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'I'yfi-TI i ,2. cl _ ,5“: #872,; '0’ 4", ::'I‘ W, filfi',‘ #1:}! iii/Inwa- (“"1'4’Mzif'. Mum} :%¥, Av Mfr!”- )Zéffl ""3"“16. a; '2... J”: u]§2{"% 1"}, r: II I 11""! If". /' f‘ '1’! l' 1’...” III” 3'17! ”PHI-1‘- “J. LIBRARY Michigan State University This is to certify that the dissertation entitled The Involvement of Viral DNA Replication and Recarbination, in the Integration Pathway of Polyoma Virus presented by David L . Hacker has been accepted towards fulfillment of the requirements for Ph . D. Microbiology degree in “4‘th n . UNA-PL» Major professor Date 11/19/87 MS U i: an Affirmative Action/Equal Opportunity Institution 0. 12771 MSU LIBRARIES .—;—. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. THE INVOLVEMENT OF VIRAL DNA REPLICATION AND RECOMBINATION IN THE INTEGRATION PATHWAY OF POLYOMA VIRUS BY David L. Hacker A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 19 87 ABSTRACT THE INVOLVEMENT OF VIRAL DNA REPLICATION AND RECOMBINATION IN THE INTEGRATION PATHWAY OF POLYOMA VIRUS BY David L. Hacker The possible contributions of viral DNA replication and recombination in the integration of viral DNA in polyoma-transformed cells were investigated. Extensive viral DNA replication was observed at early times after infection of rat cells at 33°C but not at 37°C. This was true for most strains of virus tested including wild-type AZ. By ten days postinfection, the level of viral DNA was about 10-fold higher in cells infected at 33°C than in cells infected at 37°C, but the transformation frequency was only 2-3 times higher at the lower temperature. No differences were observed in the integration patterns of wild-type A2 in transformants obtained from 33°C and 37°C infections. Viral DNA replication occurred in only a small fraction of the infected cells ((0.2%). Large T-antigen expression is also higher at 33°C than at 37°C, and this viral protein is required continuously for the synthesis of viral DNA in nonpenmissive cells. Since the increase in the level of viral DNA at 33°C did not dramatically increase the transformation frequency or alter the integration pattern in transformed cells, it is doubtful that this increase affects the integration pathway of polyoma. One of the viral strains (NGS9RA) which did not display an increased level of viral DNA at 33°C was investigated further. Hybrid viruses consisting of sequences from both A2 and NGS9RA were constructed and analyzed for their ability to synthesize viral DNA at 33°C in nonpemmissive cells. Using this approach, a mutation affecting DNA replication was mapped to large Thantigen. The fate of the viral genome in nonpermissive cells was also studied by infecting rat cells with two restriction sitedninus strains. Recombination between the parental genomes was followed by the detection of wild-type restriction fragments in the population of unintegrated viral genomes at early times after infection and in transformants resulting from the mixed infections. No recombination ‘was detected in the former, but evidence of interviral homologous recombination was observed in 38% of the transformants. These results suggest that recombination is involved in the integration pathway of polyoma virus. Since recombination was not detected at early times after infection, it may only occur in a small population of cells which ultimately become transformed. 'Ib Mom and Dad 11 ACKNOWLEDGEMENTS I thank Dr. Michele Pluck for direction, encouragement, and financial support during the course of this project. I thank my guidance committee, Drs. Larry Snyder, Jerry Dodgson, Susan Cbnrad, and Barbara Sears, for their advice during my years as a graduate student. I thank Karen Friderici, Ming-Chu Chen, and Sonya Michaud for data presented in Chapter 3; Steve Spatz and Peter Brunovskis for assistance with the in situ hybridization experiment in Chapter 2; and Claudette Priehs and Sue Kalvonjian for the maintainence of the cell cultures and virus stocks that were used in this research. Finally, I thank the other members of the Fluck laboratory for making work enjoyable. I especially thank Dr. Larry Winberry, Karen Friderici, Claudette Priehs, and Dr. Saw Yin Oh for scientific and moral support. iii TABLE OF CONTENTS List Of TableSOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOO v List Of FigureSOOOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOO... Vi Chapter 1: Chapter 2: (Shapter 3: Chapter 4: Literature Review Introduction........................................ 1 Early Gene Expression and DNA Replication in Permissive Cells................................. 3 Neoplastic Transformation by Polyomavirus........... 16 DNA Recombination in Mammalian Cells................ 23 Summary............................................. 31 References.......................................... 33 The Role of Viral DNA Synthesis in Neoplastic Transformation of Nonpermissive Rat F—lll Cells by Polyoma Virus Abstract............................................ 44 Introduction........................................ 46 Materials and Methods............................... 48 Results............................................. 52 Discussion.......................................... 78 References.......................................... 90 A Nonlethal Mutation in Large T-Antigen of Pblyoma Virus Which Affects Viral DNA Synthesis Abstract............................................ 94 Introduction........................................ 95 Materials and Methods............................... 95 Results.............................................lGG Discussion..........................................108 References..........................................ll3 The Involvement of Interviral Recombination in the Integration/Transformation Pathway of Polyoma Virus Abstract............................................116 Introduction........................................118 Materials and Methods...............................126 Results.............................................124 Discussion..........................................l46 References..........................................153 smry am ConCIuSionSOOOOOOOOOOOOOOO.OOOOOOOOOOOOOOOO000......156 iv Chapter 2: Chapter 3: Chapter 4: LI ST OF TABLES Table 1. Analysis of in situ hybridization of A2-infected F-ll'l—rat cells at 33°C and 37°C.. 63 Table 2. Transformation frequencies................ 77 Table 3. Summary of integration analysis........... 81 Table 1. Intwration aDaIYSiSOOOOOOOOOOOOO00.00.00.167 Table 1. Summary of recombination in transformed cell lines...........................132 Table 2. Transformation rates......................l45 Chapter 1: Chapter 2: Chapter 3: (:hapter 4: LIST OF FIGURES Figure 1. Physical map of polyoma virus genome........................................... 5 Figure 2. Physical map of the noncoding region of polyoma virus................................. 8 Figure 3. Proposed model for formation of duplicate junctions following nonhomologous recombination...................... 26 Figure 4. Single-strand annealing model of homologous recombination......................... 29 Figure 1. comparison of viral DNA synthesis in permissive and nonpermissive cells............ 53 Figure 2. Comparisons of various viral strains for viral DNA synthesis in rat F—lll cells at 33°C and 37°C..................... 57 Figure 3. In_situ hybridization of A2- infected F-lll cells............................. 61 Figure 4. The role of large T-antigen in viral DNA synthesis........................... 66 Figure 5. Viral DNA replication in an A2-transformed cell line......................... 70 Figure 6. western blot of whole cell extracts of polyoma-infected F—lll cells.................. 73 Figure 7. Presence of integrated tandem repeats in A2-transformed Felll cells............ 79 Figure 8. Analysis of the number of integration sites in A2-transformed cells........ 82 Figure 1. Map of polyoma virus..................... 