Nondefective Hybrid Research Articles

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.

Biosynthesis, Immunological Specificity, and Intracellular Distribution of the Simian Virus 40-Specific Protein Induced by the Nondefective Hybrid Ad2 + ND 1

Ad2(+)ND(1), a nondefective hybrid virus containing a segment of the early region of simian virus 40 (SV40) DNA covalently inserted into the human adenovirus 2 genome, enhances the growth of human adenoviruses in simian cells and induces the SV40 U antigen. This hybrid previously has been shown to code for a 28,000 (28K) molecular weight protein not present in wild-type adenovirus 2-infected cells. By radioimmunoprecipitation using sera from hamsters bearing SV40-specific tumors, we have established that the Ad2(+)ND(1)-induced 28K protein is SV40-specific. This Ad2(+)ND(1)-induced protein is synthesized as a 30K molecular weight precursor, which is detectable only when infected cells are pulse-labeled in the presence of the protease inhibitor tosylamino phenylethyl chloromethyl ketone. Upon fractionation of labeled cell extracts, about 80% of the 28K protein is found in the plasma membrane fraction, whereas the remaining 20% is associated with the outer nuclear membrane. This protein is not detectable either in the nucleus or in the cytoplasm. Blockage of proteolytic cleavage by tosylamino phenylethyl chloromethyl ketone did not alter the topographic distribution of this SV40-specific protein, although the amount of the precursor protein in the outer nuclear membrane increased fourfold while that in the plasma membrane was proportionately decreased. This result suggests that the 28K protein is transferred from the outer nuclear membrane to the plasma membrane after posttranslational cleavage of the 30K precursor polypeptide. These data offer further support to the proposal that the 28K protein contains the determinants for SV40 U antigen and is responsible for SV40 enhancement of adenovirus growth in simian cells.

The group C adenovirus tumor antigens: identification in infected and transformed cells and a peptide map analysis

Abstract Serum from hamsters bearing group C adenovirus-induced tumors can be divided into two classes: first, a broad spectrum serum that contains antibodies to several early adenovirus proteins, immunoprecipitated from virus-infected cell extracts, with molecular weights of 72,000, 58,000, 44,000 and 17,000 daltons; and second, a narrow spectrum serum that contains antibodies to the 58,000 dalton protein from virus-infected cell extracts. Both types of sera have been used to immunoprecipitate specifically the 58,000 dalton protein from a type 2 adenovirus-transformed hamster cell line and a type 2 adenovirus-SV40 nondefective hybrid (Ad2 + ND-1) transformed hamster cell line. In addition, the broad spectrum serum immunoprecipitates or co-precipitates a late adenovirus protein of 120,000 daltons from virus-infected, but not virus-transformed cells. Peptide maps of the 120,000 dalton antigen and the virus hexon structural protein (120,000 daltons) demonstrate that these proteins are closely related. The 72,000 dalton antigen has been shown to be the adenovirus single-strand-specific DNA binding protein. Peptide maps of this 72,000 dalton antigen demonstrate that it contains all the peptides found in the 44,000 dalton antigen. The 72,000 dalton antigen contains two additional peptide fragments not detected in the 44,000 dalton protein, indicating that this 44,000 dalton antigen is a proteolytic breakdown product of the 72,000 dalton protein. The 58,000 dalton adenovirus tumor antigen has a peptide map which is completely distinct from the 120,000, 72,000 and 44,000 dalton proteins. These data demonstrate that the 58,000 dalton antigen is chemically distinct from the 72,000–44,000 dalton early adenovirus proteins.

Adenovirus transcription. II. RNA sequences complementary to simian virus 40 and adenovirus 2DNA in AD2+ND1- and AD2+ND3-infected cells

