Replication in simian cells of defective viruses in an SV40-adenovirus "hybrid" population

Butel, Janet S. (Baylor University College of Medicine, Houston, Tex.), and Fred Rapp. Replication in simian cells of defective viruses in an SV40-adenovirus "hybrid" population. J. Bacteriol. 91:278-284. 1966.-An SV40-adenovirus type 7 "hybrid" virus population, previously shown to contain two viruses capable of complementation in green monkey kidney (GMK) cells, has a growth cycle in GMK cells similar to that of adenovirus type 7 in the presence of SV40. Extending previous preliminary results, the addition of adenovirus types 2, 7, or 12 to monolayers of GMK cells enhanced plaque formation by the SV40-adenovirus hybrid by as much as 200-fold. The terminal enhanced plaques, initiated by the hybrid in the presence of helper adenovirus, were found to contain progeny which could induce the synthesis of SV40 tumor antigen but which were coated with the protein of the helper adenovirus, type 2, 7, or 12, respectively. The particle carrying the SV40 tumor antigen determinant, named PARA, is defective in that it cannot direct the synthesis of capsid protein; information for the coat for PARA is supplied by the adenovirus. One-step growth curves of the hybrid virus population in monkey cells revealed that synthesis of both types of particles, adenovirus and PARA, proceeds at a similar rate, with a latent period of 16 to 20 hr being followed by an exponential increase in titer during the following 20 hr. Maximal titers for both particles were obtained 48 hr after inoculation of the cultures. Neither the PARA nor the adenovirus component replicated in GMK cells in the absence of the other.     

Variation in Properties of Plaque Progeny of PARA (Defective Simian Papovavirus 40)-Adenovirus 7

One hundred and twelve progeny from double plaque-purified clones were derived from the original PARA (defective simian papovavirus 40)-adenovirus 7 population. These progeny were found to differ in their oncogenic potential in newborn hamsters with progeny from 20 clones not inducing any tumors during 1 year of observation. The varying tumorigenicity of the individual clonal progeny was not related to the titer of PARA (particle aiding replication of adenovirus) in the inoculum. There was a perfect correlation between the tumor antigen content of the tumor cells and the antibody response of the tumor-bearing host. The tumors containing both adenovirus and simian papovavirus 40 (SV40) tumor antigens appeared earlier than those carrying only SV40 tumor antigen. Progeny from clones which induced mixed tumors also produced tumors which contained only SV40 tumor antigen. Three variants of PARA were isolated which induced the synthesis of SV40 tumor antigen in the cytoplasm of infected simian cells; all other clones yielded progeny which induced synthesis of SV40 tumor antigen in the nucleus.     

Detection of biologically active adenovirions unable to plaque in human cells

Butel, Janet S. (Baylor University College of Medicine, Houston, Tex.), Joseph L. Melnick, and Fred Rapp. Detection of biologically active adenovirions unable to plaque in human cells. J. Bacteriol. 92:433-438. 1966.-Plaque formation in green monkey kidney (GMK) cells by a defective simian virus 40-adenovirus 7 "hybrid" population (PARA-adenovirus 7) was enhanced by the addition of excess adenovirions. Adenovirus types 2, 7, and 12 were capable of providing enhancement, although none of these viruses gives rise to plaques in simian cells in the absence of PARA (particle aiding replication of adenovirus). Near maximal enhancement of the PARA plaque titer on simian cells was obtained with input multiplicities ranging from 0.02 to 0.14 plaque-forming units (PFU) of helper adenovirus per GMK cell. The PFU of helper adenoviruses tested (types 2, 7, and 12) were measured in the most sensitive assay system, human kidney cells. This input corresponded to three to nine helper virus particles per GMK cell. The majority of particles capable of enhancing plaque formation by PARA banded at a density of 1.34 in CsCl. Adenoviruses inactivated by heat or ultraviolet light were not capable of enhancing plaque formation by PARA. Highest titers were obtained when PARA and helper adenovirus were inoculated simultaneously. Inoculation of the helper adenovirus 24 hr prior to the inoculation of PARA resulted in the formation of only 50% as many plaques, and no enhanced plaques developed when the adenovirus preceded PARA by 48 hr. Conversely, the addition of adenovirus 48 hr after the inoculation of PARA initiated 56% as many plaques as simultaneous inoculation; 4% of the enhanced plaques still formed when helper virus was added as late as 5 days after inoculation of PARA. These results suggest that adenovirus particles unable to plaque on human or monkey kidney cells are nevertheless capable of interacting with PARA in simian cells, thereby facilitating replication of both particles.     