97 Figure 2. Comparison of viral DNA synthesis between A2 and RA in F—lll cells.................101 Figure 3. Transformation in mixed infections of F-lll cells...................................1G4 Figure 4. comparison of viral DNA synthesis using hybrid viruses.............................109 Figure 1. Partial restiction maps of polycma strains..................................121 Figure 2. Analysis of recombination in the unintegrated viral genomes...................127 Figure 3. Recombination in the 3.6 kbp AvaI/BamHI interval..............................130 Figure 4. Recombination in the 1.3 and 1.7 kbp AvaI/BamHI intervals.........................l34 vi Figure 5. Analysis of the 1.3 and 1.7 kbp AvaI/BamHI restriction fragments.................l38 Figure 6. Analysis of tsB-transformed cell lines.......................................l43 Figure 7. Gradient of recombination in the 1.3 and 1.7 kbp AvaI/BamHI intervals.............l49 vii Chapter 1 . Literature Review Introduction Polyamavirus (Py) is a DNA tumor virus of mice. It and the other members of the polyamavirus genus, the JC and BK viruses of humans, simian virus 40 (SV40) and B lymphotrophic virus of monkeys, and hamster papovavirus, are grouped with the papillomavirus genus to form the papovaviridae family (1) . Papovaviruses are nonenveloped viruses with a double-stranded covalently-closed circular DNA genome which, in the case of Py, is 5,292 base pairs (bp) in length and codes for six proteins (2) . The DNA is packaged in the viral capsid as a minichromosome consisting of about 24 nucleosomes (2) . The icosahedral capsid has a diameter of 45 nm and is composed of three viral structural proteins, VPl, VP2, and VP3, with VPl being the major component (2) . Py was named because of its ability to induce tumors in a wide variety of cell types after experimental infections of neonatal mice with large doses of virus. A high incidence of epithelial tumors occurs in the major and minor salivary glands, the mammary glands, the hair follicles, and the thymus (3) . Additionally, mesenchymal tumors occur at numerous sites (3) . The virus can infect over thirty cell types, with the kidney being the major site of viral replication and persistence (3) . Although the virus is prevalent in the natural population, the incidence of tumors is actually low. The two major factors that contribute to the protection of the natural population from tumors are passive immunity acquired during gestation and low doses of infecting virus. The mechanism of tumor protection by the immune system remains an interesting unsolved question. The availability of tissue culture systems for the propagation of the virus jg vi_tr_g has led to a detailed understanding of its molecular biology and genetics. Py is only able to grow lytically in mouse cell cultures, and these cells are therefore referred to as being permissive for virus growth. Lytic growth is initiated by the expression of the three early viral genes, an event which occurs prior to viral DNA replication. One of the early proteins, large T-antigen (LT), is required for the initiation of viral DNA synthesis and regulates the expression of the early genes. Following amplification of the viral genome, LT—stimnlated expression of the three late genes encoding the viral structural proteins occurs. Virions are then assembled in the nucleus and released by cell lysis. In addition to mouse cells, Py can also infect rat and hamster cells, but cells from these two species are nonpermissive and do not support replication of the virus. Rat and hamster cells, however, are useful for the study of neoplastic transformation by the virus. In nonpermissive cells the infecting virus reaches the nucleus but only the early genes are expressed. Little viral DNA synthesis or late gene transcription is observed. A small percentage ((1%) of infected nonpermissive cells become stably transformed. This state requires the continual expression of the early genes. Maintenance of the genome is ensured by integration into the host DNA. Ckowth properties of Py-transformed cells in culture include a high saturation density, a reduced serum requirement, anchorage independence, lack of contact inhibition, growth on a monolayer of normal cells, and tumor formation upon injection into susceptible animals (2) . These traits, along with some other cell surface and intracellular properties, are also transiently displayed in a larger fraction of the infected nonpermissive cells. This phenomenon is termed abortive transformation and occurs at a rate that is 10—100 times greater than stable transformation. These cells do not become stably transformed because of either a failure of the viral genome to integrate or the integration occurs in such a way as to prevent early gene expression (4) . The following review will focus on the function of the early viral proteins in both permissive and nonpermissive cells, on viral DNA replication, and on integration of the viral genome in stably transformed cells. A summary of recombination in mammalian cells is also included since this topic is relevant to the understanding of the mechanism of viral integration. Early gene expression and DNA replication _i_g permissive cells The first stage of infection of permissive cells with Py involves attachment of the virion to a cell receptor, penetration of the cell membrane, and uncoating of the viral genome. These events can be completed as early as 15 minutes after infection, but by 3 hours postinfection only about 16% of the absorbed virus has reached the nucleus (5) . The capsid protein VPl plays the major role in the adsorption of the virus to the host cell receptor, a complex which consists of at least three membrane proteins (5,6). Recently, it has been discovered that the interaction between By and the receptor induces the expression of two cellular proto-oncogenes, camyc and c-fos (7). The protein products of these two cellular genes are thought to be important in the stimulation of cellular DNA synthesis. This is advantageous to the virus since it requires host enzymes for its gene expression and DNA replication. Internalization of the virus occurs via monopinocytotic vesicles which transport the virus to the nucleus where decapsidation takes place (5,8,9). Once in the nucleus the By genome is capable of being transcribed and replicated by host enzymes. The early and late genes are transcribed in opposite directions from the 350 bp noncoding region which contains the origin of DNA replication (ori), the early and late gene promoters, and a transcriptional enhancer (Fig. 1) (10). The three early proteins are called the tumor (T) antigens because they were first defined by antibodies present in the sera of mice bearing Py-induced tumors (11). Large Teantigen (LT) is a nuclear phosphoprotein with a.molecular weight between 88 and 108 kilodaltons (kd) (12,13). Multiple forms of this protein have been detected by differences in electrophoretic mobility that can be accounted for, in part, by differences in the phosphorylation state (12,14,15). Middle Thantigen (MT) is a cytoplasmic or membrane phosphoprotein that is present in the cell as two distinct species, the 56 kd and the 58 kd forms, which differ in their phosphorylation patterns (12,13,16). The Figure 1. Physical map of polyoma virus genome. The inner circle represents the HpaII restriction map of polyoma virus with the numbering system of Soeda _e_t _a_l_; (10) beginning at the junction of fragments 3 and 5. The origin of DNA replication is marked. The boxed regions represent the protein coding sequences of the early and late genes. The jagged lines represent the introns. The nucleotides of the splice junctions and of the termini of the coding regions are also shown. The figure is taken from Soeda g a1. (10) . EcoRI (1559) Bgu(87) -°- 70 30‘ K \ BenHIM53” 58 kd species represents less than 18% of the total MT in Py-infected cells (16) . MT has a hydrophobic region of 22 amino acids at its carboxy terminus which allows it to become anchored in the plasma membrane while its amino terminus extends into the cytoplasm (10) . Small T-antigen (ST) is mainly localized in the nucleus and has a molecular weight of 22 kd (12) . The T antigens are encoded by three different mRNAs transcribed from the early region (17,18) . The polyadenylated mRNAs share common 5' and 3' termini with the major start sites of transcription being nucleotides 147 and 152 as determined by primer extension and $1 mapping (Fig. 2) (18,19). The translational start site for each of the proteins is located at nucleotide 173 (10) . The three mRNAs encode different proteins, however, as the result of differential splicing (Fig. 1) (18,20) . The noncoding region of Py includes the promoter elerents required for early and late gene transcription (19-22) . The early TATA and CAAT boxes are located at nucleotides 122 and 64, respectively (Fig. 2) (1m . The late promoter overlaps the transcriptional enhancer and does not contain TATA and CAAT consensus sequences. This may explain the extreme heterogeneity found at the 5' termini of late mRNAs (22) . Located between the promoters is an 80 bp sequence (ori) that is required for DNA replication as determined by deletion mutagenesis (Fig. 2) (23-27) . The essential nucleotides of the ori include a string of eight A:T base pairs, at least five of which are required for viral DNA replication (25) , and a 34 bp sequence of dyad symmetry (25) Figure 2. Physical map of the noncoding region of polyoma virus. The map represents the polyoma genome from the early translation start site (left side) to the late translation start site (right side). Also shown are the early and late transcription start sites (arrows). The TATA and CAAT boxes of the early promoter and the origin of replication are boxed. The large T—antigen binding sites are shown in brackets above the genome with the major sites labeled A, B, and C and the minor sites labeled 1, 2, and 3. The four enhancer eletents (A-D) are shown in brackets below the genome. The regions of homology to the bovine papilloma virus (BPV) , SV40, mouse immunoglobulin, and adenovirus Ela enhancers are shown in brackets above the genome. The DNase hypersensitive regions (DHSR) are also shown. The figure is taken from Kern g a1. (43) . 1 1i] :0: :8 :2... :2... Ex... . 3%... _ 8—: _ 2w: — a”. .4 .mnmu - obo— «mu—p o? luau—Elm:— LL— Pl 2.2.0 1— F <5 . _L .1 C _ u Fl.