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.     

Characterization of the Tumorlike (T) Antigen Induced by Type 12 Adenovirus : I. Purification of the Antigen from Infected KB Cells and a Hamster Tumor Cell Line

The T antigen induced by type 12 adenovirus was purified from KB cells infected in the presence of 10(-6)m 5-fluoro-2-deoxyuridine to inhibit synthesis of viral capsid antigens. The antigen was purified approximately 200-fold, and the purified product contained only negligible amounts of host-cell contaminants, as judged by the residual radioactivity from (14)C-labeled uninfected cells which had been added to infected cells at the initiation of the purification. Immunoelectrophoresis indicated that the purified T-antigen preparation contained a single antigenic species. The T antigen from a hamster cell line (HT-1) derived from a type 12 adenovirus-induced tumor was purified by the same procedure. The T antigens from the two different sources were shown to be immunologically similar by use of a rabbit antiserum prepared against the purified T antigen from infected KB cells and sera from hamsters bearing tumors induced by type 12 adenovirus.     

Mechanism by Which Fiber Antigen Inhibits Multiplication of Type 5 Adenovirus

Purified fiber antigen of type 5 adenovirus inhibited the multiplication of type 5 adenovirus by 50% when 35 mug of fiber antigen protein was added to 10(6) KB cells in suspension culture. Although the fiber antigen reduced the number of virions adsorbed per cell when a multiplicity of infection of 50,000 plaque-forming units (PFU)/cell was employed, the number of cells infected was not diminished under these conditions. If a low multiplicity of infection (1.1 PFU/cell) was used, viral adsorption was not detectably decreased. The fiber antigen did not reduce the capability of virions to liberate their viral deoxyribonucleic acid (DNA). The biosyntheses of DNA, ribonucleic acid (RNA), and protein were blocked about 20 to 25 hr after the addition of fiber antigen to cultures of uninfected or type 5 adenovirus-infected KB cells. Most of the fiber antigen protein became cell-associated between 22 and 36 hr after it was added to cells. The hexon antigen neither inhibited viral multiplication nor blocked the biosynthesis of DNA, RNA, or protein. Moreover, the hexon did not attach to KB cells. The profound effects of the fiber antigen were not due to the induction of an interferon-like substance, for actinomycin D did not reduce the ability of the fiber to inhibit multiplication of type 1 poliovirus.     

Biochemical Consequences of Type 2 Adenovirus and Simian Virus 40 Double Infections of African Green Monkey Kidney Cells

African green monkey kidney (AGMK) cells were nonpermissive hosts for type 2 adenovirus although the restriction was not complete; when only 3 plaque-forming units/cell was employed as the inoculum, the viral yield was about 0.1% of the maximum virus produced when simian virus 40 (SV40) enhanced adenovirus multiplication. The viral yield of cells infected only with type 2 adenovirus increased as the multiplicity of infection was increased. Type 2 adenovirus could infect almost all AGMK cells in culture; adenovirus-specific early proteins and DNA were synthesized in most cells, but small amounts of late proteins were made in relatively few cells. Even when cells were infected with both SV40 and adenovirus, only about 50% were permissive for synthesis of adenovirus capsid proteins. Approximately the same quantity of adenovirus deoxyribonucleic acid (DNA) was synthesized in the restricted as in the SV40-enhanced infection. However, in cells infected with SV40 and type 2 adenovirus, replication of SV40 DNA was blocked, multiplication of SV40 was accordingly inhibited, and synthesis of host DNA was not stimulated. To enhance propagation of type 2 adenovirus, synthesis of an early SV40 protein was essential; 50 mug of cycloheximide per ml prevented the SV40-induced enhancement of adenovirus multiplication, whereas 5 x 10(-6)m 5-fluoro-2-deoxyuridine did not abrogate the enhancing phenomenon.     

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.     

