BSC-1 Cells Research Articles

Role of Defective Simian Virus 40 Genomes in Establishment and Maintenance of Persistently Infected Primate Cell Lines

Studies are reported of persistent simian virus 40 (SV40) infections of fully permissive monkey (TC-7 and BSC-1) and human (A172) cell lines, with emphasis on the role of viral defectives in establishment and maintenance of persistence. The presence of defectives prevented complete cell killing and allowed the establishment of persistently infected cultures. Hirt supernate DNAs from these cultures showed the continuing presence of defective genomes. Restriction enzyme analysis demonstrated that altered defective genomes evolved during passage of the carrier cultures, but that they always reflected structures of the genomes used to established the cultures. There was some host cell specificity in the effectiveness of establishment of persistence, e.g., TC-7-derived defectives were more effective in preventing killing of TC-7 than of BSC-1 or A172 cells. Persistent infections of TC-7 cells could also be established in the absence of defectives, but in the presence of neutralizing anti-SV40 antiserum. In fact, defectives were eliminated from carrier cultures established in the presence of antiserum. When antiserum was withdrawn, the wild-type SV40 grew and destroyed the cells. Antiserum maintains a low level of infection by neutralizing viruses outside the cells, so that many cells do not become infected. Defectives are eliminated because spread of infection is effectively at very low multiplicity. Carrier cultures that are established and maintained by defectives result from intracellular interference by the defectives with the normal development of wild-type virus.

A survey of enteric viruses in domestic sewage.

In this second study (1979-1981) of the viral content of sewage we have demonstrated the presence of poliovirus types 1, 2, and 3 in Laval and Montreal. Several strains of poliovirus types 2 and 3 were nonvaccinal. This is in contrast with our first study (1977-1978) in which only type 1 poliovirus isolates were nonvaccinal. Coxsackievirus types B-3, B-4, and B-5 and echovirus types 1, 7, and 11 were also isolated from sewage. Interestingly, these isolations coincided with reports of isolation of the same strains during the same period by diagnostic laboratories. Our method based on Vero and BSC-1 cell cultures for virus isolation and immune electron microscopy for identification permitted the recovery not only of several strains of enteroviruses but also of some adenoviruses and reoviruses.

Serum and epidermal growth factor transiently depolarize quiescent BSC-1 epithelial cells.

Continuous intracellular recording of membrane potential with microelectrodes in BSC-1 epithelial cells has been used to study the early ionic effects of the interaction of mitogens with cell surface receptors. Initial results show that (i) there is no significant difference in membrane potential between growing and quiescent cells, (ii) addition of epidermal growth factor or serum to quiescent BSC-1 cells induces a brief and transient depolarization, (iii) the mitogenic response of BSC-1 cells to epidermal growth factor and the transient depolarization show similar concentration dependences, and (iv) serum addition to quiescent BSC-1 cells induces a sustained increase in Na+ influx that is electroneutral and amiloride sensitive. Intracellular pH changes may be a primary event triggering the response of quiescent cells to mitogenic polypeptides.

Efficient infection of monkey cells with SV40 DNA. II. Use of low-molecular-weight DEAE-dextran for large-scale experiments

With standard protocols for DNA infection, only a small percentage (about 4%) of monkey cells exposed to purified DNA of simian virus 40 (SV40) displays cytopathic effect or expresses viral T antigen. We have recently reported (Sompayrac and Danna, 1981) that by extending the time of exposure of BSC-1 cells to DNA in the presence of low concentrations of diethylaminoethyl (DEAE)-dextran, we can infect up to 50% of the cells. Because our protocol was devised for DEAE-dextran of mol. wt. 2 × 10 6, which is no longer commercially available, we have tested a low-molecular-weight polymer (mol. wt. 0.5 × 10 6) that can be purchased. Use of this reagent in our protocol resulted in marginally higher levels of infection than we found with the high-molecular-weight polymer. However, slightly more DNA (about 1.5 times as much) was needed to achieve saturation. With both reagents, the percentage of cells infected was proportional to time of exposure. Therefore, low-molecular-weight DEAE-dextran should be useful for large-scale DNA infections.

Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus.

Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.

Isolation and characterization of defective simian virus 40 genomes which complement for infectivity.

A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency.

Characterization of an herpes simplex virus type 2 mutant, which is resistant to acycloguanosine and causes fusion of BSC1 cells.