Immunofluorescence of green monkey kidney cells infected with adenovirus 12 and with adenovirus 12 plus simian virus 40

Malmgren, Richard A. (National Cancer Institute; Bethesda, Md.), Alan S. Rabson, Paula G. Carney, and Frances J. Paul. Immunofluorescence of green monkey kidney cells infected with adenovirus 12 and with adenovirus 12 plus simian virus 40. J. Bacteriol. 91:262-265. 1966.-Immunofluorescence studies of the viral antigens and tumor (T) antigens of adenovirus 12 and simian virus 40 (SV40) in green monkey kidney (GMK) cells infected with adenovirus 12 alone or in combination with the SV40 virus showed that the adenovirus 12 viral antigen was produced in detectable amounts only in the cells infected with both viruses. The adenovirus 12 T antigen, on the other hand, was formed in the GMK cells infected with the adenovirus 12 only. This antigen was formed as early as 18 hr after viral infection, and persisted for at least 48 hr after virus infection. There was a correlation between the appearance of the immunofluorescent T antigen in the nucleus and the electron microscope appearance of "nuclear stippling," which developed in the nuclei of GMK cells after infection with adenovirus 12 only, as well as after infection with both viruses.     

Quantitative Characteristics of the Transformation of Hamster Cells by PARA (Defective Simian Virus 40)-Adenovirus 7

An in vitro method for the quantitative measurement of transformation in hamster embryo fibroblasts by the PARA [defective simian virus 40 (SV40)]-adenovirus 7 hybrid has been developed. Transformation by PARA particles followed one-hit kinetics with a ratio of 1 focus-forming unit per 250 plaque-forming units. The method of viral adsorption had a direct effect upon the total number of foci which developed but not on the quantitative aspects of this assay. A fluorescent-focus assay was developed which provided a direct correlation of the observed morphological transformation and the presence of the PARA genome. This fluorescent-focus assay utilized detection of the SV40 tumor antigen, which was present in all foci transformed by PARA. Single foci induced by PARA were isolated and grown into cell lines. Two types of foci were observed and isolated; the first contained cells having a cuboidal or SV40-type morphology, and the second consisted of epithelial or adenovirus-type transformed cells. Both types contained the SV40 tumor and SV40 surface antigens as determined by the indirect fluorescence technique; however, only the epithelial cells contained the adenovirus 7 tumor antigen. All five cell lines which were injected into weanling Syrian hamsters were found to be oncogenic. These cell lines induced antibodies to both SV40 and adenovirus 7 tumor antigens in tumor-bearing animals.     

Interaction of a simian papovavirus and adenoviruses. I. Induction of adenovirus tumor antigen during abortive infection of simian cells

Feldman, Lawrence A. (Baylor University College of Medicine, Houston, Tex.), Janet S. Butel, and Fred Rapp. Interaction of a simian papovavirus and adenoviruses. I. Induction of adenovirus tumor antigen during abortive infection of simian cells. J. Bacteriol. 91:813-818. 1966.-Adenovirus types 2, 7, and 12 undergo an abortive growth cycle in green monkey kidney cells; they induce the formation of adenovirus tumor antigen, but synthesis of adeno capsid antigen and infectious adenovirus was observed only when cultures were concomitantly infected with a simian papovavirus (SV40). Several other viruses, including herpes simplex and measles which replicate in monkey cells, and rabbit papilloma and human wart papovaviruses which do not, failed to stimulate adenovirus replication in the monkey cells. Adenovirus tumor antigen was detected 8 to 10 hr postinfection by immunofluorescent techniques. The antigen induced by adenovirus types 2 and 7 appeared as intranuclear masses; adenovirus type 12 tumor antigen also appeared as cytoplasmic and nuclear flecks. Sera from hamsters bearing tumors induced by adenovirus type 12 cross-reacted with tumor antigens induced by types 2 and 7 but not with antigens induced by SV40.     

Human Papovavirus, BK Strain: Biological Studies Including Antigenic Relationship to Simian Virus 40

Some of the properties of a new human papovavirus, BK, have been examined. Host range studies of BK virus (BKV) showed human cells to be more sensitive to infection than monkey cells; human fetal brain cells appear to be highly sensitive to BKV, with the production of extensive cytopathology characterized by cytoplasmic vacuolization. The hemagglutinin of BKV is associated with the virion and is resistant to ether or heating at 56 C for 30 min. Fluorescent antibody as well as neutralization tests indicated antigenic similarities between simian virus 40 (SV40) and BKV. Cells undergoing lytic infection with BKV synthesized intranuclear T antigen(s) which reacted with SV40 T antibody demonstrable by immunofluorescence. However, BKV did not appear to induce SV40 transplantation antigens in transplantation-resistance tests. Evidence was obtained that BKV was present in humans prior to the widespread use of polio vaccines, thus ruling out the possibility that BKV is an SV40-related monkey virus, introduced into the human population by accidental contamination of poliovirus vaccines.     