Chromosomal Aberrations and Cloning Efficiency in Adenovirus Type 12-infected Hamster Cells

The fate of hamster cells, abortively infected with adenovirus type 12, has been studied by correlation of chromosomal aberrations with induction of T antigens and cloning efficiency. The incidence of chromosomal changes paralleled to some extent the T antigen formation, but was inversely related to the cloning efficiency of the cells. At an input multiplicity of 100, within 24 hr after infection, nearly all of the cells or metaphases revealed the presence of T antigens and chromosomal lesions, respectively, but no clones of cells were obtained. Inhibition of cellular deoxyribonucleic acid synthesis was not noted during this period. Increasing doses of ultraviolet irradiation reduced, successively, the capacity of the virus to induce chromosomal aberrations and correspondingly improved cloning efficiency of the exposed cells. It is concluded that most, if not all, cells revealing chromosomal lesions 24 hr after infection fail to enter further mitoses.     

Induction of Specific Chromosomal Aberrations by Adenovirus Type 12 in Human Embryonic Kidney Cells

Nonrandom chromosomal breaks in chromosomes 1 and 17 were provoked in human embryonic kidney cells 24 hr after infection with adenovirus type 12. These chromosomal changes disappeared in persistently infected cultures. Neutralization of the virus with type-specific antiviral serum prior to infection prevented the occurrence of chromosomal aberrations. No viral deoxyribonucleic acid (DNA) synthesis, as determined by autoradiography, was seen in metaphases containing adenovirus type 12-induced chromosomal aberrations. Ultraviolet irradiation of the virus reduced chromosomal aberrations linearly. This reduction in aberrations was fourfold slower than the inactivation of viral infectivity. At 24 hr after infection of cells with purified (3)H-labeled adenovirus type 12, the isotope was found to be associated with the nuclei. The uptake of isotope was reduced ninefold when the labeled virus was neutralized with type-specific antiviral serum. This difference is considered to account for neutralization of labeled virions. In metaphases infected with labeled viruses, most of the clustered grains were seen only on one arm of the chromatid, even after 72 hr. Isochromatid labeling was found, however, in a small percentage of chromosomes, and increased with time after infection. This increase was threefold between 24 and 72 hr after infection, whereas the mean grain counts decreased twofold during the same period. This has been tentatively interpreted to mean that most of the viral DNA molecules or parts thereof are merely attached to cellular chromatin, but a small fraction of them becomes gradually integrated as time proceeds. Certain chromosomal sites appeared to be preferentially labeled when chromosome 2 was used as a model for evaluation.     

Inhibition of Host Protein Synthesis in Type 5 Adenovirus-infected Cells

The effect of type 5 adenovirus infection on the synthesis of host-cell proteins by suspension cultures of KB cells was investigated. Although total protein synthesis continued at a constant rate for approximately 36 hr, net synthesis of five host enzymes (lactic dehydrogenase, acid phosphatase, deoxyribonuclease, fumarase, and phosphoglucose isomerase) was found to stop 16 to 20 hr after infection. The synthesis of alkaline phosphatase stopped 9 to 12 hr after infection. The inhibition of host protein synthesis occurred shortly after the synthesis of viral antigens had begun, accounting for the continued synthesis of total protein. An investigation of the relationship between synthesis of viral antigens and inhibition of host protein synthesis yielded results which suggest that the two processes are in some way coupled.     

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize virus nonstructural proteins.

The specificity of herpes simplex virus type 1-specific cytotoxic T cells was examined with target cells expressing either input viral structural antigens or antigens resulting from permissive infection or cells from an interrupted infection in which they expressed predominantly nonstructural immediate-early proteins. These studies indicated that only an insignificant minority of cytotoxic T cells recognized the input viral antigens, whereas a significant proportion (20 to 35%) recognized target cells that expressed the immediate-early proteins despite the absence of serologically detectable viral antigens upon the infected cell surface. The finding that a significant proportion of cytotoxic T-cell populations obtained from the draining lymph nodes of mice acutely infected with herpes simplex virus type 1 also recognized immediately-early gene-expressing target cells indicates the importance of nonstructural herpes simplex virus proteins to antiviral immunity in vivo.     

