Transformation of rat cells by cyt mutants of adenovirus type 12 and mutants of adenovirus type 5.

Several mutants with much reduced oncogenicity (spontaneous mutants H12 cyt 52 and H12 cyt 70 and UV-induced mutants H12 cyt 61, H12 cyt 62, and H12 cyt 68) of the highly oncogenic adenovirus type 12 (Ad12) were studied for their ability to transform primary baby rat kidney cells. Four of the mutants showed much reduced capacity to transform cells in vitro, while H12 cyt 61 transformed cells as efficiently as the wild-type virus. Viral gene expression in several cell lines established from cultures infected by cyt mutants was studied, and it was found that viral sequences belonging to the left 16% of Ad12 were always transcribed. These results suggest that the function of the transformed state is not defective in the cyt mutants studied. Heterotypic complementation studies showed that the defect(s) in a cyt mutant can be corrected by an Ad7 function. Ad5 dl 313, with a deletion between 3.5 and 10.5 map units, transformed rat cells only at high multiplicity. These results suggest that the region E1B of adenoviruses may be required for efficient transformation of rat cells.     

Transcription of the genome of adenovirus type 12. III. Maps of stable RNA from productively infected human cells and abortively infected and transformed hamster cells.

The adenovirus type 12-specific mRNA and the stable nuclear RNA from productively infected KB cells, early postinfection, from abortively infected BHK-21 cells, and from the adenovirus type 12-transformed hamster lines T637 and HA12/7 have been mapped on the genome of adenovirus type 12. The intact separated heavy (H) and light (L) strands of adenovirus type 12 DNA have been used to determine the extent of complementarity of the mRNA or nuclear RNA from different cell lines to each of the strands. More precise map positions have been obtained by the use of the H and L complements of the fragments of adenovirus type 12 DNA which were produced with the EcoRI and BamHI restriction endonucleases. The results of the mapping experiments demonstrate that the mRNA's isolated early from productively and abortively infected and from two lines of transformed cells are derived from the same or similar regions of the adenovirus type 12 genome. The map positions on the adenovirus type 12 genome for the mRNA from the cell lines as indicated correspond to regions located approximately between 0 and 0.1 and 0.74 and 0.88 fractional length units on the L strand and to regions between 0.63 and 0.74 and 0.89 and 1.0 fractional length units on the H strand. The HA12/7 line lacks mRNA complementary to the region between 0.74 and 0.88 fractional length units on the L strand. Similar data are found for the nuclear RNA, except that the regions transcribed are more extensive than those observed in mRNA. The polarity of the H strand has its 3'-end on the right terminus in the EcoRI A fragment, and the L strand has its 3'-end on the left terminus in the EcoRI C fragment. Thus, the H strand is transcribed from right to left (1 = leftward strand); and the L strand is transcribed from left to right (r = rightward strand). The designations H and L refer to the relative heavy and light densities of the two strands in polyuridylic-polyguanylic acid-CsCl density gradients. The EcoRI C-H and D-H complements have been shown to be part of the intact L strand; thus, there is a "reversal in heaviness" on the left terminus of the viral DNA.     

Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines.

Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.     

Complementation of adenovirus type 5 host range mutants by adenovirus type 12 in coinfected HeLa and BHK-21 cells.

We have studied the ability of adenovirus type 12 (Ad12) to complement the Ad5 transformation-defective host rang (hr) mutants during infection of human cells (HeLa) or hamster cells (BHK-21). The group I mutant hr3 (mapped within 1.3 to 3.7 map units), which is incapable of synthesizing viral DNA, was complemented for both DNA synthesis and infectious virus production in nonpermissive HeLa cells during coinfection with Ad12. Similarly, the group II mutant hr6 (6.1 to 9.4 map units), which does synthesize DNA, was also shown to be complemented for virus production. When the host cells were BHK-21, an established hamster cell line that is permissive for Ad5 but nonpermissive for Ad12 DNA synthesis and virus production, coinfection with Ad5 and Ad12 did not overcome the block to Ad12 DNA synthesis. Coinfection of BHK-21 cells with Ad12 and either hr3 or hr6 leads to the complementation of only the group I mutant (hr3). The inability of Ad12 to complement hr6 in BHK-21 cells may be due to the failure of Ad12 to express an early gene product from the region corresponding to early region 1B (4.5 to 11 map units) Ad5 where hr6 and the other group II mutations are located.     

Viral gene products in adenovirus type-2 transformed hamster cells.