— _IllL a a _ , < a u E .L. F. FL as a. 35 >2. FlL FIL «Inn stun 10 which is conserved in the SV40 ori (28). The ori is bordered on the early side by three high-affinity binding sites for LT (29,30). A common feature of these sites is the pentanucleotide 5'-G(A>G)GGC-3' that is repeated 2-4 times per site at 9-11 bp intervals (29-31) . Two additional minor binding sites are located within the ori and another one is found within the transcriptional enhancer (Fig. 2) (30). Sequences within the enhancer are not only required for transcription but are also required for DNA replication (25,27,32,33) . This does not, however, reflect a transcriptional requirement for replication (30-32) . The enhancers from SV40 and Py were the first such transcriptional elements described (34-38) . In general, enhancers act by stimulating transcription from linked promoters in an orientation- and distance- independent manner (39). The Py enhancer is located between nucleotides 5046 and 5289 (Fig. 2) (34) and is required in cis for early gene transcription (26,40-42), DNA replication (25,27,32,33), and late gene transcription (43) . Deletion mutagenesis has been used to divide the Py enhancer into several structurally distinct elerents that are, to an extent, functionally redundant (Fig. 2) (33,44,45). The BclI-PvuII restriction fragment contains the A and D elements (33) and is able to activate transcription from the chicken a-collagen promoter in mouse fibroblasts (44) and from the rabbit B-globin promoter in HeLa cells (34) . Within the A element is a sequence which is homologous to the adenovirus Ela enhancer (46) . A tandemly repeated 26-mer from the A element is sufficient to stimulate Py early gene transcription and 11 viral DNA replication in mouse fibroblasts (33,47). Several naturally occurring strains of Py, such as P16 and Tbronto large plaque, have duplications within this region which do not affect the phenotype of the virus (48,49), but Py strains with a duplication of the A domain and a deletion of the B domain (described below) can grow on mouse PCC4 embryonal carcinoma (EC) cell lines which are nonpermissive for wild-type Py (50). The PvuII restriction fragment contains the B and C enhancer elements (Fig. 2) (33). The former shares sequence homology with the core region of the SV40 enhancer (51) while the latter contains sequences homologous to the immunoglobulin G enhancer (52) . Additionally, sequence homology to the bovine papilloma virus enhancer is found between the B element and the ori (53). Py mutants selected to grow in nonpenmissive mouse F9 EC cells have a single A-G transition at nucleotide 5248 within a duplicated B domain (40,47,50,54). Cellular proteins have been shown to bind to transcriptional enhancers in order to regulate transcription in a positive or a negative manner. Several of these transcription factors have been shown to bind to the By enhancer. Two A element-binding proteins (PEAl and PEA2) (55) and two B eleient-binding proteins (PEBl and PEBZ) (56,57) have been identified in mouse 3T6 cells. Additionally, a C element-binding protein has been observed in differentiated and undifferentiated murine F9 EC cells, mouse L cells, and HeLa cells (58). PEAl also binds to the SV40 and c-fos enhancers (55) and.may be the same as activator protein 1 (APl), a HeLa cell protein which interacts with the SV40 enhancer (59). The interaction of cellular 12 proteins with the Py enhancer may lead to the formation of the DNAse I hypersensitive sites localized to the late side of the A element and to the early side of the B element (Fig. 2) (60). The expression of cellular transcription factors may be tissue— or stage-specific. Therefore, the redundant enhancer elements found in the Py genome may be necessary in order for the virus to replicate in many different cell-types in the mouse. Not only are cellular proteins important in the expression of viral genes, but LT is also a key regulatory factor. An overproduction of early mRNA occurs when cells infected with a viral mutant encoding a thermolabile LT (ts-a mutant) are shifted to the nonpermissive temperature (17). In a wild-type infection direct interaction of LT with the major binding sites near the ori results in negative autoregulation of early gene expression (31,61,62) and induction of late gene expression (43). Interestingly, LT also stimulates transcription from.cellular promoters (63-65), but this effect may be indirect since the DNA binding domain of LT is not required for the stimulation (65). Utilization of ts-a mutants has also established that LT is required for the initiation of viral DNA synthesis. When shifted to the nonpermissive terperature the ongoing round of viral DNA synthesis is completed but further initiation does not occur (66). La'may also have a mitogenic effect on host DNA synthesis (67). Phosphorylation of LT may play a significant role in its ability to perform multiple functions within the infected cell. In the comparison of infections of 13 quiescent and growing cells, LT is less phosphorylated in the growth- arrested cells early in infection. Following cellular DNA synthesis, though, the levels of LT phosphorylation are equivalent in the two populations of infected cells (68). These results can be interpreted to.mean that dividing cells provide enzymes that are required for modifications of LT. The phosphorylation state of LT may be crucial to viral DNA synthesis, since the LT of replicationrdefective ts-a mutants is underphosphorylated (68). The’mechanism.of papovavirus DNA synthesis is known in considerable detail. DNA synthesis begins with two replication forks moving in opposite directions from the ori (69,70). DNA synthesis from each of these forks proceeds in a semidiscontinuous manner (71). The two sites of initiation of continuous DNA synthesis have been mapped to a 16 bp region in the ori (72). The development of cell-free systems to replicate SV40 and Py DNA has led to a greater understanding of the roles of viral and cellular proteins in this process (73-76). For both By and SV40, LT is the only viral protein required for viral DNA synthesis ig_yi§£2_(73-76). ‘With regards to its role in DNA synthesis it should be noted that By LT is not only a DNA binding protein but it is also an ATPase (77) and a nucleotide binding protein (78). These two activities are structurally distinct and have been mapped to a region between the carboxy-terminus and the DNA binding domain located ' between amino acids 290 and 319 (79-81). Experiments reported in Chapter 3 of this thesis describe a new mutation in LT between the DNA binding region and the ATPase domain that affects DNA replication. 14 This point mutation causes a proline to alanine change that may alter the tertiary structure of the protein. In addition to the activities mentioned above, SV40 LT also has a helicase activity _ig y_i£r_g_ (82,83) . By using the cell-free systems it has been shown that LT is required prior to the initiation of DNA synthesis (84) . This step also requires ATP and cellular proteins but not dNTPs (84) . During preinitiation LT may function to unwind the ori to allow for the binding of DNA polymerase o/primase (83) . Since SV40 LT has helicase activity it has been suggested that it is involved in elongation as well as initiation. This has been shown to be the case i_n 33:53 (82,83) , but _ig .Y_il’_°. results show that LT dissociates from the viral genome after replication is 70-80% complete (85) . Both SV40 and Py have a narrow host-range with regard to productive infections. Py only replicates in mouse cells while SV40 replicates in monkey and huran cells (2) . The cell-free replication systems have allowed, in part, for an analysis of the cellular proteins involved in permissivity. SV40 DNA is replicated _i_r_1_ _v_i_tr£ in the presence of LT and extracts from either human or monkey cells but not from mouse cells (86,87) . Py DNA replication, on the other hand, proceeds in the presence of mouse cell extract but not human or monkey cell extracts (86) . Addition of the partially purified mouse DNA polo / primase complex to HeLa or monkey cell extracts allows for E M synthesis of Py DNA, suggesting that the DNA pol d/primase is a major factor in permissivity for papovaviruses (86) . Monoclonal antibodies to SV40 LT have been shown to coprecipitate LT and DNA pol a from 15 SV40-infected HeLa cell extracts, suggesting that these proteins interact directly (88). It is likely that other cellular proteins besides DNA polcx/primase are responsible for the permissivity of Py DNA replication. Experiments presented in Chapter 2 of this thesis show that a considerable amount of viral DNA synthesis occurs in established rat cell lines at 33°C as compared to 37°C. Other workers have shown that replication of the integrated viral genomes in Py-transformed cell lines occur in 1-2% of the cells (89). Other lines of evidence from SV40 infections also implicate additional factors. Infection of preimplantation mouse embryos with SV40 results in the production of infectious virions (90). Other cellular proteins known to be required for ig_yi££g' replication include topoisomerase I (91); a single—stranded DNA binding protein (91); proliferating cell nuclear antigen (PCNA), a cell-cycle regulated protein associated with cellular DNA replication (92); and topoisomerase II which is required for resolution of the two daughter DNA molecules (93) . In contrast to the case of LT, little is known about the functions of MT and ST in the lytic cycle. Hr-t