Decreased repair of gamma ray damaged DNA in progeria.

A sensitive host-cell reactivation technique was used to examine the DNA repair ability of fibroblasts from two patients with classical progeria. Fibroblasts were infected with either non-irradiated or gamma-irradiated adenovirus type 2 and at 48 hrs after infection cells were examined for the presence of viral structural antigens using immunofluorescent staining. The production of viral structural antigens was considerably reduced in the progeria lines as compared to normal fibroblasts when gamma-irradiated virus was used, indicating a defect in the repair of gamma ray damaged DNA in the progeria cells.     

Persistence of the Viral Genome in Adenovirus Type 12-infected Hamster Cells

Induction of T antigen by adenovirus type 12 was studied in growing and growth-inhibited cultures of the Nil-2 line of Syrian hamster cells. At a viral input multiplicity of 10, neoantigen was present in 100% of the cells by 24 hr. T antigen gradually disappeared in descendants of these cells so that 2 weeks after infection only 1% gave specific immunofluorescence. When cellular replication was prevented by addition of fluorodeoxyuridine, T antigen persisted in all cells for the 2-week period. Upon infection of growing cultures with purified (3)H-labeled adenovirus type 12 and autoradiographic analysis of the cells at various times thereafter, a gradual reduction in labeled nuclear loci was noted which paralleled the decrease in T antigen-containing cells. In nongrowing cultures, no change in labeled loci was noted. Correlation of T antigen and labeled loci revealed that fluorescent cells contained, on the average, about 10 times more silver grains than nonfluorescent cells. All of 92 preselected fluorescent cells showed labeled loci, whereas, of 100 nonfluorescent cells, 18 were free of silver grains. The implications of these findings are discussed.     

Fate of adenovirus type 12 genomes in nonpermissive cells

The fate of ³H-thymidine-labeled adenovirus type 12 deoxyribonucleic acid (DNA) was studied in Nil-2 cells of Syrian hamster origin. It was found that a substantial fraction of ³H-adenovirus type 12 DNA became degraded within 24 hr after infection and was released into the culture fluid. After infection of 5-bromodeoxyuridine (BUdR)-prelabeled cells with ³H-adenovirus type 12, viral DNA became readily separable from cellular DNA by equilibrium centrifugation in CsCl. Part of the viral radioactivity was found to shift gradually to the position of cellular DNA as time progressed after infection. When exponentially growing cells were exposed simultaneously to BUdR, 5-fluorodeoxyuridine, and ³H-adenovirus type 12, up to 50% of the viral radioactivity shifted within 24 hr from the density of viral DNA to that of cellular DNA after equilibrium centrifugation in CsCl. Upon denaturation of the cellular DNA, the isotope was preferentially found to be associated with the “heavy” strand which was synthesized after infection. Upon hybridization of the “heavy” and the “light” strands with sonically treated, denatured ³H-adenovirus type 12 DNA, small and nearly equal amounts of counts hybridized with both strands. The number of counts annealed was in a range similar to that of those annealed with the same amount of DNA derived from adenovirus type 12-transformed hamster cells. These results demonstrate that (i) a substantial proportion of the adsorbed virus becomes degraded within 24 hr; (ii) part of the degradation products is reutilized for cellular DNA synthesis; (iii) only a small fraction, mainly fragments, of viral DNA becomes integrated into both the newly synthesized and the parental strands of cellular DNA.     

Electron Microscopy of Adenovirus 12 Replication 1. Fine Structural Changes in the Nucleus of Infected KB Cells

The ultrastructure of KB cells infected with oncogenic adenovirus 12 was studied at various intervals from 4 to 72 hr after viral inoculation. At 12 hr after infection, the nucleus and the nucleolus became hypertrophic. At 16 hr, bundles of fibers digestable by proteolytic enzymes were seen in the nucleus; they are considered as the early viral antigens identified immunologically by others. Between 24 and 26 hr, four types of nuclear inclusions appeared. Their sequence of appearance and fine structure are described. On the basis of their sensitivity to proteolytic digestion in thin sections, and the results of immunoferritin studies made by others, some of these inclusions are believed to represent viral structural antigens. Throughout the cycle of viral replication, the nucleolus displayed prominent and constant changes in the form of focal condensations and loosening of the nucleolonema, followed by atrophy and fragmentation. It is suggested that the early nucleolar changes reflect an active participation of the nucleolus in the synthesis of adenovirus 12. A hitherto unknown striated structure with definite periodicity, which is easily digested by proteolytic enzymes, was found in the nuclei during the late stages of adenovirus 12 replication.     

