The relationship between calcium, MAP kinase, and DNA synthesis in the sea urchin egg at fertilization.

Fertilization releases the brake on the cell cycle and the egg completes meiosis and enters into S phase of the mitotic cell cycle. The MAP kinase pathway has been implicated in this process, but the precise role of MAP kinase in meiosis and the first mitotic cell cycle remains unknown and may differ according to species. Unlike the eggs of most animals, sea urchin eggs have completed meiosis prior to fertilization and are arrested at the pronuclear stage. Using both phosphorylation-state-specific antibodies and a MAP kinase activity assay, we observe that MAP kinase is phosphorylated and active in unfertilized sea urchin eggs and then dephosphorylated and inactivated by 15 min postinsemination. Further, Ca(2+) was both sufficient and necessary for this MAP kinase inactivation. Treatment of eggs with the Ca(2+) ionophore A23187 caused MAP kinase inactivation and triggered DNA synthesis. When the rise in intracellular Ca(2+) was inhibited by injection of a chelator, BAPTA or EGTA, the activity of MAP kinase remained high. Finally, inhibition of the MAP kinase signaling pathway by the specific MEK inhibitor PD98059 triggered DNA synthesis in unfertilized eggs. Thus, whenever MAP kinase activity is retained, DNA synthesis is inhibited while inactivation of MAP kinase correlates with initiation of DNA synthesis.

Host range analysis of simian virus 40, BK virus and chimaeric SV40/BKV: relative expression of large T-antigen and Vp1 in infected and transformed cells.

Simian virus 40 (SV40) persists in Rhesus monkeys and productively infects cultured simian kidney cells. In contrast to the closely related human virus BKV, SV40 is known to propagate inefficiently in human embryonic kidney (HEK) cells and human fibroblasts (HFF). We examined the growth of SV40, BKV and the chimaeric genome virus, SV40/RFV, in several types of human cells. We analysed replication, expression of T-Ag and Vp1 capsid proteins, and cytopathic effects (CPE). We also compared T-Ag and Vp1 expression in infected versus transformed HFF cells. Although SV40 DNA replicated in HFF and in one subtype of HEK cells, viral DNA accumulated slowly and did not reach high levels until six to eight weeks after transfection. In HFF or HEK cells there was little T-Ag produced but Vp1 was produced in significant amounts in HFF cells. In HFF cells the Vp1/T-Ag ratio was approximately 200: 1, and expression of the viral late region appeared to inhibit expression of the T-Ag gene. In contrast, BKV and the SV40/RFV hybrid propagated well in HEK and HFF cells. The Vp1/T-Ag ratios were also high in BKV and SV40/RFV infected HFF cells but more T-Ag was produced with BKV and SV40/RFV Because SV40/RFV contained the RFV capsid genes but a SV40 T-Ag gene and regulatory region, the human versus simian host range of SV40 was controlled by the viral late region, or one or more capsid proteins. This suggested that the production of small amounts of T-Ag could not by itself account for poor growth of SV40 in HFF cells and that very small, barely detectable amounts of T-Ag were sufficient to activate Vp1 gene expression. Also, although some feature of the SV40 late region prevented rapid growth of the virus in HFF cells, poor virus growth could not be explained by the inability to produce a significant amount of Vp1. Although little T-Ag accumulated in SV40 infected HFF and HEK cells, transformants contained large amounts of T-Ag. In transformants there was a reversal of the Vp1/T-Ag ratio, such that T-Ag was now in 10-20 fold greater amount than Vp1. The relatively large amount of T-Ag in transformants could be accounted for by the relative absence of Vp1, which may inhibit T-Ag production, or by integration of the T-Ag gene at a site in the cell DNA which allows for elevated T-Ag gene expression.

DNA sequences outside the simian virus 40 early region cause downregulation of T-antigen production in permissive simian cells.