mutations located in the LT intron affect both MT and ST and have been studied in detail. The minichromosome of hr-t mutants is deficient in the acetylation of histones H3 and H4 (94). Hr-t mutants also have an underphosphorylated VPl (98), a decreased output of viral DNA (95), and an absence of induction of c-myc and c-fos expression postinfection (7) as compared to wild-type infections. They also stimulate only one round of 16 cellular DNA synthesis as compared to the multiple rounds induced by wild-type strains (67). These observations show that either ST, MT, or both have a role in virus assembly, viral DNA replication, and virus-host interactions involving cellular gene expression and DNA replication. When ST alone is introduced into mouse NIH-3T3 cells they grow to a higher saturation density than monolayers of the same cells in the absence of ST (96,97). This result suggests that ST has a role in growth regulation of Py-infected cells (7,96). The question remains, however, whether this function is unique to ST or if it is also a function of MT since only 4 out of 196 amino acids of ST are not shared with MT. Neoplastic transformation by_polyomavirus As described above, Py is able to transform.nonpermissive rat and hamster cells is culture and to cause tumors in newborn mice (2). Early studies utilized two groups of nontransforming mutants, the ts-a (99-101) and hr-t strains (102,103), to detenmine the viral proteins involved in neoplastic transformation. Mixed infections at the nonpermissive telperatures with mutants from these two completentation groups generate stable transformants (104,105). With the knowledge that the hr-t mutations map to the LT intron and affect both ST and MT (106,107) and that the ts-a mutations map to the carboxy-terminal half of LT (14,15,106,107), it was evident that stable transformation by Py required LT and either ST, MT, or both MT and ST. Subsequent experiments revealed that transfonmation by Py can be divided into two steps called initiation and maintainence. The initiation event 17 requires the transient expression of LT and results in the integration of the viral genome (99-l0l,108). Maintainence of the transformed state is achieved with that part of the early region encoding, ST, MT, and the amino-terminal half of LT (109-112) . More recent developments using expression vectors or reconstructed Py genomes encoding only one early protein have led to a more detailed understanding of the activities of ST, MT, and LT in transformation. A Py genome encoding only MT is able to transform established rat cell lines (113) . Established or immortalized cells, as opposed to primary cells, are able to grow in long-term culture. Established cell lines are derived from rare cells within a population of primary cells that survive many passages in culture. The establisment of cells is considered to be an initial step in the pathway toward full transformation of a cell. Transformants from the experiment above are tumorigenic as demonstrated by their ability to form tumors after infection into Fischer rats (113) . DNA encoding only MT is also tumorigenic in newborn hamsters but not in newborn rats (114,115) . MT alone, however, cannot transform primary rat embryo fibroblasts (116) . These results clearly demonstrate that MT plays the major role in the maintainence of transformation by Py. The fact that it does not cause tumors in newborn rats or transform primary rat cultures, however, deionstrates a requirerent for the other early viral proteins. In contrast to MT, LT by itself cannot transform established rat cell lines (114,115). It can, however, immortalize primary mouse and rat embryo fibroblasts (117) . The imrortalizing function has been 18 mapped to a 200 bp region in the 5'-proximal half of the LT gene (118). Immortalization of cells is not understood at the molecular level, and it is not clear how LT functions in this capacity. One phenotype of LT-immortalized cells, however, is a reduced requirement for serum growth factors (117). Since LT is a DNA binding protein its action may be a regulator of cellular gene transcription. Immortalization by LT, though, does not require the DNA binding domain of this protein (81). The question of which viral proteins besides MT are required for tumorigenesis in newborn rats was answered by injecting DNA fragments encoding either ST or LT along with the MT gene. Surprisingly, these experiments revealed that either ST or LT could cooperate with MT to cause tumors in this host (114,115). In contrast to these results, transformation of primary rat embryo fibroblasts requires all three early proteins (115). As described above, established rat cells can be transformed by MT, bypassing the need for LT and ST. It should be noted that these transformed cells require serum growth factors to remain viable (116). The growth factors apparently alleviate the need for LT or ST. These results support the theory that tumorigenesis is a complex process that requires not one but multiple cellular events (119). Additional support for this theory comes from transfections of primary cells with combinations of proto-oncogenes and polyoma early genes. Either LT or cemyc can complement c-ras in the transfonmation of primary mouse embryo fibroblasts (120), while primary baby rat kidney cells are transformed by a combination of the Ela proteins of adenovirus and either MT or c-ras (121). 19 The mechanism by which MT acts to transform cells has been the subject of intense interest in recent years. MT fromlwild-type infected cells but not from hr-t infected cells is associated with a tyrosine kinase activity igwyitrg (13,122,123). It was later discovered that the kinase activity is contributed by the proto—oncogene pp60C‘SIC (c-src) (124). The kinase-active MT/c-src complex is associated with the plasma membrane (16,124,125), and monoclonal antibodies to either MT or c-src will coprecipitate the two proteins fromIPy-infected cell extracts (126). Only a small fraction of the MT in an infected cell is associated with c-src (127), and it is the level of this complex which detenmine the transformation phenotype. Cells containing the MT gene linked to an inducible promoter differ in transformation state depending upon the level of kinase-active MT/c-src (128). Anchorage-independent growth and tumor formation require higher levels of the complex than are required for focus fonmation and morphological transformation (128). It appears that the 58 kd form of MT is the one that activates c-src. MT from the hr-t mutant, N659, can be phosphorylated to produce the 56 kd species but not the 58 kd species (16) . The 56 kd form associates with c-src but is inactive in the ighyi§£g_kinase assay (129). In wild-type infections phosphorylation of the 58 kd form, but not the 56 kd species, is stimulated by the tumor promoter 12-0-tetradecanoylphorbo1-13-acetate (TPA), an activator of protein kinase C (130). The MT/c-src interaction increases the tyrosine kinase activity of c-src|ig_yit£9_ probably by altering the phosphorylation state of the protein 20 (131-133). MT-associated c-src is deficient in phosphorylation at tyrosine 527 (134). Site-specific mutagenesis of the c-src gene has shown that phosphorylation at this position decreases the tyrosine-specific kinase activity of the protein (135-137). Some points concerning the kinase activity of the c-src/MT complex are still not clear. For one thing, it is not kown if the increased kinase activity of MT/c-src seen ip_yiggg_is relevant to its activity ig_yiygL The level of phosphotyrosine in Py-transformed cells is not increased with respect to untransformed cells (138). This could be explained, however, if the MT/c-src complex has one or a few specific substrates whose increase in phosphorylation did not raise the level of total phosphotyrosine by a detectable amount. As yet, a specific substrate for MT/c-src has not been identified. The>c-src binding domain of MT has been localized to the amino- terminal end of the molecule. Mutations within this region prevent association with c-src (139-142). Tyrosine phosphorylation of MT may also play a role in its association with and activation of c-src. The phosphorylation of four tyrosines in the carboxy-terminal half of MT (amino acid positions 250, 297, 315, and 322) has been studied using site-directed mutagenesis and hr-t mutants. While the absence of phosphorylation of 297 and 322 has no effect on the transforming activity of MT, the absence of phosphorylation on either 250 or 315 decreases, but does not completely eliminate, the transforming ability of MT (143,144). Thus, the phosphorylation state of Mszay influence its ability to interact with c-src to form a kinase-active complex. 