Transformation of precrisis human cells by the simian virus 40 cytoplasmic-localization mutant pSVCT3 is accompanied by nuclear T antigen.

pSVCT3 is a cytoplasmic-localization mutant of simian virus 40 (SV40) isolated from the SV40 adenovirus 7 hybrid virus (PARA) and cloned into plasmid PBR. The large T antigen of pSVCT3 accumulates in the cytoplasm of infected monkey cells instead of being transported to the nucleus. The sole change in CT3 large T antigen is amino acid residue 128 (Lys----Asn). Transformation of precrisis rodent cells by pSVCT3 is negligible, whereas the frequency of transformation of established rodent cell lines by pSVCT3 is comparable to that of wild-type SV40. According to the model, in which transformation of precrisis cells involves the combined oncogenic action of both nuclear and cytoplasmic gene products, we predicted that pSVCT3 would localize in the cytoplasm of human cells and would therefore at most only partially and rarely transform precrisis human cells. We have found that pSVCT3 is able to transform precrisis human cells at high frequency. Furthermore, pSVCT3-transformed human precrisis cells relocalized T antigen to their nuclei. The relocalization of large T antigen was not dependent on cell growth. Wild-type and pSVCT3-transformed human cell lines both have about five copies of integrated SV40 DNA. SV40 virus-specific proteins, including the 100,000-molecular-weight super large T antigen, were expressed in pSVCT3-transformed human cells. Our results suggest that molecules in precrisis human cells, but not cells of other species, are able to complement the cytoplasmic-localization defect of the CT3 mutant large T antigen.     

Isolation of Two Plaque Variants from the Adenovirus Type 2-Simian Virus 40 Hybrid Population Which Differ in Their Efficiency in Yielding Simian Virus 40

Studies on the adenovirus type 2-simian virus 40 (SV40) hybrid population demonstrated two genetically stable variants within this population, which were isolated by plaquing in African green monkey kidney cells. These variants were similar in that each induced SV40 T antigen in human embryonic kidney cells and contained similar concentrations of nonhybrid adenovirus type 2 virions and adenovirus-encapsidated particles containing the infectious SV40 genome. These variants differed markedly, however, in their ability to produce SV40 viral antigen in human embryonic kidney cells and the efficiency with which they produce SV40 plaques in monkey cell monolayers. It is postulated that the differences in SV40-yielding efficiency between these variants lie in the nature of the recombinant deoxyribonucleic acid composing the genome of the hybrid particles.     

Isolation of Simian Virus 40 Recombinants from Cells Infected with Oligomeric Forms of Simian Virus 40 Deoxyribonucleic Acid

Oligomeric forms of simian virus 40 (SV40) deoxyribonucleic acid (DNA) were isolated from monkey kidney cells infected with two plaque morphology mutants of SV40. Recombinant, large clear-plaque-type SV40 was produced in cells productively infected with oligomeric forms of SV40 DNA.     

Conditional Lethal Mutants of Adenovirus 2-Simian Virus 40 Hybrids I. Host Range Mutants of Ad2+ND1

Human adenovirus type 2 (Ad2) grows poorly in monkey cells, although this defect can be overcome by co-infection with simian virus 40 (SV40). The nondefective Ad2-SV40 hybrid virus, Ad2(+)ND1, replicates efficiently in both human and African green monkey kidney cells, presumably due to the insertion of SV40 sequences into the Ad2 DNA. Several mutants of Ad2(+)ND1 have been isolated that grow and plaque poorly in monkey cells, although they retain the ability to replicate and plaque efficiently in HeLa cells. One of these mutants (H39) has been examined in detail. Studies comparing the DNA of the mutant with Ad2(+)ND1 either by the cleavage patterns produced by Escherichia coli R.RI restriction endonuclease digestion or by heteroduplexing reveal no differences. The pattern of protein synthesis of Ad2(+)ND1 and H39 in monkey cells is quite different with the mutant resembling Ad2, which is defective in the synthesis of late proteins. However, in human cells, the proteins synthesized by H39 and the parent Ad2(+)ND1 are very similar. The production of SV40 U antigen in H39-infected cells is different from that in Ad2(+)ND1-infected cells. Finally, the growth of H39 in monkey cells can be complemented by SV40.     

