Transient expression of hepatitis B virus surface antigen (HBsAg) gene in monkey cells by a SV40-based virus vector

Summary We have constructed a recombinant SV40-based vector carrying the S gene coding for the hepatitis B virus surface antigen (HBsAg). This vector replicates as an episome in monkey COS7 cells, producing high levels of hepatitis B virus surface antigen (HBsAg), which is liberated in the cell medium, probably as a membrane vesicule. The vector also carries the SV40-late genes and produces recombinant viruses. These viruses were used to infect fresh cell culture, with detection of HBsAg in the medium. Thus, this virus vector can efficiently transduce the gene for HBsAg.     

Mutation of the serine 312 phosphorylation site does not alter the ability of mouse p53 to inhibit simian virus 40 DNA replication in vivo.

Two mutations were introduced into the wild-type mouse p53 gene by oligonucleotide-directed mutagenesis. These mutations substituted alanine or aspartic acid for serine at position 312, which is constitutively phosphorylated. Phosphopeptide mapping of the mutant proteins, expressed in COS cells, confirmed the loss of phosphorylation at position 312. There were no changes in the ability of the mutant p53s to express the conformation-dependent epitope for monoclonal antibody PAb246 or to participate in complexes with the simian virus 40 (SV40) large T antigen. Replication of a plasmid containing the SV40 origin of replication was inhibited in COS cells by wild-type p53 and both of the phosphorylation site mutants with equal efficiency. A transforming mutant of p53, encoding valine at position 135, did not inhibit SV40 DNA replication in COS cells.     

Production of a T-antigen-related protein in mammalian cells after stable transformation with a cloned SV40 gene fragment

A recombinant plasmid based on pBR322 has been constructed which carries the replicator proximal early region of SV40 DNA, including the viral origin of replication (ORI). It lacks a major part of the tumour antigen 3'-coding region, the large T-antigen termination codon and the polyadenylation site. The recombinant plasmid was transferred together with the herpes simplex virus thymidine kinase gene, as a selectable marker into mouse LTK- cells. Integration and expression of the cloned SV40 gene fragment in TK+ transformants could be demonstrated by DNA restriction and blot hybridization and by immunofluorescence techniques.     

Regulated replication of an episomal simian virus 40 origin plasmid in COS7 cells.

The replication of a simian virus 40 (SV40) origin-containing plasmid, pSLneo, stably transfected COS7 cells has been studied. pSLneo contains the SV40 origin of replication and encodes the positive selectable marker for G418 resistance. In transient replication assays, pSLneo replicates to a high copy number in COS7 cells. Uncontrolled SV40 plasmid replication has been reported to be lethal to such transfected cells. Thus, it was anticipated that extensive plasmid replication would preclude isolation of permanent cell lines containing pSLneo. However, significant number of G418-resistant colonies arose after transfection of COS7 cells with pSLneo. Cell lines established from these drug-resistant colonies contained between 100 and 1,000 extrachromosomal pSLneo copies per cell. Episomal plasmid DNA in pSLneo/COS7 lines was stably maintained after 2 months of continuous culture in selective medium. Bromodeoxyuridine labeling and density shift experiments demonstrated that replication of pSLneo closely paralleled that of cellular DNA. On average, plasmid DNA did not replicate more than once during a single cell generation period. Regulation of pSLneo replication appeared to be negatively controlled by a cis-acting mechanism. Endogenous copies of episomal pSLneo remained at a stable low copy number during the simultaneous, high-level replication of a newly transfected plasmid encoding SV40 large T antigen in the same cells. These results indicate that regulated replication of an SV40 origin plasmid can be acquired in a cell and does not require the presence of additional genetic elements. The molecular mechanism by which cells enforce this regulation on extrachromosomal SV40 plasmids remains to be defined.     

Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen.

Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes.     

Simian virus 40 T antigen as a carrier for the expression of cytotoxic T-lymphocyte recognition epitopes.