21 Stable transformation of cells by Py requires the integration of the viral genome into the host DNA (2) . The viral DNA is usually integrated as a head-to-tail tandem (109,145,146). The generation of integrated tandems requires LT (147) and a functional viral origin of replication (148) . lbcombination between the host and viral DNA appears to be nonhomologous with only a 2-5 bp homology at the viral-host join (149-152). As analyzed by hybridization of Southern blots of DNA.from1Py-transformed cell lines, integration is apparently randomlwith respect to both the host and the viral DNA (145,146). Integration sites in the host genome, however, have not been mapped using ig_§itg hybirdization of chromosomes. Integration of Py can result in deletions, duplications, and rearrangements of host DNA at the integration site by mechanisms which have not been elucidated (152,153) . Two models have been proposed for the integration of head-to-tail tandem viral genomes. The first invokes rolling-circle replication of the viral genome to produce a linear multimer that may serve as a substrate for integration. Such a model would fit with the requirements for LT and the viral origin of replication. High molecular weight forms of SV40, proposed to have arisen by rolling-circle replication and to be the precursors to the integrated tandems, have been identified in infections of nonpermissive cells (154). Py genomes that appear to be involved in rolling-circle replication have been identified in Py-infected mouse cells by electron microscopy (155) . Evidence for a recombination step in the 22 tandemization of Py genomes comes mainly from the study of integrated viral genomes in transformed cell lines. When fused with permissive cells, transformants from mixed infections with a ts-a strain and an hr-t strain yield, in most cases, wild-type virus along with the two parents, suggesting that the two parental genomes recombined at some time during the integration process (156). Cointegration of the two parental genomes at a single site has been demonstrated in one cell line from this experiment (157). In Chapter 4 of this thesis, further evidence for a high level of recombination in the integration pathway is provided. In these experiments mixed infections with two restriction-site minus mutants of Py resulted in recombination of the two parents in about 35% of the cell lines analyzed. With regard to the role of LT in the recombination model, some evidence has emerged to suggest that this viral protein has a recombinogenic activity. Intramolecular recombination of a plasmid containing 1.03 copies of the Py genome is enhanced in murine cells if LT is provided in trans (158). In this system, replication of the By plasmid inhibited recombination, a result that may indicate that the function of LT in recombination is different than its function in DNA synthesis (159). Recombination and replication may compete for a limiting amount of LT. For tandem formation during integration, however, recombination and replication may be linked. If recombination is the favored pathway for tandem formation, then the requirement for a functional ori in this process may reflect a need for a replicating substrate. It is also possible that both rolling-circle replication and recombination are involved in 23 tandem formation. In this model a rolling-circle intermediate is first synthesized and then is capable of recombining with either viral DNA monorers or multimers. The reverse is also possible with recombination occurring prior to rolling-circle replication. After integration, the Py ori remains active. The viral DNA produced from integrated genomes ("free" viral DNA) is supercoiled and when a transformed cell population is analyzed, the viral DNA is present at the level of 10-60 copies per transformed cell (160), but the free DNA is only present in a small number of the transformed cells at any one time so that the number of copies of DNA per producing cell is actually 100-1000 times higher than this figure (89). Excision of integrated viral DNA from transformed cells also occurs at a high rate to generate cells which have reverted to the normal phenotype (89,145,146) . Excision requires regions of Py sequence hotology (161,162), LT (146), and a functional origin of replication (163). The same requireients have been found for the amplification of integrated viral genotes (161,163). DNA recombination i_n mammalian cells DNA recombination in mammalian cells is not as well understood as in the prokaryotes and fungi, but substantial progress has been made recently due to the development of recombination assays based on the introduction of exogeneous DNA substrates into cultured cells via calcium phosphate precipitation, DEAE-dextran mediated transfection, microinjection, or viral infection. Assays have been designed to measure either horologous or nonhotologous recombination using 24 substrates which undergo either intra- or intermolecular recombination. The following discussion will review these methods and some of the models proposed for DNA recombination in mammalian cells. Examples of recombination events which require little or no sequence horology are prevalent in mammalian cells and include integration of papovaviruses (149-152) , reciprocal chrotosome translocations (164) , rearrangetents of antibody and T-cell receptor genes (165,166), gene amplification events (167), and the formation of processed pseudogenes (168) . In general, the mechanism of nonhotologous recombination is thought to involve DNA breakage and end joining (169-172) . Linear DNA introduced into mammalian cells can efficiently undergo nonhomologous recombination by end joining and thus may serve as useful assay for this recombination pathway (169-172) . The mechanism of the ligation of ends in these systers is similar to that seen at the junctions found in reciprocal chromosome translocations (164) and antibody gene rearrangetents (165). The common element in these events is addition of nucleotides of unknown origin at the junctions. It has been proposed that the extra nucleotides are added to free ends by terminal transferase prior to ligation (165,169,173,174) . An extensive analysis of nonhorologous recombination junctions following recircularization of a linearized SV40 genome has been carried out. In this experiment the ends of the linearized SV 40 genore were mismatched with one being blunt and the other having a 5'-protruding strand (173) . Sequencing of the junctions of the recircularized SV40 genoies revealed that 87% of them had arisen 25 by end joining as described above, while the remaining 13% belonged to a class in which the junctions contaired duplications. In a model to explain the minor class of junctions it was proposed that the free ends mispair by way of short homologies (Fig. 3). The mispaired region could then serve as a terplate for repair synthesis restoring the duplex but leaving an unpaired region. Further repair synthesis against the strand with the unpaired region creates a duplication (173) . Homologous recombination events that occur in mammalian cells include meiotic recombination (174) , mitotic recombination (175) , sister chroratid exchange (176), and gene conversion (177). Homologous recombination of DNA introduced into mammalian cells has also been detonstrated (178-186) . These systems usually involve the transfection of two nonfunctional copies of either a selectable gene or a viral genore into the appropriate cells. Recombinationois then quantitated by assaying for a functional gene or viral genome. In these systems the frequency of recombination depends upon the length of hotology (172,182,183,187,188) and is increased by the presence of double-strand breaks in or near the homologous regions (181,182,189- 191) . In addition it has been observed that homologous recombination in these experiments is nonconservative or nonreciprocal (182,190,192, 193). To explain these observations, the double-strand break (194) and the single-strand annealing models (182,195) have been proposed. In the former a double-strand break or gap in one DNA molecule provides an end to initiate a strand invasion event in a region of hoxology on a second DNA molecule. The invading strand is used as a primer for DNA 26 Figure 3. Proposed model for formation of duplicate junctions following nonhomologous recombination. Short hotologies are shown with open and hatched boxes. As illustrated in the figure, the duplication occurs following mispairing of one of the 3' ends and repair synthesis. The figure was taken from Roth gt a_l_._ (173) . 