Comparison of methods of detection of bovine adenovirus serotype 3 in infected culture of calf kidney cells (MDBK)

The comparative study of the dynamics of the main antigen (hexon) and viral DNA of the bovine adenovirus type 3 accumulation in the established cell line MDBK under the conditions of single- or multistep cycle of infection has been undertaken. The quantitative immunoelectrophoresis and immunoenzyme assay detected the viral antigens on the late stages of infection in the period of cellular monolayer degradation. The immunofluorescence reaction and histochemical immunoenzyme method detected the antigen in the infected cells concurrently with the primary expression of the viral cytopathic effect. The reaction of the spot molecular hybridization with the [32P]-DNA probes detected the viral DNA considerably earlier than the antigen was detected by the immunological methods, before the appearance of degenerative changes in the infected cells. Preference of the immunoenzyme assay and DNA-probes in diagnosis of the virus are discussed.     

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.     

Temperature-Sensitive Mutants of Simian Virus 40: Infection of Permissive Cells

Ten temperature-sensitive mutants of simian virus 40 have been isolated and characterized in permissive cells. The mutants could be divided into three functional groups and two complementation groups. Seven mutants produced T antigen, infectious viral deoxyribonucleic acid (DNA), and structural viral antigen but predominantly the empty shell type of viral particles. Two mutants produced T antigen and infectious viral DNA, but, although viral structural protein(s) could be detected immunologically, no V antigen or viral particles were found. These two functional groups of mutants did not complement each other. A single mutant was defective in the synthesis of viral DNA, viral structural antigens, and viral particles. T antigen could be detected in infected cells by fluorescent antibody but was reduced by complement fixation assay. This mutant stimulated cell DNA synthesis at the restrictive temperature and complemented the other two functional groups of mutants.     

Ataxia telangiectasia-mutated damage-signaling kinase- and proteasome-dependent destruction of Mre11-Rad50-Nbs1 subunits in Simian virus 40-infected primate cells

Although the mechanism of simian virus 40 (SV40) DNA replication has been extensively investigated with cell extracts, viral DNA replication in productively infected cells utilizes additional viral and host functions whose interplay remains poorly understood. We show here that in SV40-infected primate cells, the activated ataxia telangiectasia-mutated (ATM) damage-signaling kinase, gamma-H2AX, and Mre11-Rad50-Nbs1 (MRN) assemble with T antigen and other viral DNA replication proteins in large nuclear foci. During infection, steady-state levels of MRN subunits decline, although the corresponding mRNA levels remain unchanged. A proteasome inhibitor stabilizes the MRN complex, suggesting that MRN may undergo proteasome-dependent degradation. Analysis of mutant T antigens with disrupted binding to the ubiquitin ligase CUL7 revealed that MRN subunits are stable in cells infected with mutant virus or transfected with mutant viral DNA, implicating CUL7 association with T antigen in MRN proteolysis. The mutant genomes produce fewer virus progeny than the wild type, suggesting that T antigen-CUL7-directed proteolysis facilitates virus propagation. Use of a specific ATM kinase inhibitor showed that ATM kinase signaling is a prerequisite for proteasome-dependent degradation of MRN subunits as well as for the localization of T antigen and damage-signaling proteins to viral replication foci and optimal viral DNA replication. Taken together, the results indicate that SV40 infection manipulates host DNA damage-signaling to reprogram the cell for viral replication, perhaps through mechanisms related to host recovery from DNA damage.