The genomes of the two nondefective adenovirus 2/simian virus 40 (Ad2/SV 40) hybrid viruses, nondefective Ad2/SV 40 hybrid virus 1 (Ad2+ND1) and nondefective hybrid virus 3 (Ad2+ND3), WERE FORMED BY A DELETION OF ABOUT 5% OF Ad2 DNA and insertion of part of the SV40 genome. We have compared the cytoplasmic RNA synthesized during both the early and late stages of lytic infection of human cells by these hybrid viruses to that expressed in Ad2-infected and SV40-infected cells. Separated strands of the six fragments of 32P-labeled Ad2 DNA produced by cleavage with the restriction endonuclease EcoRI (isolated from Escherichia coli) and the four fragments of 32P-labeled SV40 DNA produced by cleavage with both a restriction nuclease isolated from Haemophilus parainfluenzae, Hpa1, and EcoRI were prepared by electrophoresis of denatured DNA in agarose gels. The fraction of each fragment strand expressed as cytoplasmic RNA was determined by annealing fragmented 32P-labeled strands to an excess of cellular RNA extracted from infected cells. The segment of Ad2 DNA deleted from both hybrid virus genomes is transcribed into cytoplasmic mRNA during the early phase of Ad2 infection. Hence, we suggest that Ad2 codes for at least one "early" gene product which is nonessential for virus growth in cell culture. In both early Ad2+ND1 and Ad2+ND3-infected cells, 1,000 bases of Ad2 DNA adjacent to the integrated SV40 sequences are expressed as cytoplasmic RNA but are not similarly expressed in early Ad2-infected cells. The 3' termini of this early hybrid virus RNA maps in the vicinity of 0.18 on the conventional SV40 map and probably terminates at the same position as early lytic SV40 cytoplasmic RNA. Therefore, the base sequence in this region of SV40 DNA specifies the 3' termini of early messenger RNA present in both hybrid virus and SV40-infected cells.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. Transformation of hamster kidney cells by adenovirus 2 and the nondefective hybrid viruses.

Nonhybrid adenovirus 2 (Ad2) and five nondefective Ad2-simian virus 40 (SV40) hybrids (Ad2(+)ND(1)-Ad2(+)ND(5)) failed to produce tumors when injected into newborn hamsters. However, each of these agents transformed hamster kidney cells in vitro, and the transformed cell lines produced tumors when injected into suckling hamsters. Foci of cells transformed by the hybrids could not be distinguished from foci of cells transformed by nonhybrid Ad2. Histopathologically, tumors induced by the nondefective hybrid-transformed lines were of the adenovirus type with no areas of SV40-type fibrosarcoma. All of the cell lines studied contained Ad2 T antigen and Ad2 RNA. Only the B55HK(1) line contained the three early SV40 antigens, U, TSTA, and T. The ND(4)HK(1) line contained SV40 T and TSTA antigens (the latter antigen being detected only after 48 to 49 tissue culture passages) and SV40 RNA. The ND(2)HK(2) line contained SV40 RNA but no detectable SV40 antigens; the other hybrid-transformed lines contained no SV40 RNA. The transformation of hamster cells by the nondefective Ad2-SV40 hybrids seemed to be caused by the Ad2 genome and could not be attributed to the regions of the SV40 genome contained within the hybrids. Additionally, these results indicate that interactions between the genomes of the nondefective hybrid viruses and the hamster cells they transform are complex and could involve the site of integration, the size of the incorporated viral genome, the transcription or translation of the viral genome, or various combinations of these processes.

Studies of Hamster Cells Transformed by Adenovirus 2 and the Nondefective Ad2-SV40 Hybrids

Adenovirus 2 does not produce tumors when injected into newborn hamsters, and this failure is not corrected by the acquisition of the portion of the SV40 genome present in any of the nondefective Ad2-SV40 hybrid viruses. However, nonhybrid Ad2 will transform hamster cells in vitro, and these transformed cells will produce tumors when injected into newborn hamsters. This finding, taken in conjunction with other studies (Graham et al., this volume; McAllister et al., 1969; McDougall et al., this volume; Williams 1973), suggests that the categorization of Ad2 and Ad5 as "nononcogenic" viruses is a reflection of host response rather than an intrinsic property of the viruses. However, transformation of hamster cells by Ad2 is inefficient, requiring 10(7)-10(8) PFU to produce a focus of transformed cells. Transformation of hamster kidney cells by the nondefective hybrids cannot be associated with portions of the SV40 genome. During the transformation process, complex interactions must occur between the hamster cell genome and the viral genome of Ad2 or the nondefective hybrids. Thus while it appears that there is very little Ad2 DNA in one nonhybrid Ad2-transformed cell line, there is a larger amount of Ad2 DNA in all of the hybrid virus-transformed cell lines. Moreover, while one Ad2+ND2-transformed line has apparently lost the SV40 portion of the viral genome, it is present and transcribed in another line transformed by the same virus. Finally, in the lines of nondefective hybrid-transformed cells examined thus far, there is extensive transcription from the Ad2 H strand. This pattern of transcription differs from transcription of the viral genome in most hybrid Ad2-transformed hamster and rat cells in which L-strand transcripion predominates.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. VI. Characterization of the DNA from five nondefective hybrid viruses.