A mutant of herpes simplex virus type 2, which induces low levels of thymidine-kinase activity in infected BSC1 cells and consequently able to grow in the presence of acycloguanosine, was isolated. This mutant has also been shown to cause fusion of BSC1 cells. In BSC1 cells, co-infected with the wild-type strain and the mutant, the yield of each of the two viruses was normal but the rounding and aggregation of cells observed, resembled that found in wild-type infected cultures. When the mixed infection was performed in the presence of acycloguanosine (100 micrometers), the growth of the two virus strains was inhibited, as well as the cytopathic effect in the cultures. It is suggested that under these conditions, the thymidine-kinase which was induced in the infected cells by the wild-type strain, phosphorylated acycloguanosine and the activated drug formed, inhibited the growth of the two viruses by interference in their DNA syntheses.

Measurement of surface antigen by specific bacterial adherence and scanning electron microscopy (SABA/SEM) in cells infected by vesiculovirus ts mutants.

Temperature-sensitive (ts) mutants of the rhabdoviruses vesicular stomatitis virus and Chandipura virus have been used to measure the appearance of virus antigen on the surface of infected cells by the technique of surface analysis by bacterial adherence and scanning electron microscopy (SABA/SEM). The number of staphylococci specifically adhering to antiserum-treated infected PTK-2 or BSC-1 cells at permissive (31 degrees C) and restrictive (39 degrees C) temperatures was followed in time-course experiments and a close correspondence was observed between the proportion of staphylococci bound at 39 degrees C and the known phenotypic properties of the ts mutants. Virus surface antigen was undetected in cells infected by transcription- and replication-defective ts mutants with thermolabile L proteins under restrictive conditions up to an input multiplicity of infection of 50, and in cells infected by a replication-defective NS protein mutant. Some surface antigen was detected late in infection in PTK-2 cells infected by a replication-defective N protein mutant. Surface antigen accumulated normally in maturation-defective mutants with lesions in envelope proteins. These results establish the suitability of the SABA/SEM technique for quantitative estimation of virus antigen on the surface of infected cells.

Potential of intense gamma-irradiation of host cells for analysis of virus-specified transcription and replication.

Intense gamma-irradiation from a cobalt source differentially affects macromolecular synthesis of cultured mammalian cells. Exposure of monkey BSC-1 or murine fibroblastic L2 cells to 40 or 70 krad (1 rad = 1 x 10(-2) J/kg) abolishes DNA and RNA synthesis almost entirely but reduces the formation of protein much less. A dose-response analysis of irradiation shows that synthesis of total RNA and the messenger component thereof, measured as the poly(A)-containing fraction, are equally diminished. Host cells in which formation of DNA and RNA are minimal can support normal or nearly normal replication and transcription rates of vesicular stomatitis and vaccinia viruses. Therefore, use of pretreatment with gamma-irradiation, as employed here, should prove to be generally useful in examining virus-related transcription under circumstances in which application of drugs affecting gene expression, such as actinomycin D, is deemed undesirable.

Mechanism of interferon action Differential effect of interferon on the synthesis of simian virus 40 and reovirus polypeptides in monkey kidney cells

The effect of interferon (IFN) treatment on the synthesis of viral proteins was examined in BSC-1 monkey kidney cells singly and doubly infected with simian virus 40 (SV40) and reovirus. The pattern and amount of SV40 T antigen and reovirus λ, μ, and σ polypeptide synthesis were comparable in the singly and doubly infected cells in the absence of IFN treatment. As determined by Yakobson et al. ( Cell 12: 73–81, 1977) and Kingsman and Samuel ( Virology 101: 458–465, 1980) SV40-specific RNA synthesis is inhibited by treatment of cells with IFN before infection, but is not inhibited by IFN treatment after infection. IFN treatment initiated before infection inhibited the synthesis of both SV40 T antigen and reovirus polypeptides. However, SV40 T antigen synthesis was not inhibited in SV40 wt or tsA mutant-infected cells when treated with monkey kidney or human leukocyte IFN after infection. By contrast, reovirus polypeptide synthesis was markedly inhibited in BSC-1 cells treated with IFN after SV40 infection and superinfected with reovirus. SV40 infection did not rescue reovirus polypeptide synthesis from the inhibitory action of IFN, and reovirus infection did not alter the resistance of SV40 T antigen synthesis to IFN in cells IFN-treated after SV40 infection. Total cellular protein synthesis was not significantly affected by IFN treatment. These results suggest that the interferon-induced inhibitor(s) of protein synthesis discriminate between SV40 and reovirus mRNAs; the translation of SV40 mRNA, like most cellular mRNAs, is not affected under conditions where the IFN-induced inhibitor(s) significantly affect the translation of reovirus mRNA.