27 m 13—D— it. D— flL 22:23:... fixa— RENE (REPLICAYM) m (mums mum sorrow ammo) W Wv V W Addition melflON JUNCTION 28 synthesis with the intact copy of the homologous region being used as the template. This model requires that the double-strand break be located in a region of homology which is shared by the two molecules. Homologous recombination between DNA molecules in which the break occurs within a region of nonhomology cannot be explained by this model (182,192,193). To account for these results the single-strand annealing model was formulated (182,195). In this model the double-strand break is a substrate for either a 5' to 3' (182) or a 3' to 5' (195) exonuclease that produces complementary single-strands (Fig. 4). After reannealling, the molecule is repaired by DNA synthesis. Additionally, ig_yit£g systems for recombination have been deve10ped using whole cell extracts (196-198). Using this approach a recombinase from human cells has been partially-purified (199) . Such advances should be extremely beneficial in the study of the mechanism and enzymology of homologous recombination in mammalian cells. The ratio of homologous to nonhorologous recombination has been measured in several systems which utilize either intramolecular (171,172,189-200) or intermolecular recombination (187). One of the intramolecular recombination assays employed a linearized SV40 genome 'with a 131 bp duplication at its tenmini (171). A functional genome could be produced by either homologous or nonhomlogous recombination. FOllowing transfection of the DNA into monkey cells, recombination was sCored by plaque assay. The subsequent sequence analysis of functional recombinant genotes indicated that nonhorologous recombination was favored by a factor of 2-3 over hotologous recombination (171) . One 29 Figure 4. Single-strand annealing model of homologous recombination. A.) Generation of single strands by 5' exonuclease. The hatched regions represent two nonfunctional copies of the herpesvirus thymidine kinase (tk) gene. A double-strand break between regions of homology is followed by exonuclease digestion of the free 5' ends to reveal single-stranded regions of horology. Homologous pairing and repair synthesis produces a functional tk gene. The figure is taken from Lin g a_l_=_ (182) . B.) Generation of single strands by 3' exonuclease. Two non-functional copies of the herpesvirus tk gene are shown as black boxes. A double-strand break between the regions of hotology is followed by 3' exonuclease activity. The single-stranded regions are repaired as above. The figure is from Lin _et al. (195) . 30 mm ”m,“ 3 as’nc 5' f A A , 3; 37” as'c As’c . 7’6 5 (1) 10m Strand Brook . I 3! s! 4‘ J I 3.1% : 5i 3F 4‘ 41; 3' 5' Emoticon (2) lOegradation . AS'NC 3' . . 5 ll ' _# Sj 3' (3) lSInglo Strand Annealing '- ------------ H . . A3’NC ,3' ' . . 5 ” ' ‘ vs— _~ : 5:, 3' (4) lNucIeoso I ./ 3’ 5: t 1 j 3 B'jzs' I .1 ’_ ll 5 25/. (5) 160p Repair 5' ’ 3' WW 5': 3' 3711;: 3. # I; 5' A lDNA Ligation Reconstructed tk 5'7; AS'NC Hill” III AS'NC ' I If Li 3: I 3, 3 f Aslc AS'C Ifis (I) lDoubio sum Brook 5I I - 3' 5' 4. I I 37/ M 5: 34 4‘ I]; 3' 3’ Exonucloau (2) 1 Degradation 5' F3, ‘ W—fi 3: 3‘7: AIR: 5. 3 165 (3) 15inglo Strand Annealing . . “an"? 5 b3 l \L 2‘ ', , —’ 3: 3‘7: : -1-.A§'E >53 3-—/zfi5 (4) lRosyntnuis from 3' End I 3' 5'\ , 5, woman») A 4—1 3 37’]— ; \5. 514265 (5) lNucloau . . 5' a sjflw} , 4 3: 3' r fi . ‘5 f/fis (DNA Ligation Rocoutmctod tk 31 intramolecular assay involved introduced of both a bacterial plasmid with the ampicillin resistance gene and a mammalian expression vector into mouse cells. The two substrates shared regions of horology. After transfection, low molecular weight DNA was extracted from the cells and used to transform _E; ELL: Ampicillin-resistant colonies were screened by _ip _s_i_tg hybridization for the presence of the expresssion vector. Analysis of the recombination junctions revealed that nonhomlogous recombination was favored over homologous recombination by a factor of seven (187). In comparison, nonhomologous recombination is favored over homologous recombination by a factor of 104 to 105 with regard to integration of exogenous DNA into the host DNA (201) . In general, the cellular machinery is very efficient in recombining DNA introduced into cells. This is especially true for linear DNA. In all cases examined, nonhomologous recombination is favored over horologous recombination. Recombination is also very rapid. When DNA is introduced directly into the nucleus the recombination events occur within one hour (190). Experiments are described in Chapter 4 of this thesis that show that homologous recombination between Py genotes is high in cells that have been transformed by the virus. SUMVIARY Since the discovery of Py over thirty years ago, a large body of information has been obtained concerning its biology. Many of its interactions with permissive and nonpermissive cells are understood in 32 great detail. Py and SV40 have been particularly important in the discovery of RNA splicing and transcriptional enhancers. These viruses have also been beneficial in the analysis of eukaryotic DNA replication, neoplastic transformation, and control mechanisms for eukaryotic gene expression. The experiments presented in this thesis have been briefly described in the text above. In summary, the pathway of integration of the viral genome in Py-transformed cells was investigated. Evidence for the replication of viral DNA in nonpermissive cells was obtained, but this replication could not be tied to the formation of the integrated tandems. In the course of this work a new LT mutation was discovered which affected viral DNA replication in nonpenmissive cells. In another set of experiments, evidence for homologous recombination between viral genomes in the integration pathway of Py was obtained. 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Science 226:1213-1215. 197. Kenne, K. and S. Ljungquist. 1984. Nucl. Acids Res. 12: 3057-3068. 198. Kucherlapati, R.S., J. Spencer, and P.D. Moore. 1985. Mol. Cell. Biol. 5:714-720. 199. Hsieh, P., M.S. Meyer, and R.D. Camerini—Otero. 1986. Cell 44:885-894. 200. Stringer, J.R. 1985. Mol. Cell. Biol. 5:1247-1259. 201. Lin, F.-L., K. Sperle, and N. Sternberg. 1985. Proc. Natl. mad. SC]... U.S.A. 82:1391-1395. Chapter 2. The Role of Viral DNA Synthesis in Neoplastic Transformation of Nonpermissive Rat F-lll Cells by Polyoma Virusl ABSTRACT we have investigated the role of polyoma viral DNA synthesis in the formation of the precursors to the integrated viral sequences found in cells transformed by the virus: i.e. tandem repeats of the viral genome. we show that after infection of Fischer rat F-111 cells, polyoma DNA synthesis at 37°C results in a 2-3-fold increase over the input signal. ‘Ignsitu hybridization shows that synthesis is restricted to a small fraction of the population. In contrast, viral DNA synthesis at 33°C is about ten times higher than at 37°C for most strains tested, including standard wild-type A2. Exceptions to this are the strains NGS9RA and Py 1-12. Most of the viral DNA produced is supercoiled (fomm I DNA). By'ip_§itu_hybridization we show that more cells are permissive for viral DNA synthesis at the lower temperature and that the level of synthesis per permissive cell is higher at 33°C. The DNA synthesis observed is large Teantigen dependent and is paralleled by an increase in the expression of this viral protein. In the absence of large Teantigen the half-life of the de novo synthesized viral DNA is less than 12 hours. The overall levels of viral DNA 1 David L. Hacker and Michele M. Fluck, Department of Microbiology and Public Health, Interdepartmental Program.in Molecular Biology, .Michigan State University, East Lansing, MI 48824-1101. 44 45 synthesis in F-lll cells may not affect the integration of the viral genome since the patterns of integration in cells transformed by wild-type A2 at 33°C and 37°C appear similar. Furthermore, although an increase in transformation frequency of A2 is observed at 33°C (frequencies 2-3 times higher than at 37°C) , it does not parallel the increase in viral DNA synthesis. 46 INTRODUCTION The problem of cellular penmissivity in papovarirus infections has been intriguing since the discovery of these viruses. Permissivity refers to the extent of viral DNA synthesis (and virus production) in cells which display viral receptors and can support viral early gene transcription. Those host cells which are fully permissive (mouse cells for polyoma virus [Py]) undergo a cytopathic infection and are killed in the process of virus production (7,42). Nonpermissive host cells (rat and hamster cells) produce little to no viral DNA, survive the infection, and undergo abortive and stable neoplastic transformation at moderate and low frequencies, respectively (16,17,18). The existence of trans-dominant factors controlling permissivity has been suggested from the observation that hybrids between permissive and nonpermissive hosts are permissive for virus production (1,42,45). Recent evidence suggests that the permissivity factors include the host DNA polymerase01 050 Sam. o_mm_ Enn— 2n. >01 (mmmwz «PIP do ~< m::> can 59 the mutation which affects the ability of this strain to synthesize DNA at 33°C in rat cells to the central region of the large T-antigen (Hacker, et al., submitted). The bulk of viral DNA.from1the Hirt extractions shown in Fig. l and Fig. 2 is equally divided between Form I and Forms II and III DNA. Some degraded forms are also apparent even in the A2 infections, which were carried out with a recently plaque-purified stock that does not contain any detectable defective genomes. Digestion of the DNA with HpaII, which cuts seven times within the polyoma genome, produces equal proportions of each fragment indicating that, for the most part, the complete genome is being replicated (not shown). we have made extensive attempts to search in rat cells for replication intermediates of high molecular weight which might be the precursors to the integrated concatamers observed in transformed cells. For this purpose, we have isolated high molecular DNA from F-lll cells infected at a high MOI (60-100 pfu/cell) with various strains of polyota. The high molecular weight DNA was separated from polyoma DNA monomers by sucrose gradient centrifugation, digested with BglII, which does not cleave polyoma DNA, and then analyzed by either agarose gel electrophoresis on 0.4% gels or by two-dimensional gel electrophoresis (46). Cbncatameric fonms of polyoma DNA were not detected using these techniques, nor