Simian virus 40 (SV40) large T antigen can immortalize a wide variety of mammalian cells in culture. We have taken advantage of this property of T antigen to use it as a carrier for the expression of cytotoxic T-lymphocyte (CTL) recognition epitopes. DNA sequences corresponding to an H-2Db-restricted SV40 T-antigen site I (amino acids 205 to 215) were translocated into SV40 T-antigen DNA at codon positions 350 and 650 containing EcoRI linkers. An H-2Kb-restricted herpes simplex virus glycoprotein B epitope (amino acids 498 to 505) was also expressed in SV40 T antigen at positions 350 and 650. Primary C57BL/6 mouse kidney cells were immortalized by transfection with the recombinant and wild-type T-antigen DNA. Clonal isolates of cells expressing chimeric T antigens were shown to be specifically susceptible to lysis by CTL clones directed to SV40 T-antigen site I and herpes simplex virus glycoprotein B epitopes, indicating that CTL epitopes restricted by two different elements can be processed, presented, and recognized by the epitope-specific CTL clones. Our results suggest that SV40 T antigen can be used as a carrier protein to express a wide variety of CTL epitopes.     

Constitutive expression of simian virus 40 large T antigen in monkey cells activates their capacity to support polyomavirus replication.

Polyomavirus DNA replication is normally restricted to rodent cells, and simian virus 40 (SV40) DNA replication is restricted to primate cells. We demonstrate that DNAs containing the polyomavirus origin can be replicated in monkey cells which constitutively express SV40 large T antigen. Permissivity is most likely caused by SV40 T antigen modification of cellular protein(s) required to replicate the polyomavirus origin. A possible target for the T-antigen-induced modification is DNA polymerase alpha-DNA primase.     

Assembly of nascent DNA into nucleosome structures in simian virus 40 chromosomes by HeLa cell extract.

A soluble system was developed that could support DNA replication in simian virus 40 (SV40) chromosomes. DNA synthesis in this system required the presence of purified SV40 large tumor antigen, SV40 chromosomes prepared from virus-infected monkey cells, a crude extract from HeLa cells, and several low-molecular-weight components. In comparison to the replication of purified SV40 form I DNA, the rate of DNA synthesis was 15 to 20% in this system. DNA synthesis started near the replication origin of SV40 and proceeded bidirectionally in a semiconservative manner. Micrococcal nuclease digestion experiments revealed that the replicated DNA produced in this system became organized into a regularly spaced array of nucleosome core particles when an appropriate amount of purified HeLa core histones was added to the reaction mixture. SV40 form I DNA replicating under the same conditions was also assembled into nucleosomes, which were arranged in a rather dispersed manner and formed an aberrant chromatin structure.     

Expression of Hepatitis B Virus Surface Antigen Gene in Cultured Cells by Using Recombinant Plasmid Vectors

By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.     

Increased disulphide dimer formation of latent associated peptide fusions of TGF-β by addition of l-cystine

The development of novel protein therapeutics relies on the ability to express appreciable amounts of correctly folded recombinant proteins. Latent IFN-β is engineered using the latency-associated peptide (LAP) of transforming growth factor β1 (TGF-β1) to maintain IFN-β in a biologically inactive form until such time as it is released at sites of inflammation by matrix metalloproteinase activity (see Adams et al., 2003). CHO cells cultured in suspension were used for expression of latent IFN-β to allow medium scale transient transfection. However, the recombinant protein expressed in this system consisted of a mixture of properly linked disulphide dimers and monomers. The ratio of dimer:monomer produced could be significantly altered towards increased dimer production by the addition of l-cystine to the CHO culture medium. The total yield of latent IFN-β was increased by co-transfection of plasmid coding for the simian virus (SV) 40 large T antigen to the plasmid with the SV40 origin of replication expressing latent IFN-β DNA. These results provide valuable new insights for developing protocols to produce substantial quantities of latent cytokine dimers in CHO cells in suspension.     

High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells.     

Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants.

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.     

An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors

We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes.