Frank J. O'Neill

Amplification of papovavirus defectives during serial low multiplicity infections

Abstract Many human cell lines share with A172 glioblastoma cells the ability to support lytic growth of SV40 and to accumulate defectives on low multiplicity passage. The latter is also characteristic of quite a number of host cells for BKV; thus, the need for high multiplicity infection to accumulate defectives is not as stringent as previously envisioned. Very few lines share with A172 the retention of reiterated viral termination regions in the defectives.

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 excesss 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.

The archetype enhancer of simian virus 40 DNA is duplicated during virus growth in human cells and rhesus monkey kidney cells but not in green monkey kidney cells

Archetype SV40, obtained directly from its natural host, is characterized by a single 72-bp enhancer element. In contrast, SV40 grown in cell culture almost invariably exhibits partial or complete duplication of the enhancer region. This distinction has been considered important in studies of human tumor material, since SV40-associated tumor isolates have been described having a single enhancer region, suggesting natural infection as opposed to possible contamination by laboratory strains of virus. However, the behavior of archetypal SV40 in cultured cells has never been methodically studied. In this study we reengineered nonarchetypal 776-SV40 to contain a single 72-bp enhancer region and used this reengineered archetypal DNA to transfect a number of simian and human cell lines. SV40 DNA recovered from these cells was analyzed by restriction endonuclease analysis, PCR, and DNA sequencing. Reengineered archetype SV40 propagated in green monkey TC-7 or BSC-1 kidney cells remained without enhancer region duplication even after extensive serial virus passage. Archetype SV40 grown in all but one of the rhesus or human cell lines initially appeared exclusively archetypal. However, when virus from these cell types was transferred to green monkey cells, variants with partial enhancer duplication appeared after as little as a single passage. These findings suggest (1) that virus with a single 72-bp enhancer may persist in cultured cells of simian and human origin; (2) that variants with partially duplicated enhancer regions may arise within cell lines in quantities below limits of detection; (3) that these variants may enjoy a selective advantage in cell types other than those from which they arose (e.g., green monkey kidney cells); and (4) that certain cell lines may support a selective growth advantage for the variants without supporting their formation. Our data indicate that enhancer duplication may also occur in human as well as rhesus kidney cells. Thus, detection of enhancer region duplication may not, a priori, indicate laboratory contamination, nor does detection of a single 72-bp enhancer exclude the possibility that contamination may have occurred. These findings may be of relevance to studies attempting to detect SV40 DNA in human tumors or other clinical specimens.

Complex chromosome aberrations in continuous mammalian cell lines

Complex chromosome aberrations (CCA) are described, occurring spontaneously in low frequency, in numerous mammalian cell lines. These aberrations appear similar to those reported in leukocyte cultures of some Yanamama Indians. In some cell lines the frequency of CCA is increased by the administration of cytochalasin B (CB) a drug which prevents cytoplasmic division. The frequency of CCA may also be increased by the protease inhibitor tosyl lysyl chloro methyl ketone (TLCK). TLCK may also produce binucleate cells but unlike CB does not result in high degrees of multinucleation. In one cell line, 3T12, the simultaneous administration of CB and TLCK resulted in high frequencies of CCA. Thus the induction of CCA in cell culture is reproducible. However the etiology of CCA remains unknown.

Propagation of archetype and nonarchetype JC virus variants in human fetal brain cultures: demonstration of interference activity by archetype JC virus.

In immunologically normal individuals, the polyomavirus, JC virus (JCV), produces an asymptomatic primary infection followed by lifelong persistence of the virus in renal tubular epithelial cells. In some immunocompromised patients, however, in particular acquired immunodeficiency syndrome (AIDS) patients, JCV causes an opportunistic central nervous system (CNS) disorder, progressive multifocal leukoencephalopathy (PML). JCV DNA as it persists in kidneys (archetypal JCV) and JCV DNA isolated from PML lesions show differences in their regulatory regions in which transcription and replication are controlled. Archetypal JCV DNA has a single enhancer and no rearrangements or deletions in the regulatory region. In contrast, JCV DNA from PML isolates is characterized by alterations in the regulatory region. Some PML-associated JCVs can be grown in cultures of human fetal brain (HFB) cells. Growth of archetypal JCV in cultured cells has not been reported, however. Here we demonstrate successful propagation of the archetypal JCV, strain GS/K, in HFB cells. Growth occurred more slowly and to lower titers than is seen with the prototypical PML JCV strain Mad-1, with relatively few cells containing viral T antigen (T-Ag) or viral capsid protein, Vp1. Interestingly, GS/K growth could be enhanced, with a large increase in viral DNA and cytopathic effect, by coinfection with GS/B, a nonarchetypal brain-derived JCV variant isolated from the same PML patient as GS/K. The amount of GS/KDNA was also greatly enhanced when it was cotransfected with Mad-1 JCV DNA, the prototypical PML isolate. In contrast to GS/K plus GS/B—cotransfected cells, in GS/K plus Mad-1-infected cells, cytopathic effect was not increased. On subsequent passage of culture lysates to naïve cells, however, the infection produced by either combination of viral DNAs slowed, no cytopathic effect (CPE) was present, and the amount of GS/B or Mad-1 viral DNA was greatly reduced as compared to that of GS/K DNA. These data suggest that GS/K was able to use either GS/B or Mad-1 as a helper and that GS/K was in turn able to interfere with the growth of either helper virus. Archetype JCV can be successfully propagated in HFB cells, although infection develops much more slowly than that caused by the PML JCV variant Mad-1. The ability of archetypal and variant JCVs to enhance or retard each other’s replication may have implications in vivo for the maintenance of JCV persistence and the growth of JCV variants.

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.

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.

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.

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.

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.