Compartmentalization of polyamines in mammalian cells

A rapid method for the separation of cytoplasm and nuclei has been adapted for polyamine analysis of the different compartments of BSC-1 cells. The whole cells contain putrescine, spermidine and spermine at concentrations of 7.3, 6.5, and 12.4 nmol/10 6 cells respectively. By separating the cytoplasm and the nuclei within 20 seconds we found strikingly different polyamine distributions in the cell compartments. In the cytoplasm putrescine and spermine are present in almost equal amounts, whereas in the nucleus spermine is in 5-fold excess over putrescine and spermidine. Fluctuations of polyamines in the different cell compartments were investigated during the cell cycle after synchronizing the cells with thymidine/hydroxyurea treatment. An increase of putrescine in the cytoplasm parallels the initiation of DNA synthesis.

Shope fibroma virus. I. Biological and molecular properties of a cytocidal and a noncytocidal strain.

The biological and molecular properties of two strains of Shope fibroma virus (SFV) were compared. SFV-I was highly cytocidal to most of the cell lines tested and produced pocks in the chorioallantoic membrane of chick embryos. By contrast, SFV-W did not produce cytopathic effects in any of the cell lines or in the chorioallantoic membrane, but it induced characteristic foci 3 to 4 days after infection. Both strains produced tumors when inoculated into the skin of susceptible rabbits. Maximal infectivity in BSC-1 cells was reached by both strains between 24 to 48 h after inoculation. Viral DNA synthesis also took place at the same time, although cells infected with SFV-I incorporated three times more [(3)H]thymidine than cells infected with SFV-W. Sedimentation analysis and hydroxylapatite chromatography of the two viral DNAs indicated that their molecular weights were similar and that both were naturally cross-linked. Digestion with three restriction endonucleases, however, revealed that they had different restriction sites. When SFV-I and vaccinia DNA were compared, the restriction patterns were more alike. Analysis of the virion structural proteins by gel electrophoresis indicated that SFV-I, SFV-W, and vaccinia virus had many polypeptides in common, although there were distinctive differences among the three viruses. Finally, the results of plaque neutralization tests with different antisera showed that SFV-I and SFV-W shared common antigens and that vaccinia antiserum inhibited SFV-I but not SFV-W. We conclude that the SFV-I genome contains information for both cytolysis and tumorigenesis. This unusual virus may be a recombinant between an orthopoxvirus and a leporipoxvirus.

Action of cytochalasin D on cytoskeletal networks.

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end-to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.

DNA and histone synthesis in butyrate-inhibited BSC-1 cells infected with SV40

The effect of butyrate on DNA synthesis, histone synthesis, and virus production in SV40-infected monkey cells has been studied. When cells in which DNA and histone synthesis have been blocked by treatment with butyrate are infected with SV40, DNA synthesis is induced to a level at least equal to that in untreated infected cells. Both viral and cellular DNA sequences are replicated. Viral DNA synthesized is predominantly SV40 Form I. The amount of viral DNA which accumulates is reduced by 10-fold in the presence of butyrate, and the yield of infectious virus is lowered 100-fold. Histone synthesis in these cells is stimulated concomitantly with DNA synthesis. The hyperacetylation of histones characteristic of butyrate-treated cells is unchanged by SV40 infection; the newly synthesized histones are hyperacetylated. Thus the mechanism by which butyrate blocks DNA and histone synthesis is not directly related to the hyperacetylation of histones.

Comparison of bovine rotavirus strains by plaque assay

Using plaque assay on MA 104 cells, four strains of bovine rotavirus were compared: the attenuated American strain NCDV, and three virulent strains isolated in Canada (PQ) or in Belgium (S14 and S 77). The attenuated strain (NCDV) formed smaller plaques than the three others. The plaque size may thus be used as a marker for this strain. The Belgian strain S 77 formed the largest plaques and no significant differences could be found between the Canadian strain PQ and the second Belgian strain S 14. On BSC-1 cells, the Canadian strain PQ formed much smaller plaques than on MA 104 cells.

Expression of Epstein-Barr viral early antigen in monolayer tissue cultures after transfection with viral DNA and DNA fragments.

