Isolation and characterization of a specific deletion mutant of human adenovirus type 2.

A specific deletion mutant of human adenovirus type 2 (Ad2) was detected when pools of wild-type Ad2 were grown at high multiplicity of infection in HeLa cells. This deletion mutant, designated dl-Ad2, was enriched when the defective Ad2-SV40 hybrids of the Ad2⁺⁺HEY population were cloned in monkey cells in the presence of an added excess of wild-type Ad2. Electron microscope heteroduplex analysis and restriction endonuclease examination established the Ad-specific nature of the dl-Ad2 DNA and revealed a single, homogeneous deletion of about 0.08 to 0.09 fractional genome length situated between 0.78 and 0.87 Ad2 map unit. This genome structure is very similar to that of several other incomplete adenoviruses already described. The deleted segment encompasses one of the four early genome regions of Ad2. dl-Ad2 particles are not infectious in both human and monkey cells, although the mutant DNA is replicated in these cell types. dl-Ad2 is able to interfere efficiently with SV40 DNA replication in coinfected monkey cells. Furthermore, virus populations could be cloned consisting exclusively of Ad2⁺⁺HEY hybrid viruses and dl-Ad2 “helper” viruses, indicating that dl-Ad2 can complement sufficiently for the large Ad2 DNA deletion in the Ad2⁺⁺HEY hybrid genomes.     

Induction of simian virus 40-specific tumour rejection by the Ad2+ND2 hybrid virus.

Immunization of BALB/c mice with Ad2+ND2, a non-defective hybrid virus containing about half of the early region of simian virus 40 (SV40) DNA covalently integrated into the human adenovirus 2 (Ad2) genome, can confer protection against subsequent challenge by syngeneic SV40 tumour cells. Analysis of subcellular fractions from Ad2+ND2-infected cells shows a close correlation between the tumour rejection activity and the presence of the two SV40-specific proteins induced by this hybrid virus. These two proteins, with mol. wt. of 56 000 (56K) and 42 000 (42K), can be specifically immunoprecipitated using sera obtained from hamsters bearing SV40-induced tumours. Such immunoprecipitates, which contain no detectable contaminating components as determined by polyacrylamide gel electrophoresis, can efficiently immunize mice against SV40 tumour challenge, suggesting that the 56K and 42K proteins are directly responsible for the induction of tumour rejection. Moreover, we have found, by immunoprecipitation, a novel antigen in SV40-transformed BALB/c cells, also of 56 000 mol. wt.; possibly, this 56K protein is responsible for induction of transplantation immunity in SV40-transformed cells.     

Inhibition of measles virus replication by cyclic AMP

In some hybrid cell lines between human and mouse cells the ribosomal RNA (rRNA) genes of both species are present but only the rRNA of the dominant species is expressed. The silent rRNA genes can be reactivated if the hybrid cells are infected with simian virus 40 (SV40). The role of the SV40 early gene, the A gene, in the reactivation of the silent rRNA genes was investigated. A temperature-sensitive mutant of the SV40 early region, tsA58, could not reactivate the silent mouse rRNA genes at the nonpermissive temperature of 39° in human > mouse hybrids, although it could do so at 32°. Wild-type SV40 did induce the expression of mouse rRNA at both 32° and 39°. Adenovirus 2 (Ad2) could not reactivate the silent rRNA genes in these hybrid cells. Infection by either Ad2+D?1 or Ad2+D?2, hybrid adenoviruses which contain portions of the A region of the SV40 genome, led to expression of both species of 28 S rRNA. However, infection by the nondefective adenovirus Ad2+ND-1 (H71), which contains an incomplete SV40 A region, failed to reactivate silent rRNA genes. Hybrid cells infected with the small t deletion mutant of SV40, dl-2005, also showed reactivation. Finally, both human and mouse rRNA were found in SV40-transformed mouse-human hybrid cells and in polyoma virus-infected hybrids. These results indicated that SV40 reactivation of silent rRNA genes in human-mouse hybrids requires the major product of the A gene, the large T antigen.     

