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.     

Variants of Defective Simian Papovavirus 40 (PARA) Characterized by Cytoplasmic Localization of Simian Papovavirus 40 Tumor Antigen

Three isolates of PARA (particle aiding replication of adenovirus)-adenovirus 7 out of a total of 112 clonal progeny derived by two successive plaque purifications in green monkey kidney cells (GMK) were found to induce the synthesis of simian papovavirus40 (SV 40) tumor (T) antigen in the cytoplasm of infected cells. The variant viruses induced plaque formation in human embryonic kidney cells which followed one-hit kinetics. In GMK cells, plaque formation followed two-hit kinetics which converted to first-order kinetics in the presence of additional helper adenovirus type 7. Analysis of plaque progeny from human cells showed that the progeny could replicate only in human cells, whereas progeny from monkey cells could multiply in both human and monkey cells. Heterologous human adenoviruses were able to enhance plaque formation by the variant viruses in monkey kidney cells. Neutralization tests indicated that both components of the populations had a type 7 adenovirus capsid. All three viruses were capable of inducing SV40 transplantation immunity in weanling hamsters. These results indicate the three variants are PARA-adenovirus 7 populations. Response of the induction of the synthesis of the cytoplasmic antigen to metabolic inhibitors was the same as for the synthesis of the nuclear SV40 T antigen. Different pools of sera which reacted with the intranuclear SV40 T antigen also detected the cytoplasmic antigen induced by the variant viruses. An adsorption experiment with cells containing either nuclear or cytoplasmic T antigen to remove tumor antibody from hamster sera also indicated that it is probably SV40 T antigen which is responsible for the cytoplasmic reaction. The species of the host cell-human, simian, or rabbit-appeared to play no role in the altered localization of this antigen. It is postulated that these PARA variants are further defective in some virus-mediated transport mechanism which shifts the T antigen from the cytoplasm to the nucleus.     

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.     

Synthesis of virus deoxyribonucleic acid during abortive infection of simian cells by human adenoviruses

Rapp, Fred (Baylor University College of Medicine, Houston, Tex.), Lawrence A. Feldman, and Manley Mandel. Synthesis of virus deoxyribonucleic acid during abortive infection of simian cells by human adenoviruses. J. Bacteriol. 92:931-936. 1966.-Inoculation of green monkey kidney cells (GMK) with adenovirus types 2 or 12, under conditions where neither infectious virus was synthesized, resulted in an increase in the uptake of H(3)-thymidine into deoxyribonucleic acid (DNA). Extraction of the DNA from infected cells, followed by identification by isopycnic analysis in CsCl gradients, revealed the presence of virus DNA. Cells infected with adenovirus type 2 yielded DNA giving bands with peak densities of 1.699 g/ml [GMK DNA with 40 moles% guanine + cytosine (GC)] and 1.714 g/ml (adenovirus type 2 DNA with 55 moles% GC). Cells infected with adenovirus type 12 also yielded the GMK DNA and a band at 1.706 g/ml (adenovirus type 12 DNA with 47 moles% GC). The rate of synthesis of adenovirus type 2 DNA in KB cells (productive cycle) and in GMK cells infected only with adenovirus (nonproductive cycle) or with adenovirus and simian virus 40 (adeno-productive cycle) was not significantly different.     

Characterization of the Strain of Adenovirus Type 7 Carrying the Defective Monkey Cell-adapting Component

The strain of adenovirus type 7 carrying the defective monkey cell-adapting component (MAC) has been further characterized. MAC is more sensitive to inactivation by ultraviolet light than the associated adenovirus, which, in turn, is more rapidly inactivated than complete simian virus 40 (SV40). The 37% dose was 16 sec for MAC, 60 sec for adenovirus, and 84 sec for SV40. Filtration through membranes revealed that both MAC and adenovirus were retained by 100-mmu filters. MAC equilibrated in cesium chloride at the same buoyant density as the complete adenovirions (1.34 g/ml). Deoxyribonucleic acid extracted from green monkey kidney cells infected with MAC-adenovirus 7 did not hybridize in vitro with SV40 complementary ribonucleic acid. The two components (MAC and adenovirus) of the virus population possess such similar biophysical properties that their separation has not yet been achieved. Evidence to date indicates that MAC is probably not derived from SV40.     

The growth of simian virus 40 (SV40) host range/adenovirus helper function mutants in an African green monkey cell line that constitutively expresses the SV40 agnoprotein.