Using a series of modified wtSV40 and early region SV40 DNAs we assayed the effect of viral late region sequences on T-antigen production by the SV40 early region. We found that SV40 late region (L-SV40) DNA sequences reduced T-antigen (T-Ag) production by the SV40 early region (E-SV40) when both viral regions were linked as they are in wtSV40 DNA. This was demonstrated by Western analysis which showed that E-SV40 DNA produced 10 times more T-Ag than wtSV40 DNA L-SV40, with its own promoter but unlinked to E-SV40 DNA, also greatly inhibited T-Ag production when it was contrasfected with E-SV40. Therefore, L-SV40 DNA inhibited T-Ag production by E-SV40 DNA when present in cis or in trans. We have shown that expression of the SV40 late transcription unit dominated that of the early (T-Ag gene) transcription unit because late region RNA accumulated to much higher levels than early viral RNA. However, in contrasfected cells L-SV40 DNA did not replicate to higher levels than E-SV40 DNA. We offer a model for control of T-Ag expression in which a relatively small amount of T-Ag activates late transcription at the expense of T-Ag gene transcription and that this represents a switch from early to late viral gene expression. We suggest that when activation of the late transcription unit occurs at the late promoter, expression of the T-Ag gene is greatly reduced. The L-SV40 promoter may inhibit T-Ag gene transcription by sequestering cellular factors required for early transcription, factors which may be present in limited amounts. We suggest further that activation of late transcription allows for the necessary production of large amounts of capsomeres and virions and downregulation of early transcription prevents the early region from interfering with capsid synthesis. We tested the model using a construct with a wild-type T-Ag gene but with mutations in the SV40 major late promoter which prevent the promoter from being bound by cellular repressors of late transcription. We found that this construct, which overproduces late SV40 RNA, was defective for T-Ag production. This indicates that activation of the late promoter results in repression of T-Ag gene expression.

Mutations of pma-1, the gene encoding the plasma membrane H+-ATPase of Neurospora crassa, suppress inhibition of growth by concanamycin A, a specific inhibitor of vacuolar ATPases.

Concanamycin A (CCA), a specific inhibitor of vacuolar ATPases, inhibited growth of Neurospora crassa in medium adjusted to pH 7 or above. Mutant strains were selected for growth on medium containing 1.0 microM CCA. Sixty-four (of 66) mutations mapped in the region of the pma1 locus, which encodes the plasma membrane H+-ATPase. Analysis of V-ATPase activity in isolated vacuolar membranes from the mutant strains showed wild-type activity and sensitivity to CCA. In contrast, plasma membrane H+-ATPase activity in isolated plasma membranes from the mutants was reduced as compared with wild-type, and in four strains the activity showed increased resistance to vanadate. The most interesting change in the plasma membrane H+-ATPase was in kinetic behavior. The wild-type enzyme showed sigmoid dependence on MgATP concentration with a Hill number of 2.0, while the seven mutants tested exhibited hyperbolic kinetics with a Hill number of 1.0. One interpretation of these data was that the enzyme had changed from a functional dimer to a functional monomer. Mutation of the plasma membrane H+-ATPase did not confer resistance by preventing uptake of CCA. In the presence of CCA both wild-type and mutant strains were unable to accumulate arginine, failed to concentrate chloroquine in acidic vesicles, and exhibited gross alterations in hyphal morphology, indicating that the CCA had entered the cells and inactivated the V-ATPase. Instead, we hypothesize that the mutations conferred resistance because the altered plasma membrane H+-ATPase could more efficiently rid the cell of toxic levels of Ca2+ or protons or other ions accumulated in the cytoplasm following inactivation of the V-ATPase by CCA.

Identification of p53 unbound to T-antigen in human cells transformed by simian virus 40 T-antigen.

In several clones of SV40-transformed human cells, we investigated the relative amounts of large T-Antigen (T-Ag) and p53 proteins, both unbound and associated within complexes, with the goal of identifying changes associated with transformation and immortalization. Cells were transformed by wild type (wt) T-Ag, a functionally temperature sensitive T-Ag (tsA58) and other T-Ag variants. Western analysis showed that while most of the T-Ag was ultimately bound by p53, most of the p53 remained unbound to T-Ag. Unbound p53 remained in the supernatant after a T-Ag immunoprecipitation and p53 was present in two to fourfold excess of T-Ag. In one transformant there was five to tenfold more p53 than T-Ag. p53 was present in transformants in amounts at least 200-fold greater than in untransformed human cells. In wt and variant T-Ag transformants, including those generated with tsA58 T-Ag, large amounts of unbound p53 were present in both pre-crisis and immortal cells and when the cells were grown at permissive or non-permissive temperatures. We also found that in transformants produced by tsA58, an SV40/JCV chimeric T-Ag and other variants, T-Ag appeared to form a complex with p53 slowly perhaps because one or both proteins matured slowly. The presence in transformed human cells of large amounts of unbound p53 and in excess of T-Ag suggests that sequestration of p53 by T-Ag, resulting from complex formation, is required neither for morphological transformation nor immortalization of human cells. Rather, these results support the proposal that high levels of p53, the T-Ag/p53 complexes, or other biochemical event(s), lead to transformation and immortalization of human cells by T-Ag.

