Density Heterogeneity of Simian Virus 40 Ribonucleic Acid Late After Infection of Permissive Cells

Ribonucleic acid (RNA) was isolated from CV-1 cells 44 hr after simian virus 40 (SV40) infection. The molecules containing SV40 base sequences were characterized with respect to their buoyant density distribution. The density of these molecules was compared to that of single- and double-stranded RNA synthesized by using SV40 DNA and Escherichia coli RNA polymerase. The results suggest that about 20% of the SV40 RNA in the infected cells consisted of partially double-stranded molecules.     

Isolation and Characterization of Simian Virus 40 Ribonucleic Acid

Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization in formamide was used to isolate simian virus 40-specific RNA. Early in the lytic cycle, a 19S viral RNA species was observed. Late in the lytic cycle, 16S and 19S viral species were found. The 16S and 19S species of viral RNA were localized in the cytoplasm. High-molecular-weight heterogeneous RNA, containing viral sequences, was isolated from the nuclear fraction of infected cells late in the lytic cycle. This RNA may contain non-viral sequences linked to viral sequences. The formamide hybridization technique can be used to isolate intact late lytic viral RNA which is at least 99% pure.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.     

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.     

Integration of Simian Virus 40 Deoxyribonucleic Acid into the Deoxyribonucleic Acid of Permissive Monkey Kidney Cells

Integration of simian virus 40 (SV40) deoxyribonucleic acid (DNA) into cellular DNA occurred when permissive African green monkey kidney (CV-1) cells were infected at a low multiplicity of SV40 in the presence of cytosine arabinoside.     

Virogenic Properties of Bromodeoxyuridine-sensitive and Bromodeoxyuridine-resistant Simian Virus 40-transformed Mouse Kidney Cells

When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells.     

Properties of Somatic Cell Hybrids Between Mouse Cells and Simian Virus 40-Transformed Rat Cells

Hybrids between mouse cells and simian virus 40 (SV40)-transformed rat cells were made, and their properties and chromosome constitution were investigated over many generations. Their hybrid nature was confirmed by enzyme studies. During a period of 1 year a loss of 10 to 20% of the total number of chromosomes was observed. The SV40 tumor antigen was present and remained present in the hybrids. The parental and hybrid cells were studied for agglutination with concanavalin A, for growth in soft agar, and for serum requirement. These growth and surface characteristics of the transformed cells appeared in the hybrids.     

Characteristics of the Syrian Hamster Ribonucleic Acid Present in Cells Transformed by Polyoma, Simian Virus 40, or Adenovirus 12

Ribonucleic acids of known specific activities were separately prepared from hamster cells transformed by polyoma, simian virus 40 (SV40), and adenovirus 12. When increasing amounts of these labeled ribonucleic acids were added to nitrocellulose filters containing immobilized (14)C-hamster deoxyribonucleic acid (DNA), about 5% of the DNA sites were saturated in each case. An additive saturation-hybridization experiment indicated that a portion of the 5% of the hamster DNA transcribed in SV40-transformed cells was also transcribed in polyoma-transformed cells. In addition, a separate region of the hamster genome was also transcribed in polyoma-transformed cells.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature.     

Pattern of Protein Synthesis in Monkey Cells Infected by Simian Virus 40

After infection of several permanent monkey cell lines by simian virus 40 (SV40), four additional protein bands can be detected by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. These bands appear only after the onset of viral deoxyribonucleic acid (DNA) synthesis, and inhibitors of DNA synthesis prevent their appearance. Three of them correspond to three previously identified capsid components, VP1, VP2, and VP3. The fourth protein band, which does not correspond to a previously identified virion component, is induced by SV40 infection of CV-1 and BSC-1 cultures but not by infection of MA-134 cultures.     

Genetic Analysis of Simian Virus 40 III. Characterization of a Temperature-Sensitive Mutant Blocked at an Early Stage of Productive Infection in Monkey Cells

A temperature-sensitive mutant of simian virus 40 (SV40), ts(*)101, has been characterized during productive infection in monkey kidney cells. The mutant virion can adsorb to and penetrate the cell normally at the restrictive temperature, but cannot induce the synthesis of cellular deoxyribonucleic acid (DNA) nor initiate the synthesis of SV40-specific tumor, virion, or U antigens or viral DNA. First-cycle infection with purified ts(*)101 DNA is normal at the restrictive temperature, but the resulting progeny virions are still temperature-sensitive. The mutant neither complements nor inhibits other temperature-sensitive SV40 mutants or wild-type virions. The affected protein in the ts(*)101 mutant may be a regulatory structural protein, possibly a core protein, that is interacting with the viral DNA.     