I have analyzed viral gene products expressed in five adenovirus type 2 (Ad2)- cytoplasmic, viral RNA which was selected by hybridization to cloned restriction endonuclease fragments of Ad2 DNA. Proteins synthesized in vitro were analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and compared with those directed by RNAs prepared from productively infected cells. The early regions E1 and E4 of adenovirus type 2 (Ad2) were found to be expressed in all of five Ad2-transformed hamster embryo cells lines. RNA transcribed from early region E2, which codes for the 72,000-molecular-weight (72K) DNA-binding protein was detected in cell line HE1 only, and early region E3 was expressed exclusively in cell line HE4. RNA transcribed from the region between approximately 12 and 35 map units, coding for immediate early (13.5K, 52/53K) and immediate early proteins (13.6K, 16K, 17K, 87K), as well as RNA from late genes, was not found in any of the cell lines HE1 to HE5 had electrophoretic mobilities similar to those programmed by RNA from productively infected cells.     

The unmethylated state of the promoter/leader and 5'-regions of integrated adenovirus genes correlates with gene expression.

An inverse correlation has been established between the levels of DNA methylation at 5'-CCGG-3' (MspI/HpaII) sites in specific genes of integrated viral DNA in adenovirus type 12 (Ad12)-transformed hamster cell lines and the extent to which these genes are expressed ( Sutter and Doerfler , 1979, 1980). In general, early genes are transcribed into mRNA, while late genes are permanently switched off in these cell lines. Adenovirus type 2 genes methylated in vitro at 5'-CCGG-3' sites are not transcribed upon microinjection into nuclei of Xenopus laevis oocytes - unmethylated genes are expressed ( Vardimon et al., 1982a ). The MspI sites in the early and in some of the late Ad12 genes in cell lines HA12 /7, T637 , and A2497 -3 have now been precisely mapped. The data presented here reveal that the promoter/leader and 5'-regions of the early genes are unmethylated both at MspI sites and at 5'-GCGC-3' (HhaI) sites. In some instances, e.g., in the E2a regions in all three lines, the main parts of the early genes are partly methylated, even though the genes are expressed. In cell line HA12 /7, the early region E3 is not expressed, and the promoter/leader and 5'-regions of this segment are fully methylated. All late regions are completely methylated. The results suggest that the state of methylation in the promoter/leader and 5'-regions of integrated adenovirus genes is important in the control of gene expression.     

Physical organization of subgroup B human adenovirus genomes.

Cleavage sites of nine bacterial restriction endonucleases were mapped in the DNA of adenovirus type 3 (Ad3) and Ad7, representative serotypes of the "weakly oncogenic" subgroup B human adenoviruses. Of 94 sites mapped, 82 were common to both serotypes, in accord with the high overall sequence homology of DNA among members of the same subgroups. Of the sites in Ad3 and Ad7 DNA, fewer than 20% corresponded to mapped restriction sites in the DNA of Ad2 or Ad5. The latter serotypes represent the "nononcogenic" subgroup C, having only 10 to 20% overall sequence homology with the DNA of subgroup B adenoviruses. Hybridization mapping of viral mRNA from Ad7-infected cells resulted in a complex physical map that was nearly identical to the map of early and late gene clusters in Ad2 DNA. Thus the DNA sequences of human adenoviruses of subgroups B and C have significantly diverged in the course of viral evolution, but the complex organization of the adenovirus genome has been rigidly conserved.     

Establishment and characterization of hamster cell lines transformed by restriction endonuclease fragments of adenovirus 5.

We have established a library of hamster cells transformed by adenovirus 5 DNA fragments comprising all (XhoI-C, 0 to 16 map units) or only a part (HindIII-G, 0 to 7.8 map units) of early region 1 (E1: 0 to 11.2 map units). These lines have been analyzed in terms of content of viral DNA, expression of E1 antigens, and capacity to induce tumors in hamsters. All cells tested were found to express up to eight proteins encoded within E1A (0 to 4.5 map units) with apparent molecular weights between 52,000 (52K) and 25K. Both G and C fragment-transformed lines expressed a 19K antigen encoded within E1B (4.5 to 11.2 map units), whereas an E1B 58K protein was detected in C fragment-transformed, but not G-fragment-transformed, lines. No clear distinction could be drawn between cells transformed by HindIII-G and by XhoI-C in terms of morphology or tumorigenicity, suggesting that the E1B 58K antigen plays no major role in the maintenance of oncogenic transformation, although possible involvement of truncated forms of 58K cannot be ruled out. Sera were collected from tumor-bearing animals and examined for ability to immunoprecipitate proteins from infected cells. The relative avidity of sera for different proteins was characteristic of the cell line used for tumor induction, and the specificity generally reflected the array of viral proteins expressed by the corresponding transformed cells. However, one notable observation was that even though all transformed lines examined expressed antigens encoded by both the 1.1- and 0.9-kilobase mRNAs transcribed from E1A, tumor sera made against these lines only precipitated products of the 1.1-kilobase message. Thus, two families of E1A proteins, highly related in terms of primary amino acid sequence, appear to be immunologically quite distinct.     