Immortalization of human cells by mutant and chimeric primate polyomavirus T-antigen genes.

Human fibroblasts were morphologically transformed with wild type and mutant SV40 T-antigens (T-Ags) and with SV40/JCV and SV40/BKV chimeric T-Ags. The transformants were then assayed for the attainment of immortal cell growth. Several observations relating T-Ag and T-Ag domains to immortalization were made. Approximately 10% of SV40-transformants became immortal. Transformants generated by transfection or infection of cells with C-terminal T-Ag deletion mutants of SV40 did not immortalize. SV40/JCV and SV40/BKV chimeric T-Ags, containing C-terminal sequences from JCV or BKV, immortalized cells more efficiently than did the intact SV40 T-Ag, suggesting that the C-termini of the JCV and BKV T-Ags contain an enhanced immortalization function. However, chimeras in which the N-terminal or proximal-central portions of T-Ag were composed of JCV sequences failed to immortalize but did induce transformation. Constructs in which the JCV T-Ag Rb binding domain was replaced with SV40 sequences transformed human cells, but again the cells failed to immortalize. Transformants and immortalized cell lines produced by some SV40/JCV chimeras, contained p53 which was unbound by T-Ag. This occurred under conditions where p53 from SV40 and SV40/BKV transformants was bound to T-Ag. This may reflect the reduced stability of the SV40/JCV T-Ags.

Persistence of the SV40 early region without expression in permissive simian cells.

SV40 containing recombinant vectors were introduced into permissive simian, non-permissive rodent and semi-permissive human cell lines, and assayed for transformation. All mouse and human cell clones expressed T-antigen (T-Ag) and were morphologically transformed when they contained only the wt T-Ag gene (E-SV40) or the entire wt viral genome with an interrupted late region. However, of 63 simian clones with these recombinant vectors, none became morphologically transformed and T-Ag containing cells were rare or absent. Nearly all simian cell lines made either no detectable early SV40 RNA or only small amounts of viral RNA but contained viral DNA restriction fragments similar to those in the original recombinant vectors. Functional T-Ag genes were recoverable from several cell clones and used to regenerate infectious virus. Hence, T-Ag gene expression had been suppressed. We found two conditions where T-Ag expression was activated. In a BSC-1 cell line containing E-SV40 DNA, subsequent introduction of a vector with a functional viral late coding region (L-SV40) resulted in the appearance of T-Ag and transformation. These findings suggest that L-SV40 sequences activate or enhance T-Ag expression and that this activation requires a functional Vpl gene. We found also, that vectors with E-SV40 DNA from the bipartite variant EL-SV40 consistently transformed simian CV-1 cells. Transformation was shown to be effected by the multiple alterations present in the regulatory region of this variant.

Reconstitution of wild type viral DNA in simian cells transfected with early and late SV40 defective genomes.