Specific DNA binding activity of T antigen subclasses varies among different SV40-transformed cell lines

Large tumor antigen (T antigen) occurs in at least three different oligomeric subclasses in cells infected or transformed by simian virus 40 (SV40): 5-7 S, 14-16 S, and 23-25 S. The 23-25 S form is complexed with a host phosphoprotein (p53). The DNA binding properties of these three subclasses of T antigen from nine different cell lines and free p53 protein were compared using an immunoprecipitation assay. All three subclasses of T antigen bound specifically to SV40 DNA sequences near the origin of replication. However, the DNA binding activity varied between different cell lines over a 40- to 50-fold range. The 23-25 S and 14-16 S forms from most of the cell lines tested bound much less SV40 origin DNA than 5-7 S T antigen. The free p53 phosphoprotein did not bind specifically to any SV40 DNA sequences.     

Differential effect of adenovirus 2 E3/19K glycoprotein on the expression of H-2Kb and H-2Db class I antigens and H-2Kb- and H-2Db-restricted SV40-specific CTL-mediated lysis

The E3/19-kDa glycoprotein (E3/19K) coded by adenovirus type 2 (Ad2) is known to inhibit the cell-surface expression of major histocompatibility complex (MHC) class I antigens by binding to the MHC antigens intracellularly, and thus reduces recognition of antigens by MHC-restricted cytotoxic T lymphocytes (CTL). We have studied the effect of the E3/19K expression in SV40-infected monkey cells, TC-7/H-2Kb and TC-7/H-2Db expressing transfected H-2Kb and H-2Db antigens, respectively, on the cell-surface H-2 class I antigens and on lysis of the cells by SV40 large tumor (T)-antigen-specific H-2Kb- and H-2Db-restricted CTL clones. H-2Db antigen expression on TC-7/H-2Db cells was drastically reduced by infection with Ad2 but not with an E3/19K-negative SV40-Ad2 hybrid virus, Ad2+ND1, as early as 12 hr postinfection. However, H-2Kb antigen expression on Ad2-infected TC7/H-2Kb cells remained unaltered, even at 24 hr postinfection. Specific lysis of SV40-infected TC-7/H-2Db cells by H-2Db-restricted SV40 T-antigen-specific CTL clones, Y-1 and Y-3, was strongly reduced by coinfection of the target cells with Ad2 but not with Ad2+ND1. Lysis of SV40-infected TC-7/H-2Kb cells by a H-2Kb-restricted SV40 T-antigen-specific CTL clone Y-4 was also reduced significantly by Ad2 infection, but not Ad2+ND1. These results indicate that the E3/19K protein affects cell-surface expression of H-2Db antigen but not H-2Kb antigen.     

Effect of inhibitors on induction of SV40 tumor antigen by an adenovirus-SV40 hybrid

Summary A type 7 adenovirus-SV40 hybrid induces the synthesis of SV40 tumor or T antigen in hamster, rabbit, human, and monkey cells. Actinomycin D inhibits synthesis of the antigen suggesting that development of the antigen requires DNA-dependent RNA. Puromycin allows synthesis of T antigen which is therefore, apparently a low molecular weight protein or polypeptide. Synthesis of the antigen is not inhibited by p-fluorophenylalanine. Neither mitomycin C, fluorouracil, iododeoxyuridine, nor cytosine arabinoside prevent development of T antigen. It therefore appears probable that for synthesis of T antigen to proceed the synthesis of new virus DNA is not required regardless of whether this is virus DNA of SV40 or of adenovirus.Antiserum prepared against type 7 adenovirus neutralizes the ability of the hybrid to induce SV40 tumor antigen. Antibody against the SV40 tumor antigen or against whole SV40 virus failed to do so. The evidence therefore suggests that the adenovirus hybrid is carrying a piece of SV40 tumor-inducing genome which replicates with the adenovirus. Adenovirus-SV40 hybrids have been found not only with type 7 but also with many other types (24, 25).Dedicated to the Honor of the 60th birthday of ProfessorSven Gard.     