were they detected using orthogonal-field-alternation gel electrophoresis (OFAGE) (35). The absence of high molecular weight viral DNA species, however, does not eliminate the possibility of rolling circle-type replication as has been proposed for SV40 (8) and 60 observed in rare instances in polyoma infections of permissive cells (5,25) . If rolling circle replication is occurrin , recombination and conversion to monoreric viral DNA must be very efficient. Despite the high levels of DNA synthesis in A2-infected cells at 33°C, late gene expression as determined by indirect immunofluorescence using an anti-virion antiserum was not detected. Additionally, plaque assays of cell lysates from A2-infected rat cells were negative for infectious virions (not shown). _I_n_ situ wridization analysis o_f_ polyoma-infected rat cells. No possibilities could account for the difference in viral DNA synthesis at 33°C and 37°C. The tetperature might affect either the number of cells that are permissive for viral DNA synthesis or the rate of synthesis per permissive cell or both. To differentiate between these possibilities, we investigated the distribution of viral DNA in single A2-infected F-lll cells using _ip gig hybridization. No viral DNA was detected at one day postinfection, which indicates that the input level of viral DNA (the amount corresponding to the fraction of viral DNA that is taken up by the cells following infections at 10 pfu/cell) is below the detection level of this technique (data not shown). A positive signal for viral DNA was first detected at 3 days postinfection for cells both at 33°C and 37°C (Fig. 3A) . The number of DNA positive cells at both terperatures was determined for several time points. As is evident from the data presented in Table l, more viral DNA positive cells are observed at 33°C than at 37°C for any 61 Figure 3. _I_n fl hybridization of A2-infected F-lll cells. F-lll cells were infected with A2 at an MOI of 10 pfu/cell and grown at either 33°C or 37°C. Infected cells were fixed (see Materials and Methods) at the times indicated below. The hybridization probe was the HpaII-S fragment of polyoma virus labeled with (355)-acrp. A.) A2-infected r-111 cells grown at 33°C or 37°C. B.) Controls. NIH-3T3 cells were infected with A2 at an MOI of 10 pfu/cell. Cells were fixed at 72 hours postinfection. Uninfected rat F-lll cells are shown in the panel marked F-lll. 62 . a a .. .. \ \. :7“. . fl . . H., sums . . ohm 1.2 . .i. a; «I... 3:... 22:30 goo @- n>ao m goo m 63 Table 1. Analysis of in situ hybridization of A2-infected F-lll cal—s at 33°C and 37°C Day No. viral DNA positive cellsa 33°C/37°C Post-infection 33°C 37°C 3 55 14 3.9 6 58 mob - 9 119 19 6.3 11 122 29 4.2 13 196 60 ‘3.3 aThe number of viral DNA positive cells is based on the number of cells in 20 microscopic fields which exhibited a hybridization signal. bNot done. 64 time point chosen up to 13 days postinfection (see Fig. 3A). For time points between 3 and 13 days, the difference in the number of DNA positive cells at the two temperatures varied between 3- and 6-fold (Table 1). By 13 days after infection, transformed foci were visible on the coverslips at both temperatures (Fig. 3A). For the 37°C incubation, 94% of the DNA positive cells at this time were localized to the focal areas. In contrast, only 69% of the DNA positive cells were found in focal areas on the 33°C coverslips. Throughout the time course of the experiment, it appears that a greater number of cells are supporting detectable levels of viral DNA replication at 33°C than at 37°C. The difference between the number of viral DNA replication positive cells at the two temperatures (3- to 6-fold) is slightly lower than the difference observed on Southern blots (l0-fold). This may indicate that there is also an increase in the rate of synthesis per cell at the lower temperature. Both of these results could be accounted for by higher rates of initiation of viral DNA synthesis at 33°C. However, since the proportion of DNA positive cells remains small even at 33°C, it is unlikely that the increase in DNA synthesis per cell can be accounted for by statistical considerations alone (increased probability of reinitiation per cell). It is also conceivable that the same number of cells produce high levels of viral DNA at 33°C and 37°C, and that these cells are killed at 37°C before they can reach levels of DNA equivalent to those at 33°C. Considerations on the behavior of transformed cells at 33°C make this an unlikely explanation (see below). 65 The level of detectable DNA positive cells is low (0.2%). The frequency of DNA positive cells is slightly higher than previous results obtained for polyota infections of hamster BHK cells at 37°C (19). As had been noted previously in the case of infections of the BHK cell line, the proportion of DNA positive cells and the proportion of cells which become transformed are equivalent. It must be noted, however, that the ig_§itg_hybridization technique only scores cells with a relatively high level of viral DNA sequences. Since the bulk of DNA replication appears to be contributed by a small percentage of the population, it appears that in those cells the level of DNA replication may be as high as the average level produced in permissive cells. The sametconclusions have been reached for the production of unintegrated viral genomes in polyota transformed rat cells (47). The fate 9§_viral DNA ig_the absence g£_large T-antigen. To investigate the role of large T-antigen in viral DNA synthesis in nonpenmissive cells, F-lll cells were infected with a temperature-sensitive mutant, ts-a (23), which encodes a thenmosensitive large Teantigen. The infected cells were grown at either 33°C or 39°C or shifted from.the lower to the higher temperature at various times after infection. Viral DNA was extracted and analyzed as described above, and the results are presented in Fig. 4. For cells infected at 33°C with ts-a or A2, the amount of viral DNA increases throughout the time course of the experiment. At 39°C, however, little ts-a DNA remains by 11 days postinfection while the 66 Figure 4. The role of large Teantigen in viral DNA synthesis. A2 and ts-a infected F-lll cells were grown at either 33°C or 39°C. Viral DNA was isolated from.these conditions at the times indicated in the figure. For the ts-a infections, cells were also shifted from 33°C to 39°C at times between 1-10 days postinfection as indicated (lanes marked ts-a shift). Viral DNA from all of the 33°C to 39°C shift samples was isolated at 11 days postinfection. Gel electrophoresis and hybridization were conducted as in Fig. l. .67 an own mMNFF-mww-MF 4... >00 can some 5:3 can one can one arm; mum... mime N< For; 68 level of viral DNA in A2-infected cells is approximately one-third of the input level. Viral DNA from cells that were shifted from 33°C to 39°C was rapidly degraded as seen in the lanes marked "shift." In ts-a mutant-infected cells shifted to 39°C at 10 days postinfection and maintained at 39°C for 24 hours, less than 25% of the level of viral DNA remains as compared to the ts-a infected cells maintained at 33°C for the entire 11 day time course. This indicates that the half-life of polyoma DNA in the absence of large T-antigen is less than 12 hours in rat F-lll cells at 39°C. It should also be pointed out that the viral DNA in the 33°C to 39°C shift experiment appears to degrade faster than the parental ts-a genomes in the cells maintained at 39°C. This may signify different stabilities at 39°C for input DNA and DNA synthesized de novo in F-lll cells. From these results we conclude that large T-antigen is required continuously for the replication of polyora DNA in F-lll cells. Large T-antigen may also be required for the stabilization of the viral genome in nonpermissive cells. Viral DNA replication 9: exogenous and endogenous viral sequences _ig mlyota transformed cells. Multiple explanations are possible for the occurrence of a small subfraction of infected cells with a high level of viral WA. For example, there may exist a small fraction of cells in which large T-antigen is produced at a high level, or there may be a stall fraction of the population engaged in the transformation pathway. To attempt to analyze these possibilities, we tested the replication of the polyora viral genore introduced by infection into a polyota transformed cell lire; that is, in a population of cells in 69 which all cells are transformed and express high levels of large T-antigen. Results from this experiment indicate that the superinfecting genome is replicated less than the endogenous viral genome at either temperature, but the replication of the superinfecting genote is greater in transformed cells than in F-lll cells (data not shown). The'results do not support the hypothesis that either transformation or high levels of large T-antigen are sufficient for high levels of viral DNA replication. Similar results have been observed for SV40 (26). Previous results have shown that integrated tandems of polyoma DNA undergo large T-antigen-mediated DNA replication in a small fraction of the cell population (47). Excision of the replication products leads to the formation of free viral DNA within the transformed cell. Since we find a large difference in DNA replication