The simian virus 40 T-antigen carboxy-terminal mutants, dlA2459 and dlA2475, are cell line and temperature dependent for growth and plaque formation in monkey kidney cells. Although these mutants did form plaques on BSC-1 cells at 37 degrees C, they were about fivefold less efficient for plaque formation than wild-type simian virus 40. These mutants did not grow in CV-1 cells and did not synthesize agnoprotein in those cells. CV-1 cells which constitutively express the agnoprotein were permissive for mutant plaque formation. However, late mRNAs, virion proteins, and progeny virion yields did not accumulate to wild-type levels during mutant infection of the agnoprotein-producing cells.     

Hybrid of Monkey and Human Adenoviruses

Following joint replication of monkey SA7 adenovirus (C8 strain) and human adenovirus type 2 in green monkey kidney tissue culture, a virus possessing the properties of a hybrid was obtained. It was designated Ad2C8. Ad2C8 preparations contained two types of viral particles: human adenovirus type 2, and hybrid particles. The hybrid virions multiplied in green monkey kidney cells in the presence of human adenovirus types 1, 2, and 3, but not 3 and 7, and acquired the capsid of the helper adenovirus. The hybrid can serve as a helper for human adenoviruses. It can apparently induce T antigen of the C8 virus but, in contrast to the latter, does not induce tumors in hamsters.     

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain.     

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.     

Stimulation of adenovirus replication in simian cells in the absence of a helper virus by pretreatment of the cells with iododeoxyuridine.

Pretreatment of African green monkey kidney cells with 50 mu g of 5'-iododeoxyruidine (IUdR) per ml can modify their susceptibility to the replication of human adenovirus type 7 in the absence of simian virus 40 (SV40) although this enhancement of adenovirus replication is not as efficient as that of the helper SV40 virus. Since the number of infectious centers remains unchanged after IUdR pretreatment whereas the burst size of virus from each infected cell increases, the IUdR appears to allow each infected cell to produce more virus. Cell DNA synthesis appears to be stimulated in IUdR pretreated cells infected with adenovirus 7, but the host cell DNA synthesized is small enough to remain in the Hirt supernatant fluid. The modification of susceptibility to adenovirus replication and the changed pattern of cell DNA synthesis is stable for at least two additional cell passages of the pretreated cells.     

Equilibrium Density Gradient Studies on Simian Virus 40-Yielding Variants of the Adenovirus Type 2-Simian Virus 40 Hybrid Population

The simian virus 40 (SV40)-yielding variants of the adenovirus type 2 (Ad.2)-SV40 hybrid (Ad.2(++)) population were studied by means of fixed-angle equilibrium density gradient centrifugation in cesium chloride. The hybrid virions of the Ad.2(++) high-efficiency yielder population banded at densities of 0.004 g/cm(3) lighter than the nonhybrid Ad.2 virions. The degree of separation of the hybrid particles was sufficient to permit greater than 100-fold relative purification by two cycles of centrifugation. Hybrid particles that produce adenovirus plaques in African green monkey kidney cells by two-hit kinetics (one-hit kinetics when assayed on lawns of nonhybrid adenovirus) were not separable from the particles that yield SV40 virus. The hybrid particle in the Ad.2(++) low-efficiency yielder population was not separable from the nonhybrid Ad.2 virions.     

Influence of SV40 Genome on the Replication of an Adenovirus-SV40 "Hybrid" Population.

Feldman, L. A. (Baylor University College of Medicine, Houston, Tex.), J. L. Melnick, and F. Rapp. Influence of SV40 genome on the replication of an adenovirus-SV40 "hybrid" population. J. Bacteriol. 90:778-782. 1965.-Replication of a type 7 adenovirus-SV40 hybrid population in primary African green monkey kidney cells was accompanied by the formation of SV40 tumor antigen, adenovirus antigens, and cytopathic changes characteristic of adenovirus infection. Prior infection of the cultures with SV40 stimulated replication of nonintegrated adenovirus 7 but did not enhance the replication of the hybrid virus. These results suggest that the population of the adenovirus-SV40 hybrid studied contains many particles carrying SV40 information. Replication of SV40 virus was not enhanced by co-infection with nonintegrated adenovirus 7 or with the adenovirus-SV40 hybrid. Cytosine arabinoside strongly inhibited replication of the adenovirus-SV40 hybrid population in African green monkey kidney cells. Enhanced replication of nonintegrated adenovirus 7 by SV40 was blocked by cytosine arabinoside; this block could be reversed by 2-deoxycytidine or deoxycytidine triphosphate.     

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.     

Search for inhibitors against herpes simplex virus type-I in cell extracts derived from human lymphoblastoid cell lines.