Fate and expression of simian virus 40 DNA after introduction into murine cells under nonselective conditions

When SV40 infects mouse cells, it does not replicate but instead causes neoplastic transformation of a small percentage of the cells. It is unknown, however, what happens to the virus in those cells that do not become transformed. We introduced SV40 into mouse cells by nonselective means, either by cotransfection of SV40 DNA with a selectable marker or by random cloning of SV40-infected cells. We analyzed the fate of viral DNA sequences, expression of T antigens, and transformation properties of these cells. We found that, upon infection, viral DNA integration occurs at a frequency that is at least 10-fold higher than the frequency of transformation. The majority of these cells are not transformed due to lack of expression of T antigen. One cell line which expresses a truncated T antigen is not transformed. We have mapped the viral sequences in the genome of these cells and find that integration in the large T intron is probably responsible for the defect. Lack of transformation can therefore be attributed to both cellular and viral factors, namely, introduction of viral DNA into cells that are resistant to transformation or integration of viral DNA in such a way that T antigen expression is prohibited.     

Genetic expression of human adenoviruses in simian cells. Evidence for interserotypic inhibition of viral DNA synthesis

Most simian cells are permissive for SV40 and adenovirus-SV40 hybrids but nonpermissive for human adenoviruses, and the defect has been shown to take place at the level of processing of late viral mRNAs (Klessig and Grodzicker, 1979). Viral DNA synthesis and virus progeny production were studied in simian cells infected with different adenovirus serotypes. Adenoviruses belonging to oncogenic subgroups A and B (Ad31 and Ad3) failed to replicate their DNA in CV1 cells, whereas DNA replication occurred for all the other serotypes. Co-infection of CV1 cells with SV40 and Ad3 (or Ad31) resulted in the inhibition of SV40 DNA synthesis, as well as cellular DNA synthesis. The inhibition was not related to adenovirus DNA replication, since SV40 did not complement the Ad3/Ad31 replication defective function. Similar results were obtained in coinfected BSC and MK2 simian cell lines. Inhibition of Ad2ND1 DNA synthesis and gene expression also occurred in co-infection of simian cells with nondefective Ad2ND1 hybrid and defective Ad3/Ad31. In permissive human cell lines (HeLa or KB) co-infected with Ad2 and Ad3 (or Ad31), a dominant, inhibitory effect of Ad3 (or Ad31) over Ad2 was also observed. The inhibition appeared to function stoichiometrically and not catalytically, and to involve early adenovirus gene products. In both simian and human cells a hierarchy of dominance appeared between serotypes belonging to different subgroups. The degree of inhibitory effect occurred in the following decreasing order: Ad3 and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad31 (A), Ad2 and Ad5 (C).     

Evolutionary variants of simian virus 40: replication and encapsidation of variant DNA.

The genomes of evolutionary variants of simian virus 40 (SV40) contain reiterations of DNA sequences derived from the origin of replication of viral DNA. To determine the role of such reiterated DNA segments in the evolution of variants, the relative rates of replication and the encapsidation efficiencies were determined for cloned variants with three, five, six, or nine copies of the SV40 origin sequence. The rate of variant DNA replication relative to that of the helper virus in the variant stock was an exponential function of the number of origin segments per genome. In contrast, encapsidation efficiency was not correlated with the number of origin sequences in the variant genome. We conclude that a major factor in the evolution of SV40 variants is the replicative advantage conferred by the reiteration of the origin of DNA replication.     

Biological and biophysical characterization of SV 40 cytoplasmic T-antigen-inducing mutants of PARA-adenovirus 7

Summary Mutants of PARA-adenovirus 7 which induce the synthesis of SV 40 T antigen in the cytoplasm of cells were characterized biologically and biophysically. The following properties were found to be similar to those of the parental virus which induces nuclear SV 40 T antigen: kinetics of replication in monkey kidney cells, intracellular site of particle morphogenesis, relative kinetics of inactivation by ultraviolet light, and buoyant densities of the virions and viral DNA. Transcapsidation of the mutant PARA particles to heterologous adenovirus serotypes resulted in populations which retained the ability to induce SV 40 T antigen in the cytoplasm of cells, indicating that the localization of the SV 40 T antigen was under the control of the defective SV 40 genome. It was also concluded that the cytoplasmic localization of SV 40 T antigen did not influence the enhancement of adenovirus replication in simian cells by SV 40 information. Attempts to detect an altered localization of cytoplasmic or nuclear SV 40 T antigen after coinfection of cells with different virus mixtures, by sequential harvests of infected cells, or after treatment with cycloheximide were not successful.     