Identification of the Simian Virus 40 Which Replicates When Simian Virus 40-Transformed Human Cells Are Fused with Simian Virus 40-Transformed Mouse Cells or Superinfected with Simian Virus 40 Deoxyribonucleic Acid

Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.     

Effect of Ultraviolet Irradiation on the Survival of Simian Virus 40 Functions in Human and Mouse Cells

The relative sensitivities to ultraviolet light of various simian virus 40 (SV40) functions were studied in human and mouse cells. Transformation appeared to be less ultraviolet (UV)-sensitive than either T or V antigen when all functions were compared in the same cell. However, the time course of both T- and V-antigen appearance was delayed with UV-irradiated virus, so that the survival curves of these functions changed with time. Mouse and human cells which were transformed by UV-SV40 all contained SV40 T antigen. Infectious virus could be recovered from many more transformants than would be expected from the infectivity in African green monkey kidney cells of the irradiated virus. The results suggest that human and mouse cells are capable of reactivating UV-damaged SV40.     

Differential phosphorylation of cytoplasmic and nuclear variants of simian virus 40 large T antigen encoded by simian virus 40-adenovirus 7 hybrid viruses.

The phosphorylation patterns of cytoplasmic and nuclear forms of simian virus 40 large T antigen encoded by simian virus 40-adenovirus 7 hybrid viruses were analyzed by two-dimensional peptide mapping. The PARA(cT) mutant which encodes a large T antigen defective for nuclear transport was used as source for cytoplasmic large T antigen. The data suggest that the large T antigen is phosphorylated in a sequential manner at a subset of sites in the cytoplasm and at additional sites in the nucleus.     

Development of antigens in human cells infected with Simian virus 40

Bissett, Marjorie L. (University of Michigan, Ann Arbor), and Francis E. Payne. Development of antigens in human cells infected with simian virus 40. J. Bacteriol. 91:743-749. 1966.-An explanation for the apparent infrequency with which human cells transform in response to exposure to simian virus 40 (SV40) was sought by following the development of virus-induced antigens in human euploid cells, strain CR. For about 8 weeks after exposure to a high multiplicity of SV40, only a small proportion of the cells produced tumor (T) or viral (V) antigen detected by immunofluorescence. Double-tracer staining techniques revealed that the development of T and V antigen in about 1% of the CR cells resembled that in green monkey kidney cells, strain BS-C-1, in which SV40 replicates and destroys all the cells. T antigen was detected before V antigen; both antigens were detected in the nucleus, but only V antigen appeared later in the cytoplasm. All intact cells that contained V antigen also contained T antigen. Infected CR cell cultures, before and after transformation or when in "crisis," contained only 0.1 to 1.0% of cells with both V and T antigen. Some CR cells contained only T antigen, and by 8 days after exposure to virus these cells were present as loose foci associated with an occasional cell containing V antigen. The proportion of CR cells with only T antigen increased from about 1% during the first 4 weeks to 8% at 7 weeks, and to nearly 100% at 11 weeks, when essentially all of the cells were epithelioid. Foci of epithelioid cells were first recognized in the 9th week. It was concluded that those CR cells that contained T antigen at any given time represented (i) a few cells that subsequently produced V antigen and lysed, and (ii) a progressively increasing population that produced only T antigen. If the latter population, in whole or in part, gave rise to the epithelioid transformed cells, then its initial size could account, at least in part, for the apparent infrequency with which human cells transform in response to SV40.     

Properties of Simian Virus 40 Rescued from Cell Lines Transformed by Ultraviolet-Irradiated Simian Virus 40

Simian virus 40 (SV40) strains have been rescued from various clonal lines of mouse kidney cells that had been transformed by ultraviolet (UV)-irradiated SV40. To learn whether some of the rescued SV40 strains were mutants, monkey kidney (CV-1) cells were infected with the rescued virus strains at 37 C and at 41 C. The SV40 strains studied included strains rescued from transformed cell lines classified as "good," "average," "poor," and "rare" yielders on the basis of total virus yield, frequency of induction, and incidence of successful rescue trials. Four small plaque mutants isolated from "poor" yielder lines and fuzzy and small plaque strains isolated from an "average" and a "good" yielder line, respectively, were among the SV40 strains tested. Virus strains rescued from all classes of transformed cells were capable of inducing the transplantation antigen, and they induced the intranuclear SV40-T-antigen, thymidine kinase, deoxyribonucleic acid (DNA) polymerase, and cellular DNA synthesis at 37 C and at 41 C. With the exception of four small plaque strains rescued from "poor" yielders, the rescued SV40 strains replicated their DNA and formed infectious virus with kinetics similar to parental SV40 at either 37 or 41 C. The four exceptional strains did replicate at 37 C, but replication was very poor at 41 C. Thus, only a few of the rescued virus strains exhibited defective SV40 functions in CV-1 cells. All of the virus strains rescued from the "rare" yielder lines were similar to parental SV40. Several hypotheses consistent with the properties of the rescued virus strains are discussed, which may account for the significant variations in virus yield and frequency of induction of the transformed cell lines.     