The DNAs of polyomaviruses ordinarily exist as a single circular molecule of approximately 5000 base pairs. Variants of SV40, BKV and JCV have been described which contain two complementing defective DNA molecules. These defectives, which form a bipartite genome structure, contain either the viral early region or the late region. The defectives have the unique property of being able to tolerate variable sized reiterations of regulatory and terminus region sequences, and portions of the coding region. They can also exchange coding region sequences with other polyomaviruses. It has been suggested that the bipartite genome structure might be a stage in the evolution of polyomaviruses which can uniquely sustain genome and sequence diversity. However, it is not known if the regulatory and terminus region sequences are highly mutable. Also, it is not known if the bipartite genome structure is reversible and what the conditions might be which would favor restoration of the monomolecular genome structure. We addressed the first question by sequencing the reiterated regulatory and terminus regions of E- and L-SV40 DNAs. This revealed a large number of mutations in the regulatory regions of the defective genomes, including deletions, insertions, rearrangements and base substitutions. We also detected insertions and base substitutions in the T-antigen gene. We addressed the second question by introducing into permissive simian cells, E- and L-SV40 genomes which had been engineered to contain only a single regulatory region. Analysis of viral DNA from transfected cells demonstrated recombined genomes containing a wild type monomolecular DNA structure. However, the complete defectives, containing reiterated regulatory regions, could often compete away the wild type genomes. The recombinant monomolecular genomes were isolated, cloned and found to be infectious. All of the DNA alterations identified in one of the regulatory regions of E-SV40 DNA were present in the recombinant monomolecular genomes. These and other findings indicate that the bipartite genome state can sustain many mutations which wtSV40 cannot directly sustain. However, the mutations can later be introduced into the wild type genomes when the E- and L-SV40 DNAs recombine to generate a new monomolecular genome structure.

Host range analysis of a chimeric simian virus 40 genome containing the BKV capsid genes.

Simian virus 40 (SV40) propagates poorly in cells from human embryonic kidney (HEK) and human fetal fibroblasts (HFF) while BK virus grows well in many human cell types. It has been suggested that sequences within the SV40 late region but not within the BKV late region may act to inhibit growth of virus in HEK and HFF cells. In order to test this and to identify a late region host range function, we have replaced the late region of wtSV40 DNA with the late region of RFV (a variant of BKV) to produce an intermolecular hybrid or chimera. The constructed SV40/RFV chimeric genome contained approx. 5900 base pairs, more than 650 base pairs greater than wtSV40. Nevertheless, when introduced by transfection the chimera appeared to be infectious. Three chimeric genomes were recovered from infected cells and all contained deletions of nearly 600 base pairs, exclusively at the region of the 3' terminal junction. Since all three chimeras propagated in human HFF and HEK cells, the RFV late region and not the RFV regulatory region possesses a host range function required for growth in human cells. Analysis of T-antigen gene expression suggests that the replacement of the SV40 late region with the BKV late region leads to full expression of the SV40 early region in human cells. Two chimeras exhibited a BKV-like host range and the third exhibited both a BKV and an SV40-like host range. We determined precisely which sequences were deleted in each chimera and we exchanged 3' terminal junction fragments containing these deletions, between two chimeras with different host ranges. From these experiments we demonstrated that: (1) The 3' terminus of the SV40 large T-antigen gene is required for growth of SV40/RFV in TC-7 and CV-1 simian cells but not for growth in human cells; (2) while the SV40 late region is refractory for growth in human cells, the RFV late region is not refractory for growth in simian cells; (3) the 3' terminus of the RFV T-antigen gene is not required for growth in human cells. The results of the 3' terminal junction exchanges and studies of early gene expression also demonstrate that BKV and SV40 can penetrate human and simian cells, even when they failed to grow in one cell type.

Host range determinant in the late region of SV40 and RF virus affecting growth in human cells.

WtSV40 and its variant EL-SV40 (contains two complementing defective genomes) fail to productively infect human embryonic kidney cells or human fibroblasts. However, early SV40 (E-SV40) genomes can propagate in human cells when complemented by a particular late RF virus (L-RFV) genome or the closely related wtBKV genome. The L-RFV genome (L-RFV clone H) contains a deleted early region, a complete set of BKV capsid genes, and a single SV40 regulatory region (acquired by recombination). In contrast, it was not possible to make the reciprocal genome cross in human cells; late SV40 genomes containing a deleted early region do not complement early RFV or early BKV DNAs. The L-RFV clone H genome was also shown to complement wtSV40 in human cells. However, wtSV40 DNA was rapidly lost and replaced by a defective SV40 genome. The SV40 defective (E-SV40 alpha) contained a deletion of the late region, an intact early region, and paired with L-RFV clone H DNA to form a new hybrid virus. In human cells wtSV40 was also complemented by wtBKV DNA, but after two serial passages SV40 DNA disappeared. These findings indicate that SV40 late or capsid gene sequences, but not SV40 early sequences, generate a block to SV40 growth in human cells. When the SV40 late region is replaced by a RFV or a BKV late region, E-SV40 DNA propagates efficiently in human cells and in some cases more rapidly than wtBKV. Northern blot hybridization indicates that SV40 DNA is poorly transcribed in human cells when the SV40 late region is present.

