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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., (Copyright 2000 Academic Press.)

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.

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.

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.

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.

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.

Low-passage BHK21/C13 cells respond to cytochalasin B (CB) by undergoing limited or controlled nuclear division. These cells respond to CB as normal cells do since nuclear division usually occurs only once. Premature chromosome condensation, a result of mitoses in highly multinucleate cells, occurs in less than 0.5% of the cells. When they are inoculated into weanling hamsters, s. c. tumors appear within 3 to 4 weeks with as few as 10(3) cells. When these cells are returned to cell culture they respond to CB with uncontrolled nuclear division and premature chromosome condensation. All cultured tumors respond in this manner regardless of the number of cells originally inoculated into animals. This suggests at least two possibilities: (a) that loss of controlled nuclear division in BHK cells is closely associated with or required for tumorigenicity, with in vivo passage selecting for a rare tumorigenic variant or (b) that loss of controlled nuclear division is secondary to tumorigenicity and results when cells are passed in vivo, i.e., in vivo passage has the direct effect of causing cells to lose controlled nuclear division. If the first possibility is operating, then it would be expected that a very small fraction of BHK21/C13 cells show uncontrolled nuclear division (approximately 1 of 1000 CB-treated cells). Also, clones of C13 should be nontumorigenic if only 1 of 1000 cells is tumorigenic. Extensive examination of CB-treated C13 cells shows 1 of 1250 cells to be highly multinucleated although not as highly multinucleated as tumor cells. This provides some evidence in support of the first possibility. However, three separate clones of C13 cells were found to be tumorigenic providing evidence supporting the second possibility. BHK21/C13 and various BHK21 tumors all appeared to grow to concentration densities markedly higher than hamster embryo fibroblasts. However, the tumor cells usually grew to the same density as did BHK21/C13 or only slightly higher. This suggests that loss of contact inhibition is not sufficient for loss of controlled nuclear division. It also suggests that the hypothetical 1 of 1000 tumorigenic cells and 1 of 1250 cells with uncontrolled nuclear division do not overgrow the "normal" cells because all cells grow to similar densities. The relationship between the ability to grow in agar and uncontrolled nuclear division was also examined. Approximately 1 of 2500 C13 cells were able to form colonies in agar. All three colonies isolated showed normal control of nuclear division. These results show that the ability to grow in agar may be separate from the expression of uncontrolled nuclear division. They also suggest a fundamental difference between tumor cells and cells that have been grown in agar.

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.

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.

Cultures of normal human and hamster and malignant human and hamster cells respond to cytochalasin B (CB) differently. The neoplastic cells become highly multinucleated with continuous nuclear division, while cytoplasmic division is prevented. These cells exhibit uncontrolled nuclear division. The normal cells show control of nuclear division, since CB treatment results in only binucleation, although cytoplasmic division is prevented. The CB-treated normal cells also show reduced incorporation of [3H]thymidine. When rapidly growing normal or neoplastic cells of either species are treated with cytosine arabinoside (ara-C), all cells are killed within 10 to 20 days. If the normal cells are treated with ara-C in the presence of CB, the cells survive for at least 35 to 40 days, suggesting that CB can protect normal cells from the destructive effects of ara-C. However, if malignant cells are treated with ara-C in the presence of CB, all cells are destroyed in 25 days. Although CB affords some protection to tumor cells, it is relatively small and appears only to effect a delay in ultimate cell death. Treatment of any of the cell lines with CB alone results in some loss of viability, but the drug is reversible although less so for the malignant cells, In additional experiments, are-C plus CB-treated normal and neoplastic cells were reversed from the drugs and propagated in normal medium at various periods after the initiation of ara-C treatment. ara-C plus CB-treated normal cells could be freely reversed at any time after the start of ara-C treatment up to at least 35 days. After reversal, cell growth resumed, and the total number of cells returned to that present prior to drug treatment. The tumor cells could not be freely reversed from ara-C plus CB, even when the ara-C was present for only 5 days. In this case the cells seemed to be destroyed at the same rate as in cultures where both drugs were kept in the medium. These results suggest that CB can protect normal but not malignant cells from the toxic effects of ara-C and offer a method to specifically destroy tumor cells.