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 Two Simian Virus 40-Specific RNA Molecules from Infected BS-C-1 Cells

Two discrete simian virus 40 (SV40) RNA species sedimenting at 19 and 16S, respectively, that are present in infected BS-C-1 cells were characterized with respect to the base composition and the ribonuclease T1 fingerprints. The base composition of the 19S SV40 RNA was found to be cytidylic acid (C), 23.0; adenylic acid (A), 28.3; guanylic acid (G), 23.9; and uridylic acid (U), 24.8; that of the 16S SV40 RNA was C, 19.3; A, 34.0; G, 22.0; and U, 24.7 mol%. Analysis of the ribonuclease T1 fingerprints indicated a difference in the base sequence of the 19 and 16S SV40 RNA. The presence of long sequences of adenylic acid residues (poly A) in these viral RNAs was confirmed.     

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.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: II. Detection and Characterization of Simian Virus 40 Pseudovirions

Purified simian virus 40 (SV40) virions, grown in primary African green monkey kidney cells labeled prior to infection with (3)H-thymidine, contain a variable quantity of (3)H-labeled deoxyribonucleic acid (DNA). This DNA is resistant to deoxyribonuclease, sediments at 250S, and is enclosed in a particle that can be precipitated with SV40-specific antiserum. DNA-DNA hybridization experiments demonstrate that this (3)H-labeled component in purified SV40 virions is cellular DNA. When this (3)H-labeled DNA is released from purified virus with sodium dodecyl sulfate, it has an average sedimentation constant of 14S. Sedimentation through neutral and alkaline sucrose gradients shows that this 14S DNA is composed of a collection of different sizes of DNA molecules that sediment between 11 and 15S. As a result of this size heterogeneity, SV40 virions containing cellular DNA (pseudovirions) have a variable DNA to capsid protein ratio and exhibit a spectrum of buoyant densities in a CsCl equilibrium gradient. Pseudovirions are enriched, relative to true virions, on the lighter density side of infectious SV40 virus banded to equilibrium in a CsCl gradient. Little or no cellular DNA was found in purified SV40 virus preparations grown in BSC-1 or CV-1 cells.     

Initial Site of Synthesis of Virus During Rescue of Simian Virus 40 from Heterokaryons of Simian Virus 40-Transformed and Susceptible Cells

Simian virus 40 (SV40) can be rescued from certain SV40-transformed hamster cells by fusion with susceptible African green monkey kidney (CV-1) cells, in the presence of ultraviolet-irradiated Sendai virus. We have determined the sites in which SV40 is produced during rescue in these heterokaryons. To determine the sequence, nuclei were isolated from fused cells at various times after fusion, separated on sucrose-density gradients, and assayed for infectious center formation and virus content on CV-1 monolayers. Virus was first detected in the transformed nucleus (40 hr postfusion), and later associated with both transformed and susceptible nuclei (68 to 72 hr). Viral rescue apparently does not depend upon the transfer of SV40 deoxyribonucleic acid to a susceptible CV-1 nucleus, since the transformed nucleus is the primary site of virus production. The time course of certain cytological events in the rescue process and in productive infection was found to be similar.     

Initial Stage of Transformation of Permissive Cells by Simian Virus 40: Development of Resistance to Productive Infection

A quantitative assay has been used to determine the conditions leading to acquisition of resistance of permissive cells to lytic infection. The number of cell colonies surviving infection depends on the occurrence of several cell divisions after infection. High yields of resistant colonies were obtained when infected, confluent cultures were released from contact inhibition 10 to 14 hr after infection. Infection of actively growing cells produced similar results, but halting further division by seeding these growing cells on confluent monolayers prevented the development of colonies. Colony formation was a direct function of multiplicities lower than 5. An inverse killing response was observed with higher multiplicities, yet colonies were produced at a multiplicity of infection as high as 50. Brief exposure of input simian virus 40 to ultraviolet light stimulated colony formation. Irradiation of the virus for longer periods of time led to reduction of colony formation at a rate slower than the rate of inactivation of viral infectivity. It was concluded that resistance is induced by simian virus 40 and that this alteration represents one of the earliest detectable characteristics of the transformation of permissive cells.     

Replication of Simian Foamy Virus in Monkey Kidney Cells

The structure of foamy virus, its mode of maturation, and the origin of vacuoles in monkey kidney cells are described.     

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.