Antigens associated with the Epstein-Barr virus (EBV) replicative cycle were found in the nucleus and cytoplasm of human placental, Vero, BSC-1, and owl monkey kidney cells transfected with EBV DNA prepared from several different strains of virus. The number of antigen-positive nuclei increased when transfection was followed by cell fusion induced by inactivated Sendai virus. About 1,200 antigen-positive foci were induced per micrograms of EBV DNA. On the basis of their reactivity with various well-characterized human sera, it appears that the antigens are part of the early antigen complex. None of the four restriction endonucleases, EcoRI, HindIII, SalI, and BamHI, destroyed the ability of EBV DNA to induce early antigen. However, only SalI seemed to leave intact the full spectrum of antigen expression by the HR-1 and FF41 strains of EBV DNA. By means of transfection with recombinant DNA plasmids containing different EBV (FF41) DNA fragments regenerated by EcoRI, we showed that the coding region for early antigen was at least partially contained on the 17.2-megadalton EcoRI B fragment.

Efficient infection of monkey cells with DNA of simian virus 40.

With standard protocols for DNA infection, only a small fraction (about 4%) of monkey cells exposed to purified DNA of simian virus 40 (SV40) exhibits signs of infection. We have devised a protocol by which we can extend the time of exposure of BSC-1 cells to DNA in the presence of low concentrations of DEAE-dextran. The efficiency of infection is proportional to the time of exposure. With an 8-hr exposure, we are reproducibly able to infect 25% of the cells, and we have been able to achieve levels of infection as high as 50% with a 16-hr exposure. The percentage of cells infected was measured either by scoring for nuclei positive for SV40 tumor antigen or by an infectious centers assay. We also report the use of ethidium bromide as a nonspecific nuclear counterstain in the immunofluorescence assay for SV40 tumor antigen.

Stabilization and the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition.

Treatment of epithelial BSC-1 cells with low concentrations of the detergent Brij 58 results in partial or complete removal of the plasmalemma and partial extraction of internal membrane-bound organelles without causing massive release of "cytosolic" proteins from the cytoplasmic ground substance. Stereoscopic high-voltage electron microscopy of such extracted and fixed cells demonstrates a system of slender (4-20 nm) strands in a three-dimensional "microtrabecular" arrangement similar to that observed in unextracted whole-mount preparations. Extraction of Brij-extracted cells with Triton X-100 dissolves many of the microtrabecular strands, leaving, as a more stable structure, a characteristic cytoskeletal network composed of various filaments and microtubules. Two-dimensional polyacrylamide gel electrophoresis of 35S-labeled polypeptides performed concurrently with the morphological studies demonstrates that Triton extraction of Brij-extracted cells releases a large number of polypeptides. This release parallels the loss of structural components observed by electron microscopy. Labeling of Brij-extracted cells with heavy meromyosin subfragment 1 decorates actin filaments with characteristic arrowhead complexes which are readily visualized only after subsequent Triton extraction. These observations support the concept that many cytoplasmic proteins are structure-bound and, in addition to the components comprising the cytoskeleton, are structure-forming. We conclude that a metastable association of various proteins of the cytoplasmic ground substance exists whose morphological integrity is maintained, at lest temporarily, after removal of the plasmalemma in solutions containing Brij 58.

Mechanism of interferon action: Stability and translation of simian virus-40 early mRNA coding for T-antigen is comparable in untreated and interferon-treated monkey cells

The effect of interferon treatment on the translation and the stability of simian virus 40 (SV40) early mRNA coding for T-antigen was examined in tsA-infected monkey kidney BSC-1 cells at 40°. Neither the translation nor the stability of SV40 early mRNA was altered by interferon under cellular conditions where the synthesis of reovirus polypeptides was significantly inhibited by interferon. SV40 early mRNA decayed with a half-life of about 3 hours as measured by T-antigen synthesis; the decay rate was indistinguishable between untreated and interferon-treated cells.

The metabolism of SV40 RNA is associated with the cytoskeletal framework

We prepared the cytoskeletal framework of SV40-infected BSC-1 cells late after infection by extracting the cells with Triton X-100. The cytoskeletal framework obtained by this procedure carries more than 80% of the newly synthesized viral polyribosomes. Almost all the viral-specific cytoplasmic RNA is engaged in the formation of cytoskeletal-associated polyribosomes. The cytoskeletal framework harbors both the poly(A) + 19 S and 16 S late viral RNAs, as well as most of the poly(A) − viral RNA. The poly(A) − viral RNA sediments in sucrose gradients between 4 and 18 S representing heterogeneous species and comprises about 30–50% of the total virus-specific RNA in the cytoplasm. Based on pulse-chase experiments it is concluded that: (i) the poly(A) + viral RNA emerges from the nucleus and becomes associated with the cytoskeletal framework, (ii) the decay rate of the poly(A) + RNA species on the cytoskeletal framework is faster than that of the poly(A) − viral RNA, and (iii) both the poly(A) + and poly(A) − viral RNAs detach from the cytoskeletal framework and move to the soluble fraction.