Cytochalasin B (CB) prevents cytoplasmic cleavage without directly affecting nuclear division. Secondary cultures of mouse embryo fibroblasts or 3T3 cells show controlled nuclear division when treated with CB: only binucleated cells are formed. Many CB-treated transformed cells show uncontrolled nuclear division and become highly multinucleated. When CB-treated normal cells are concurrently infected with high inputs of SV40, many of these cells become highly multinucleated. It is suggested that these highly multinucleated cells represent abortively transformed cells since the actual number of transforming units (focus-forming units) of simian virus 40 (SV40) is too low to account for the appearance of these cells. Also, if the CB treatment is begun 6 days after SV40 inoculation, the large increase in highly multinucleated cells is not observed. Most cells stably transformed by SV40 or adenovirus show uncontrolled nuclear division when treated with CB. However, 3T3 cells transformed by SV40 or adenovirus and analyzed shortly after transformation are an exception and show controlled nuclear division. This property of 3T3 cells is apparently overcome by high inputs of SV40 since abortively transformed cells become highly multinucleated.

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.

Hamster BHK21 tumor cells and human rhabdomyosarcoma (RD) cells responded to cytochalasin B (CB) by becoming highly multinucleated. Nearly 80% of the BHK cells contained four or more nuclei after 7 days of exposure to CB, and 35% of these had at least seven nuclei. Similar frequencies of multinucleation were obtained with RD cells. Administration of 10(-3) M theophylline and dibutyryl adenosine 3',5'-cyclic monophosphate (Bu2cAMP) at 5X10(-4) M or 10(-3) M to CB-treated RD or BHK cells greatly reduced the frequency of cells with five or more nuclei. The frequency of cells with seven or more nuclei was reduced to less than 5%. Along with this reduction in highly multinucleated cells was an increase in the incidence of binucleated cells. Bu2cAMP was toxic and caused many CB-treated BHK cells to detach from the culture surface, but not all cells were killed. Bu2cAMP had little toxic effect on CB-treated RD cells. These observations indicated that the inhibition of high degrees of multinucleation were not the result of nonspecific toxic effects of Bu2cAMP but that nuclear division was limited by it. The effect of Bu2cAMP on density-dependent inhibition of growth was also studied. Addition of only theophylline and Bu2cAMP to either BHK or RD cells resulted in growth to significantly lower saturation densities. The toxicity of Bu2cAMP on cells in crowded cultures apparently caused the limited propagation. Bu2cAMP resulted in significant cell killing or detachment but, once the lower saturation densities were reached, cell death was minimized. Thus Bu2cAMP did not restore contact inhibition per se. It was also found that untreated RD cells grew to lower concentration densities than expected from the microscopic inspection of cells in situ. Microscopic inspection revealed high concentration densities and numerous mitoses. This apparent contradiction was due to the ability of RD cells to fuse upon the attainment of confluence and to produce multinucleated cells without the aid of CB.

Clonal derivatives of C3HMT murine mammary cell lines in culture demonstrate conversion of mammary tumor virus (MMTV) expression at a rate of appriximately 6 per 100 clones. This alteration is largely unidirectional from a relatively high level (MMTV(H)) to a 10 fold lower level (MMTV(L)). This high rate of MMTV(L) variant conversion is in apparent contrast to the presumably mutational rate (approximately 3 per million cells) that governs development of resistance to 6-thioguanine (TG) in the same mammary cells. In somatic cell hybrids between different MMTV TGr clones and mouse or hamster TK- cells, expression of constitutive levels of MMTV and responsiveness to dexamethasone induction is dominant. Thus MMTV expression is regulated by at least two levels of positive control, constitutive expression and glucocorticoid stimulation, but the former is subject to a high rate of variant formation.

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.

Mouse, hamster, rat, human, and chick cells were transformed by RNA and DNA tumor viruses: simian virus 40, adenovirus type 7, Kirsten mouse sarcoma virus (Ki-MuSV), Moloney mouse sarcoma virus, and Rous sarcoma virus. All cultures of transformed cells grew to high concentration densities. Normal and transformed cells were treated with cytochalasin B (CB) at concentrations preventing cytoplasmic cleavage. Cells altered by DNA tumor viruses responded to CB with numerous nuclear divisions resulting in highly multinucleated cells. All but one line of cells transformed by RNA tumor viruses responded to CB with usually only one and occasionally two nuclear divisions. Only binucleated cells were formed. One clone of CB-treated BALB/c mouse embryo fibroblasts transformed by Ki-MuSV showed numerous cells with four and five nuclei. HOWEVER, IN CONTRASt to cells transformed by DNA viruses, few cells had seven or more nuclei. These results suggest that, in the presence of CB, cells transformed by DNA tumor viruses show uncontrolled nuclear division, whereas cells tranformed by RNA tumor viruses show controlled nuclear division.

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.