Genome maps of simian virus 40 defectives propagated in human glioblastoma cells.

The DNA sequences present in four defective SV40 genomes propagated on human glioblastoma cells (A172) have been characterized by analysis of restriction enzyme digests and by hybridization to the wild-type genome. Like defectives grown on monkey cells, these molecules are 10 to 20% shorter than wild-type DNA and contain reiterations of specific segments of the nondefective genome, including multiple copies of the viral replication origin. However, unlike the monkey cell defectives, those grown on A172 cells have also retained multiple copies of the region of viral DNA around the BamHl site at map position 0.15. These defectives consist only of sequences derived from SV40 and contain no detectable host cell sequences.     

The isolation of defective variants of simian virus 40 whose genomes contain sequences derived from adenovirus 2 DNA.

A new set of hybrid viruses has been isolated whose closed circular genomes 5 to 6 kB in size, contain DNA sequences derived in part from adenoviruses 2 and in part from SV40. The structure of these genomes is complex, but in the simplest case, analyses by restriction endonuclease digestion and hybridization indicate that the adenovirus 2 DNA is present as a continuous block, of maximum size 2.8 kB. Different hybrids contain sequences derived from different segments of the adenovirus 2 genome.     

Complexity of EBV homologous DNA in continuous lymphoblastoid cell lines

The complexity of Epstein-Barr Virus (EBV) homologous DNA in 11 EBV-infected lymphoblastoid cell lines which have been passaged for several years in culture was determined by hybridization of lymphoblastoid cell DNA to DNA extracted from EBV purified from HR-1 cells and labeled in vitro. Five of the cell lines analyzed contained no early (EA) or viral capsid (VCA) antigens which have been associated with the replication of EBV. Two other cell lines contained EA but not VCA. Of the seven VCA-negative cell lines, those which contained some early antigen or in which early antigen could be induced with IUDR had 56, 48, and 25 copies per diploid cell genome of more than 90% of the sequences of EBV DNA. Five cell lines which did not contain EA even after induction had 23, 8, 8, 6, and 2 copies per cell of the EBV genome. The data indicate that there is a correlation between the number of copies of EBV DNA in nonpermissive cells and the ability of these cells to express EA. Three of the five EA and VCA negative nonpermissive cell lines contained DNA homologous to more than 90% of the sequences of EBV DNA. The kinetics of hybridization of the DNA of two other nonpermissive cell lines, Namalwa and SKL, which contain two and eight copies, respectively, of some EBV DNA sequences, suggest but do not prove that these cell lines may contain incomplete viral genomes. The retention of the full complexity of viral DNA in most nonpermissive lymphoblastoid cell lines may be related to the relatively large number of copies of the viral genome in these cells.     

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.     

Simian virus 40 specific cytotoxic lymphocyte clones localize two distinct TSTA sites on cells synthesizing a 48 kD SV40 T antigen

Simian virus 40 specific antigenic sites (TSTA) which react with SV40 specific cytotoxic lymphocytes (CTL) were localized on the surface of mouse embryo fibroblasts (H-2b) transformed with a recombinant plasmid which contain SV40 large T antigen coding DNA sequences (0.65-0.37 map units). These cells (B6/pSV-20-GV) synthesize a large T antigen polypeptide of 48 kD and could be lysed with two independently isolated CTL clones which recognize distinct antigenic sites on SV40 transformed cell surface. These results suggest that at least two distinct TSTA sites are present in cells synthesizing only the amino terminal half of SV40 T antigen.     