Cell extracts obtained from KB cells and 5 human lymphoblastoid cell lines including 2 from Burkitt's lymphoma (P3HR-1 and Raji), one each from nasopharyngeal carcinoma (no.223), acute lymphatic leukemia (MOLT-4) and a healthy person (NC-37) were tested for their inhibitory effects on the growth of herpes simplex virus type-1 (HSV-1) in green monkey kidney (GMK) cells by the plaque titration method. The relationship between the production of HSV-1 inhibitors and the degree of Epstein-Barr virus (EBV) genome repression in lymphoblastoid cells were also examined. Among the cell lines used P3HR-1 and no.223 cells produced a few EBV particles, Raji and NC-37 cells contained EBV genomes only, and MOLT-4 as well as KB cells were EBV genome-negative. The results revealed that P3HR-1 cell extract showed a tendency to inhibit HSV-1 growth in GMK cells but the other 4 lymphoblastoid cell lines and KB cells did not produce HSV-1 inhibitors, indicating that EBV genomes governing the formation of EBV structural antigens were not related to the production of HSV-1 growth inhibitors. The extracts from MOLT-4 cells, which are only a T lymphocyte cell line used in this study, stimulated HSV-1 growth in GMK cells significantly.     

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.     

Penetration of Host Cell Membranes by Adenovirus 2

Highly purified human adenovirus type 2 directly penetrated the plasma membranes of KB cells. The process of membrane penetration resulted in the appearance of large numbers of adenovirions free in the cytoplasm of the infected cells. The virions underwent a morphological change as they penetrated the cell surface. Penetration of the plasma membranes and the accompanying alteration in virion morphology was dependent on a function associated with the intact cells, because neither event occurred when purified virions were added to isolated cell membranes. Inactivation of the adenovirions with heat or antibodies before inoculation of the cells reduced the infectivity of the virus population and prevented the appearance of virions free in the cytoplasm. The inactivation of the virions did not significantly reduce the number of virus particles which were found in cell vacuoles and pinocytotic vesicles.     

Cell killing by simian virus 40: variation in the pattern of lysosomal enzyme release, cellular enzyme release, and cell death during productive infection of normal and simian virus 40-transformed simian cell lines.

Simian virus 40 (SV40) growth on rhesus kidney cells and on the T-22 line of SV40-transformed green monkey kidney (GMK) cells is largely limited by the low plating efficiency of SV40 on these cells. In addition, a fraction of the rhesus kidney and T-22 cells are resistant to infection by SV40. Nevertheless, 72-h viral yields per infected rhesus kidney and T-22 cell are nearly equivalent to that obtained on normal GMK cells and are independent of the multiplicity of infection. Despite the production of high viral yields, infected rhesus kidney and T-22 cells are killed slowly by SV40. Monolayers of these cells are also refractory to plaque formation by SV40. SV40 induces the release of lysosomal N-acetyl-beta-glucosaminidase into the cytoplasmic fractions of rhesus kidney and T-22 cells to an extent equal to that observed during infection of rapidly killed normal GMK cells. In contrast, damage to the plasma membrane, as indicated by the release of the cellular enzymes lactic dehydrogenase and glutamic oxaloacetic transaminase into the overlay media, occurred to a much greater extent in the normal GMK cells than in the rhesus kidney or T-22 cells. Neither a lysosomal hydrolase mechanism nor viral release appear to be responsible for this phenomenon. The different rates and extents of the SV40 cytocidal process on these cells do not result from the differences in the viral plating efficiency on them.     

Physical Assay and Growth Cycle Studies of a Defective Adeno-Satellite Virus

Electron microscopic particle counting of the defective adeno-satellite virus (ASV), by use of pseudoreplication and negative staining with phosphotungstic acid, was shown to be a reproducible quantitative assay procedure. Particles of satellite type 4 that were counted in fluids from infected cultures had the same morphology as particles that banded at a buoyant density of 1.43 g/cc in cesium chloride. Other satellite virus serotypes examined in the same manner had a buoyant density of 1.37 to 1.38 g/cc. A comparison of satellite titers obtained by complement fixation and by particle counting demonstrated that an increase in satellite particles resulted in a corresponding increase in CF titers; however, electron microscopy was at least 10 times more sensitive than complement fixation for detecting satellite virus. Growth cycle studies of satellite virus in cells co-infected with adenovirus, as assayed by particle counting, indicated that the kinetics of satellite virus production closely followed the kinetics of its helper adenovirus production, with an eclipse period of 12 to 16 hr. The eclipse period of the satellite remained the same when cultures were preinfected with satellite 24 hr prior to adenovirus inoculation. However, when cultures were infected with adenovirus 12 hr before satellite virus, the eclipse period of the satellite was shortened to between 4 and 6 hr. Thus, satellite virus replication seems dependent upon a relatively late event in the adenovirus replication cycle. When cells were co-infected with adenovirus and its defective satellite, the yield of adenovirus was markedly reduced from that obtained in cells singly infected with adenovirus.