Replication and Complementation of Human Adenoviruses and Simian Papovavirus at an Elevated Temperature

The simian papovavirus SV40 replicated as well in simian cells incubated at 41 C as in cells incubated at 37 C, although the latent period was shortened at the elevated temperature. Human adenoviruses differed in their responses to the elevated temperature. Some serotypes, such as 3, 4, 5, 7, 8, 16, and 21, replicated as well, or almost as efficiently, in human cells incubated at 41 C as in cells incubated at 37 C, whereas with other serotypes, such as 1, 2, 6, 12, and 14, maximal yields in cultures incubated at 41 C were much lower than the yields from companion cultures incubated at 37 C. This difference was also detected in simian cells co-infected with SV40 and a human adenovirus; maximal complementation occurred with some serotypes at the elevated temperature but not with other serotypes. The degree of complementation observed in the simian cells at 41 C was directly correlated with the ability of the adenovirus to replicate at 41 C in human cells. Therefore, the capacity of SV40 to serve as a helper virus is not affected by the elevated temperature, showing that the complementation event supplied by the simian virus is heat-stable between 37 and 41 C. Maximal complementation appeared to depend upon a characteristic present in the adenovirus genome.     

Characterization of simian cells tranformed by temperature-sensitive mutants of simian virus 40.

Seven lines derived from primary African green monkey kidney cells, which had survived lytic infection by wild-type simian virus 40 (SV40) or temperature-sensitive mutants belonging to the A and B complementation groups, were established. These cultures synthesize SV40 tumor (T) antigen constitutively and have been passaged more than 60 times in vitro. The cells released small amounts of virus even at high passage levels but eventually became negative for the spontaneous release of virus. Virus rescued from such "nonproducer" cells by the transfection technique exhibited the growth properties of the original inoculum virus. Four of the cell lines were tested for the presence of altered growth patterns commonly associated with SV40-induced transformation. Although each of the cell lines was greater than 99% positive for T antigen, none of the cultures could be distinguished from primary or stable lines of normal simian cells on the basis of morphology, saturation density in high or low serum concentrations, colony formation on plastic or in soft agar, hexose transport, or concanavalin A agglutinability. However, the cells could be distinguished from the parental green monkey kidney cells by a prolonged life span, the presence of T antigen, a resistance to the replication of superinfecting SV40 virus or SV40 viral DNA, and, with three of the four lines, an ability to complement the growth of human adenovirus type 7. These properties were expressed independent of the temperature of incubation. These results indicate that the presence of an immunologically reactive SV40 T antigen is not sufficient to ensure induction of phenotypic transformation and suggest that a specific interaction between viral and cellular genes and/or gene products may be a necessary requirement.     

Mapping of Simian Virus 40 Early Functions on the Viral Chromosome

The simian virus 40 (SV40) DNA segment in the nondefective adenovirus 2-SV40 hybrid, Ad2(+)ND(4), is colinear with the segment between 0.11 and 0.59 SV40 fractional length from the site at which the R(1) restriction endonuclease cleaves SV40 DNA. This specifies the region of the SV40 DNA molecule which induces the early SV40 antigens: U antigen, tumor specific transplantation antigen, and T antigen. A variant of Ad2(+)ND(4), called Ad2(+)ND(4del), was found which has a deletion of the DNA segment between 0.50 and 0.57 SV40 fractional length from the R(1) endonuclease cleavage point.     

Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells

Gilden, R. V. (Wistar Institute, Philadelphia, Pa.), and R. I. Carp. Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells. J. Bacteriol. 91:1295-1297. 1966.-Synthesis of the simian virus 40 (SV40) T antigen in primary African green monkey kidney cells was abolished when cycloheximide was added up to 10 hr postinfection. In contrast, puromycin, another inhibitor of protein synthesis, did not suppress antigen production. The basis of this differential effect was the inability of puromycin to inhibit protein synthesis in the cells used. This was shown by the failure of the drug to depress the incorporation of labeled amino acid into protein and also failure to inhibit poliovirus synthesis. The puromycin preparation used was very effective in inhibiting poliovirus synthesis in HeLa cells. Thus, appearance of the SV40 T antigen is dependent on protein synthesis in infected cells.