecular weight of 110 x 10(6) for virion DNA. Large Eco RI DNA fragments of special interest were also cloned in cosmids using another source of EBV DNA, that is, to circular viral DNA derived from Raji cells. In order to provide a set of overlapping sequences, all the 29 internal Bam HI fragments of B95-8 virion DNA were cloned in pBR322. The map location within the viral genome of each cloned DNA fragment was identified by hybridizing to blots of virion DNA cleaved with several different restriction endonucleases.     

Epstein-Barr virus intrastrain recombination in oral hairy leukoplakia.

In laboratory lymphoblastoid cell lines and in natural human infections, Epstein-Barr virus (EBV) strains have been identified by DNA restriction fragment length polymorphisms of the BamHI H fragment. Multiple, heterogeneous BamHI H fragments have been detected in oral hairy leukoplakia (HLP), raising the question of EBV coinfection with multiple strains. To investigate whether the heterogeneous BamHI H fragments represent different EBV strains or recombinant variants of the same strain, EBV DNA from HLP lesions was analyzed to characterize the viral strains and determine the source of possible recombinant variants. Clones of heterogeneous BamHI H fragments from a single HLP lesion were determined to have strain identity on the basis of sequence identity of the EBNA-2 genes. Intrastrain homologous recombination within the IR2 internal repeat region and nonhomologous recombination of other sequences accounted for the heterogeneity of the BamHI H fragments. PCR amplification from additional HLP specimens detected similar recombinant variants. A possible example of site-specific recombination joining the BamHI Y portion of the EBNA-2 gene to sequences within the BamHI S fragment was also detected in multiple HLP specimens. These data indicate that intrastrain recombination during productive replication confounds the use of restriction fragment length polymorphism analysis of the BamHI Y and H fragments to identify EBV strains in HLP. In patients with permissive epithelial EBV infections, EBV strains could be more accurately distinguished by sequence identity or divergence within known regions of genetic strain variation.     

Integration and expression of several molecular forms of Rous sarcoma virus DNA used for transfection of mouse cells.

To assess the factors required for integration and expression of retroviral DNA, we have examined viral DNA, RNA, and protein in NIH/3T3 mouse cells transformed by transfection with various forms of cloned Rous sarcoma virus (RSV) DNA. Linear RSV DNA molecules, derived from circular DNA containing two long terminal repeats (LTRs) and permuted by cleavage at the SacI restriction endonuclease site in the leader sequence, were integrated near the ends of the linear molecule, with the LTRs on the 3' side of the src gene. Integration of a subgenomic RSV DNA fragment containing the viral src gene without intact LTRs also occurred near the ends of the linear molecule. Head-to-tail tandem arrays of RSV DNA species were observed in some transformed cell lines that received fully digested DNA and in all cell lines that received DNA ligated to produce oligomers before transfection. Closed circular RSV DNA, with one or two LTRs, integrated without apparent specificity within several regions of the viral genome. After transfection with SacI-permuted RSV DNA still linked to arms of the lambda bacteriophage vector DNA, bacteriophage sequences were joined to host DNA. Transformed cell lines produced by transfection with the various forms of RSV DNA produced similar levels of viral src protein, although the efficiency of successful transformation varied by at least two orders of magnitude. Analyses of viral polyadenylated RNA, together with the patterns of viral DNA in transformed cells, indicated that viral DNA can be integrated and expressed without regard to LTR sequences, with adjacent host DNA presumably supplying signals required for the promotion and processing of functional src mRNA.     

Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells

The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.     

The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells.

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support latent replication of Epstein-Barr virus (EBV). To assess the likelihood that EBNA-1 regulates the amount of EBV DNA in a cell, we measured the average numbers of EBNA-1 molecules and EBV DNA molecules per cell in different clones of cells. The amount of EBNA-1 protein present in recently established lymphoblastoid cell lines was measured with affinity-purified anti-EBNA-1 antibodies, and viral DNA was measured by nucleic acid hybridization. The average levels of EBNA-1 protein varied little between these cell lines, whereas the average amount of viral DNA present varied substantially; consequently, these numbers were not correlated. There is no apparent relationship between amounts of EBNA-1 and viral DNA.     

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.     

Cleavage of Epstein-Barr virus DNA by restriction endonucleases EcoRI, HindIII and BamI.

The cleavage of the DNAs of the B95-8 and P3HR-1 virus strains of Epstein-Barr virus by the restriction endonucleases EcoRI, HindIII and BamI was investigated using a new technique for quantitative evaluation of the fluorescence of ethidium stained DNA fragments separated on agarose gels. The results obtained with B95-8 DNA showed that in addition to the limited repetitions of nucleotide sequences observed in the EcoRI and HindIII cleavage patterns, the molecule contained a BamI fragment with a molecular mass of 2.0 megadaltons which was present in a total of about 11 copies and localized to a limited part of the DNA molecule. The same sequences were also present in the P3HR-1 DNA albeit in a lower molar ratio. P3HR-1 DNA yielded restriction enzyme cleavage patterns suggesting DNA sequence heterogeneity of P3HR-1 virus. No fragment was present in more than about 4 copies per molecule of P3HR-1 DNA. Comparison of the restriction enzyme cleavage patterns of P3HR-1 and B95-8 DNA revealed a high degree of structural homology emphasized by nucleic acid hybridization experiments with EBV complementary RNA synthesized in vitro.