Isolation of a nondefective recombinant between adenovirus type 5 and early region 1A of adenovirus type 12.

A nondefective recombinant between adenovirus type 5 (Ad5) and type 12 (Ad12), rc-1 (Ad5 dl312, carrying the Ad12 E1A gene), was isolated from hamster cell foci transformed by a defective recombinant, rcB-1 (dl312, carrying the Ad12 E1 gene). The recombinant rc-1 grew in human embryo kidney and KB cells in the absence of helper and synthesized Ad12 T antigen g, the product of the E1A gene. The genome of rc-1 has a deletion between 79.9 and 82.5 map units of Ad5 dl312 DNA with an insertion of 0.1 to 5.5 map units of Ad12 DNA at the deletion site. The mRNAs of Ad12 E1A were transcribed from the Ad12 E1A promoter, and unusual RNAs were abundantly transcribed from the Ad5 E3 promoter on the opposite strand. The frequency of cell transformation with rc-1 was lower than those with Ad5 and Ad12 wild types.     

Mappings of adenovirus type 7 cytoplasmic RNA species synthesized early in lytically infected cells and synthesized in transformed cells.

Early virus-specific RNA synthesized in KB cells infected with adenovirus type 7 and virus-specific RNA synthesized in rat embryo cells (71JY1-2) transformed by the adenovirus type 7 HindIII-I.J fragment (left-hand 8.1% of the viral genome) have been mapped on the viral genome. About 25% of the viral genome, four discrete regions, two on each strand of the viral genome, are expressed as "early" mRNA. Almost similar regions in the left-hand 8.1% of the viral genome are transcribed both in KB cells at early times after infection and in 71JY1-2 cells.     

Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome.

We have isolated as recombinant DNA clones, in the plasmid pBR322, regions of the herpesvirus type 1 genome spanning the region between 0.53 and 0.6 on the prototypical arrangement. This 11,000-base-pair region corresponds to 10% of the large unique region and encodes five major and several minor mRNA species abundant at different times after infection, which range in length from 7 to 1 kilobase. In this report, we have used RNA transfer blots and S1 nuclease digestion of hybrids between viral DNA and polyribosomal RNA to precisely localize (+/- 0.1 kilobase) these mRNA's. Comparison of neutral and alkaline gels of S1 nuclease-digested hybrids indicates no internal introns in the coding sequences of these mRNA's, although noncontiguous leader sequences near (ca. 0.1 kilobase) the 5' ends of any or all mRNA's could not be excluded. The 5' ends of several late mRNA's that are encoded opposite DNA strands map very close to one another, and the 3' ends of a major late and a major early mRNA, which are partially colinear, terminate in the same region. In vitro translation of the viral mRNA's isolated by hybridization with DNA bound to cellulose and fractionation of mRNA species on denaturing agarose gels allowed us to assign specific polypeptide products to each of the mRNA's characterized. Among other results, it was demonstrated unequivocally that two major late mRNA's, which partially overlap, encode the same polypeptide.     

Structure of viral DNA in a rat cell line transformed by the cloned EcoRI-C fragment of adenovirus 12.

A DNA segment carrying viral DNA was cloned from a rat cell line transformed by the cloned EcoRI-C fragment (0 to 16.4 map units) of human adenovirus type 12(Ad12), and the viral sequence in the clone was analysed. The cloned segment contained the region from nucleotide positions 118 to 3520 of the Ad12 genome in the middle. No unique structure was found at the viral and non-viral DNA junctions. When examined the transforming activity, the conserved viral sequence was able to transform rat 3Y1 cells efficiently. Southern blotting analysis of the viral sequence in five re-transformed cell lines showed that the viral sequence was inserted at different sites of cellular DNA. These results indicate that (I) the Ad12 DNA moiety from the enhancer-promoter region of the E1A gene to the end of the E1B gene contains enough information for efficient transformation of the rat cell, and (II) integration of the viral sequence at unique cellular sites is not prerequisite for transformation.