Transformation of human cells by a polyomavirus containing complementing JCV and RFV genomes.

Molecularly cloned viral DNA from late RFV (L-RFV) and early JCV (E-JCV) were transfected into human fetal brain (HFB) cells and complementation was demonstrated. A new infectious virus (E-JCV/L-RFV) was produced. Infection resulted in partial transformation of HFB and human embryonic kidney cells. No transformation was observed with EL-JCV or wtJCV. The transformants contained T-antigen and had a lifespan similar to SV40-transformed human cells but failed to express some phenotypes of transformation. All transformants contained E-JCV viral DNA, usually both integrated and episomal. Although no L-RFV DNA was present in the transformants, L-RFV appears to play a role in the initiation of transformation.

Late coding region sequences required for competition by SV40 defectives.

SV40 defectives containing the complete early coding region (E-SV40) or the complete late region (L-SV40) were separately transfected into green monkey cells. They were analyzed for their ability to compete with wtSV40 (introduced by infection) or to undergo replication in the presence of constitutively produced SV40 T-antigen. L-SV40 competed very strongly. It appeared rapidly in infected cells, overgrowing wt genomes by at least 10:1. In addition, it slowed the growth of wt virus and reduced its ability to kill cells. L-SV40 DNA, as expected, replicated continuously in Cosl cells. E-SV40 genomes were poor competitors. They appeared slowly and by themselves did not overgrow wtSV40. When transfected into Cosl cells, E-SV40 genomes replicated efficiently for the first few days and disappeared within a week. Deletion or insertion mutations were introduced into a molecular clone of L-SV40, within the Vp1 gene or the Vp2 gene. All mutants were unable to form infectious virus in two different assays. The mutants were then assayed for competition against wtSV40 and for replication in Cosl cells. The Vp1 mutants competed very poorly with wt genomes and were rapidly lost from coinfected cells. These mutants, like E-SV40, replicated for only a few days in Cosl cells. In contrast, the Vp2 mutant competed with wtSV40 nearly as well as L-SV40. It also replicated continuously rather than transiently in Cosl cells. Next, we determined whether L-SV40 could effectively compete with other evolved SV40 defectives, not containing the late region but containing up to nine SV40 origin regions. We have shown that within five serial passages, L-SV40 became the predominant viral DNA species and the other defectives were lost. Although the Vp1 mutants and E-SV40 were weak competitors, they were shown to recombine with wtSV40 genomes to generate new L-SV40 genomes which again became the predominant species of viral DNA. These results demonstrate that L-SV40 is a potent competitor and that the Vp1 gene or a part of Vp1 plays an important role in this extraordinary competition. We suggest that the Vp1 gene functions to allow L-SV40 genomes to persist rather than generating a product which directly interferes with wtSV40 replication.

Complementation between SV40 and RFV defectives and acquisition of SV40 origins by late RFV genomes.

EL SV40 and RFV are variants of SV40 and BKV which contain bipartite or dual genomes. One molecule contains all the early viral sequences (E-SV40, E-RFV) and the other all the late viral sequences (L-SV40, L-RFV). Early and late genomes complement one another during productive infection. Experiments were designed to determine if E-genomes of one virus could complement L-genomes of another virus. If complementation did occur, intermolecular recombination events which lead to a more efficient infection or an altered host range might occur, and the sequences involved could than be identified. Two combinations were generated by direct transfection of BSC-1 green monkey cells. E-RFV and L-SV40 DNA complementation resulted in hybrid virus growth and cell killing. The hybrid demonstrated a narrow host range. Following serial passage, some E-RFV genomes contained SV40 origin region sequences but these recombinants did not overgrow prototype E-RFV genomes, even after many virus passages. In addition, no significant alterations in host range could be detected. Complementation between E-SV40 and L-RFV yielded a virus with a relatively wider host range. Virus growth and cell killing appeared very slowly at first. However, with each passage of E-SV40/L-RFV, cell killing occurred progressively more rapidly, until passage 7 when it became extensive in 7 days rather than 6-8 weeks. Infected cells contained 10-20 times more E-SV40 than L-RFV DNA during the first passage. However, by passage 7, both genomes were equally represented. During serial passage, L-RFV DNA acquired SV40 sequences from around the origin and the terminus of replication, such that recombinant (r) L-RFV genomes contained three SV40 origins [corrected] (including the 72-bp repeat) and 2 termini, and prototype L-RFV DNA was lost. E-SV40/rL-RFV demonstrated an altered host range propagating in some cell lines which did not support E-SV40/L-RFV growth. Both the host range change and the increased growth of rL-RFV genomes were shown to be at least partly caused by the acquisition of the SV40 sequences.