Certain biophysical characteristics of the DNA from each of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND(1), Ad2(+)ND(2), Ad2(+)ND(3), Ad2(+)ND(4), Ad2(+)ND(5)) have been determined. The guanine plus cytosine content varied from 55 to 57% and was not significantly different from that of nonhybrid Ad2 (56%), and the hybrid DNA molecules had mean molecular lengths which were similar to that of the standard, Ad2. The Ad2 and SV40 components of each hybrid were linked by alkali-resistant, presumably covalent bonds. The percentage of SV40 DNA in each hybrid virus was determined by hybridization with SV40 complementary RNA in a calibrated system. The results indicate that each hybrid virus DNA contains a different percentage of SV40 nucleotide sequences. The estimated size of the SV40 DNA component varies from 48,000 daltons for Ad2(+)ND(3) to 840,000 daltons for Ad2(+)ND(4), the latter being equivalent to between one-fourth and one-third of the SV40 genome.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. VII. Characterization of the simian virus 40 RNA species induced by five nondefective hybrid viruses.

Five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated and found to contain segments of SV40 DNA covalently linked to Ad2 DNA. The quantity of SV40 DNA present is a stable characteristic of each hybrid virus, and varies from less than 5% (in Ad2(+)ND(3)) to more than 30% (in Ad2(+)ND(4)) of the SV40 genome. We have characterized the SV40 portions of these hybrids by relating the SV40-specific RNA sequences transcribed in cells infected with each hybrid virus to those transcribed in cells infected with each of the other hybrid viruses and with SV40 itself. RNA-DNA hybridization-competition experiments indicate that the number of unique SV40 RNA sequences transcribed in infected cells is proportional to the size of the SV40 DNA segment contained within each hybrid and, in the case of the three hybrids which induce detectable SV40-specific antigens, to the number of SV40 antigens induced. Furthermore, the SV40-specific RNA sequences transcribed from any one of the hybrids are completely represented in the RNA transcribed from all other hybrids with longer SV40 segments. Thus, the SV40 DNA regions in the five hybrid viruses appear to contain some nucleotide sequences in common. The SV40-specific RNA transcribed from Ad2(+)ND(4), the hybrid containing the largest SV40 segment, is qualitatively similar to the SV40-specific RNA transcribed early (i.e., prior to viral DNA replication) in SV40 lytic infection. Thus, it appears that no significant amount of late SV40 DNA is transcribed during infection by any of the five nondefective Ad2-SV40 hybrid viruses.

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. IV. Characterization of the simian virus 40 ribonucleic acid species induced by wild-type simian virus 40 and by the nondefective hybrid virus, Ad2 + ND 1 .

Ad2(+)ND(1), a nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, has been previously shown to contain a small segment of the SV40 genome covalently linked to Ad2 deoxyribonucleic acid (DNA). The SV40 portion of this hybrid virus has been characterized by relating the SV40-specific ribonucleic acid (RNA) sequences transcribed from the Ad2(+)ND(1) DNA to those transcribed from the DNA of SV40 itself. RNA-DNA hybridization-competition studies indicate that the SV40 component of Ad2(+)ND(1) consists of some, but not all, of that part of the SV40 genome which is transcribed early, i.e., prior to viral DNA replication, in SV40 lytic infection.

A nondefective (competent) adenovirus-SV40 hybrid isolated from the AD.2-SV40 hybrid population.

A new nondefective hybrid virus has been plaque-isolated from the Ad.2-SV40 hybrid population. This virus replicates efficiently with one-hit kinetics in both human embryonic kidney and African green monkey kidney cells, induces an SV40 specific antigen which is detectable by immunofluorescence and complement-fixation using sera from SV40 tumor-bearing hamsters, and produces SV40-specific RNA detectable by DNA-RNA hybridization. The SV40-specific antigen induced by this virus is heat-stable, sensitive to inhibitors of DNA synthesis, serologically different from SV40 T and viral antigens, and is an unrecognized SV40 antigen.