Real-time, quantitative PCR assays for the detection of virus-specific DNA in samples with mixed populations of polyomaviruses

Mixtures of polyomaviruses can be present in the central nervous system, the gastrointestinal tract, the genitourinary tract, blood, and urban sewage. We have developed 12 primer/probe sets (four per virus) for real-time, quantitative PCR assays (TaqMan) that can specifically detect BKV, JCV, and SV40 genomes present in mixtures of these viruses. The specificities of these primer/probe sets were determined by evaluating their level of interaction with the DNA from other polyomaviruses and their ability to estimate the number of copies of homologous viral DNA in blinded samples of defined mixtures of three polyomaviral DNAs. Three early region and three late region primer/probe sets determined, within a two-fold range, the number of copies of their respective DNAs. Four sets of SV40 primer/probes also detected 1.1-2.4 copies of SV40 DNA per COS-1 cell, cells estimated to contain a single copy of SV40 DNA. Three JCV primer/probe sets detected 3.7-4.2 copies per cell of JCV DNA in the JCV-transformed cell line M1-HR, cells estimated to contain between 0.5 and 1 copy of the JCV genome. We suggest that the virus-specific primer/probe sets in this study be considered sufficiently characterized to initiate the quantification of polyomavirus DNA in biological samples.     

Immortalization of xeroderma pigmentosum cells by simian virus 40 DNA having a defective origin of DNA replication

A simian virus 40 (SV40) DNA fragment, encompassing the whole early region and having a defective origin of DNA replication, has been used to transform human fibroblast cells derived from two xeroderma pigmentosum (XP) patients. Two of the SV40-transformed XP cell lines, belonging to complementation group C, had acquired the characteristic of indefinite life-span in culture. These XP cell lines synthesize T antigen as shown by immunofluorescence and retain the high sensitivity to UV irradiation. Detailed karyotype analysis shows very few chromosomal changes, while the transfecting SV40 DNA is integrated into cellular DNA sequences. These are the first immortalized XP cell lines derived from complementation group C. In view of the extreme difficulty in obtaining immortalized human fibroblasts, we suggest a possible advantage of replication defective SV40 DNA molecules for immortalizing human fibroblast cells of any source.     

Altered DNA binding and replication activities of JC virus T-antigen mutants.

Ten mutations were introduced into the JC virus (JCV) T antigen within a region corresponding to the SV40 T-antigen DNA binding domain (SV40 amino acids 131 to 220); nine of these increased homology between the two proteins in sequences critical for SV40 T antigen DNA binding. All mutant JCV T antigens bound to JCV and SV40 origins of DNA replication. Binding efficiency relative to the of wild-type JCV T antigen ranged from 83 to 301% for the JCV binding sites and from 44 to 240% for the SV40 binding sites. Nine mutant proteins promoted viral DNA replication in primary human fetal glial (PHFG) and CV-1 cells. In PHFG cells, promotion of DNA replication ranged from 26 to 220% relative to that of wild-type T antigen; in CV-1 cells it ranged from 14 to 522%. Coding sequences for five mutant proteins were transferred into the hybrid virus M1 (SV40) [M1(SV40) contains coding sequences from JCV and regulatory sequences from SV40]. Wild-type T antigen promoted replication weakly from the SV40 origin in these hybrid viruses in CV-1 cells (2% that from the JCV origin); replication driven by the mutant proteins ranged from 110 to 412% of that induced by the wild-type protein. Efficient specific DNA binding by a mutant T antigen was not a reliable indicator of that mutant protein's ability to promote DNA replication.     

Identification and biochemical analysis of DNA replication-defective large T antigens from SV40-transformed cells

Nine commonly studied Simian virus 40 (SV40)-transformed rodent cell lines were screened for tumor (T) antigens defective in SV40 DNA replication using a simple polyethylene glycol-mediated cell fusion assay. Each line contained a functional origin of SV40 DNA replication, as shown by fusion with Cos 1 cells. Fusion with uninfected monkey cells revealed that T antigens from two lines lacked detectable replicative activity, while T antigens from five other lines exhibited only very weak replicative activity. One line, and a tumor cell line derived from it, expressed T antigen with wild-type replication activity. Biochemical analysis of these proteins revealed defects in DNA binding activity and ATPase activity. One line expressed large T antigen defective in both activities. All of the lines contained complexes of T antigen with the cellular protein p53 and all of the T antigens exhibited nucleotide-binding activity. The results indicate that some of these lines may constitute a useful source of new replication-defective T antigens.