Isolation of a papovavirus with a bipartite genome containing unlinked SV40 and BKV sequences.

Wild-type (wt) BK virus was introduced into permissive BSC-1 cells along with either early or late defective SV40 genomes. The defectives contained all of the late (L-SV40) or all of the early (E-SV40) coding sequences. Persistently infected (PI) BSC-1 cultures were established and contained wt BKV DNA and E- or L-SV40 DNA in Hirt supernatants. Each of the BKV/SV40 combinations could be serially passed in BSC-1 cells. Also, DNase I digestion of virus stocks from BKV/E-SV40 infections did not eliminate E-SV40. This suggested that (1) E-SV40 genomes could be packaged in BKV capsids and (2) BKV T antigen acted to stimulate the growth of L-SV40 genomes. During continuous culture of PI BSC-1 cells containing BKV and L-SV40, wt BKV genomes were lost and replaced by a BKV defective. The BKV defective (E-BKV) contained a deletion in the late region, an intact early region, and a duplication of the origin. This combination represents a new papovavirus with a bipartite genome in which the early region is derived from BKV and the late region from SV40, and both are present in separate molecules. The BKV and SV40 defectives complement each other for infectivity. Infectious virus is formed with the E-BKV genomes packaged in SV40 capsids. It is hypothesized that this kind of recombination (reassortment) is a way in which papovaviruses may generate variation. The host range for the new BKV/SV40 is narrow. It propagates well in BSC-1 cells, relatively poorly in fetal human brain cells, and not at all in green monkey TC-7 or human embryonic kidney cells. However, it transforms fetal human brain cells at a frequency 25-50 times greater than wt BKV does.

In vitro growth control phenotypes of transformed rodent cells prior to and following tumorigenesis.

A number of virus and chemical carcinogen-transformed cell lines were generated in tissue culture and analyzed for growth control phenotypes prior to and following tumorigenesis in appropriate hosts. The cell lines include those of mouse, rat, human, and Syrian hamster, transformed by papovaviruses and adenoviruses (DNA) or murine (RNA) tumor viruses. Cell lines were assayed for: (a) multinucleation or uncontrolled nuclear division (UND+) and uncontrolled DNA synthesis in cytochalasin B (CB) medium; and (b) the continuation of DNA synthesis in media containing reduced (0.5%) amounts of serum. All or nearly all lines of DNA virus transformants exhibited UND+ and high frequencies of DNA-synthetic cells in CB medium. Two lines of SV40-transformed hamster cells also showed UND+ following tumorigenesis in weaning hamsters. In addition, DNA virus transformants showed the ability to continue DNA synthesis unabated in low-serum medium. In contrast, the mouse sarcoma virus (MSV)-transformed lines exhibited varying degrees of controlled nuclear division and reduced DNA synthesis in CB medium, both prior to and following tumorigenesis. However, the reduction in DNA-synthetic cells was often not as great as that found in untransformed cells. Results similar to the RNA virus transformants were observed with hamster cells transformed by chemical carcinogens. Nearly all of the MSV-transformed lines showed significantly reduced levels of DNA synthesis in low-serum medium as was found in untransformed cells. One cell line, KA31, was followed through three consecutive in vivo tumorigenic passages, but these cells did not acquire UND+ or the ability to continue DNA synthesis in low-serum medium. These results suggest that many MSV- and carcinogen-transformed rodent cells exhibit transformation phenotypes at levels barely above those of normal cells and markedly less than those of DNA virus transformants, and yet they are tumorigenic.

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.

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.