between cells infected at 33°C and 37°C, a study of the viral DNA replication in a polyoma-transformed cell line was undertaken. For this experiment we used an A2-transformed F-lll cell line, FA37-l4, which contains mutliple tandem integrations of the viral genote. FA37-l4 cells were grown at both 33°C and 37°C for two weeks prior to the extraction of low'molecular weight DNA. The analysis of viral DNA from.an equal number of cells grown at 37°C (Fig. 5, lane 1) and at 33°C (lane 4) shows that viral DNA synthesis is greater at the lower temperature. Lanes 2 and 3 represent dilutions of 1:4 and 1:2, respectively, of the DNA from the 33°C cells. By comparing the polyoma DNA concentrations in lanes 1 and 2, we conclude that viral DNA synthesis in FA37-l4 is 70 Figure 5. Viral DNA replication in an A2-transformed cell line. The A2-transfonmed cell line FA37-14 was:maintained at both 33°C and 37°C for two weeks prior to viral DNA isolation. For the experiment, two plates were seeded with FA37-l4 at each temperature. One of these was used to determine the cell number and the other was used for isolation of viral DNA. An aliquot of the isolated DNA from an equal number of cells from the 37°C plate (lane 1) or the 33°C plate (lane 4) was digested with EcoRI and electrophoresed, transferred, and hybridized as in Fig. 1. Also presented are 1:4 (lane 2) and 1:2 (lane 3) dilutions of the DNA aliquot in lane 4. 1 71 234 72 approximately 15 times greater at 33°C than at 37°C. This difference is similar to that seen in A2-infected F-lll cells by 10 days postinfection (see Fig.1). Expression 9§_1arge T-antigen at_§§°C and 32°C. As shown in Fig. 4, large Teantigen is required for viral DNA synthesis in rat cells. It is conceivable that the level of expression of large Tbantigen is greater at 33°C than at 37°C, and that increased levels of large T-antigen account for the increased viral DNA synthesis at 33°C. Another possibility is that the protein is more stable at the lower temperatures The accumulation of large Teantigen in A2-infected cells at early times after infection was investigated by immunoblot analysis of whole cell extracts as described in Fig. 6 and Materials and Methods. From this experiment, it is clear that the accumulation of large T-antigen by 12 days postinfection in F-lll cells is much greater at 33°C than at 37°C (compare B and C of Fig. 6). In the 37°C infection, large T-antigen can only be detected at the 12 day time point if the gel is overloaded (data not shown). By 12 days postinfection the level of large T-antigen at 33°C is approximately 10 times greater than at 37°C (data not shown). Previously, we attempted to use metabolic labeling of infected cells with 355-methionine, but we were not able to detect any large T-antigen. This result may indicate that the rate of synthesis of large Teantigen is not increased at 33°C, but that it is more stable at this tetperature. Using indirect immunofluorescence large T-antigen was detected as early as three days postinfection in A2-infected F-lll cells at 33°C and 37°C. 73 Figure 6. western blot of whole cell extracts of polyoma-infected F3111 cells. Felll cells were infected at an MOI of 10 pfu/cell. At the times indicated in the figure a plate of infected cells was harvested and processed as described in Materials and Methods. SOS-PACE analysis of 1/30 of each sample was performed on‘a 10% acrylamide gel. A.) Control lanes. Whole cell extracts are from uninfected F-lll cells (Co) and from A2-infected mouse NIH-3T3 cells (MOI of 10 pfu/cell) at 48 hours postinfection (NIH). B.) Whole cell extracts from A2-infected F-lll cells at 33°C. Time points are 1, 4, 8, and 12 days postinfection. C.) Same as in B.) except the infection was carried out at 37°C cells. The position of large Teantigen is marked LT. D.) Whole cell extracts from A2-transformed cell line, FA37-4. “— 44- O U 74 '1': dayl4Il2 40114.12 75 Also included in this experiment are whole cell extracts from an A2-transformed cell line, FA37-4. This cell line was maintained at both 33°C and 37°C for one week prior to the protein extraction. The results again show a higher level of large Thantigen in cells grown at 33°C compared to cells at 37°C (Fig. 6D). This difference, however, is only 3 to 4-fold compared to the l0-fold difference seen at early times after infection of rat cells. Immunoblotting has not allowed in the detection of the other early proteins, middle and small T-antigens. Therefore, it is not known if these proteins are also expressed at higher levels in cells grown at 33°C. Thelother proteins visible on the blot are probably cellular proteins which react with the anti-polyoma tumor ascites fluid. In summary, an increase in large Teantigen expression corresponds to a similar increase in viral DNA.synthesis. However, it cannot be determined from this experiment whether the increased DNA synthesis provides more templates for early gene transcription or if the increase in large Teantigen synthesis is required for the increase in viral DNA synthesis. we note that no large Teantigen can be detected in infected F—lll cells at early times postinfection, a time when experiments show that large Teantigen is required for transformation. Indeed, in infections with ts-a mutants, incubation at the permissive temperature is required only during the first 2-3 days postinfection (24). Thus, if synthesis of large Teantigen is required at these times, them very low levels may be sufficient. 76 Effects g viral DNA synthesis g1 transformation. As mentioned above, we had previously noted that transformation frequencies with polyoma are sorewhat higher in rat cells maintained at 33°C compared to those maintained at 37°C. The observed increase in viral DNA synthesis :may cause an increase in transformation in at least two ways. One is that processes associated with viral DNA synthesis per se are required for integration (for example, unwinding or nicking of DNA during replication). The other is that the number of templates per cell is increased and consequently the integration probability is increased. Representative results from transformations at 33°C and 37°C are shown in Table 2. These results show that the large overall increase in viral DNA levels (l0-fold) at 33°C compared to 37°C is not paralleled by an equivalent increase in transformation frequency (2-fold). The increase in transformation frequency at 33°C may not correlate with the increase in viral DNA synthesis since it is also observed in infections with N659RA in which no increase in viral DNA synthesis at 33°C is observed. Effect 93 the number pf viral terplates o_n integration @tterns. If viral DNA replication is required for the tandem integration of viral genores in polyoma-transformed rat cells, then the difference in viral DNA replication at 33°C and 37°C may lead to differences in the integration patterns of cells transformed at the two temperatures. The increase in the number of genores at 33°C may also lead to an increase in the number of integration sites. These points were investigated by isolating total DNA from cell lines 77 Table 2. Transformation frequenciesa Percent transformation Strain 33°C 37°C 33°C/37°C A2 0.25 (r=0.22-0.29) 0.11 (r=0.07—0.15) 2.3 NG59RA 0.11 (r=0.06—0.15) 0.05 (r=0.02-0.07) 2.2 aF-lll cells at a density of 1 x 105 cells/60 mm culture dish were infected with either A2 or N659RA at an MOI of 10 pfu/cell. After the infection the cells were passed 1:4 and grown at either 33°C or 37°C. The transformation frequencies presented are the average of three experiments for A2 and two experiments for N659RA. The frequencies are based on the number of transformants per 1 x 105 cells. 78 transformed by A2 at the two temperatures. Digestion of DNA with EcoRI was used -to determine the presence or absence of tandem repeats, and digestion with 89111 was used to detenmine the number of integration sites per cell line. Using this approach we analyzed 19 A2-transformed cell lines. The results of representative EcoRI digests are shown in Fig. 7. Of the 19 lines analyzed only three did not have an integrated tandem repeat (see Fig. 7, lane 4; and Table 3). Two of these originated from the 33°C infection and one from the 37°C infection. Analysis of the number of integration sites in these cell lines by digestion with BglII also failed to detect differences between the 33°C and 37°C cell lines (Fig. 8). Only a few of the A2-transfonmed lines, 2/10 from 33°C and 2/9 from 37°C, contain a single integration site (see Table 3). Furthermore, the overall number of integration sites from the 33°C cell lines does not appear to be increased compared to that found in the 37°C cell lines, and the number of genomes per site as determined by the sizes of the BglII restriction fragments is not affected by temperature. Overall, the integration pattern of polyoma virus appears bimodal regardless of the temperature at which the cells were transformed and maintained: either many sites of integration with tandem repeats or a single site without tandem repeats. DISCUSSION we have analyzed viral DNA synthesis in nonpermissive cells during the early stages of neoplastic transformation by polyoma virus. In F—lll cells, little overall net synthesis is observed at 37°C. Higher levels of synthesis are seen in FR-3T3 and BHK cells at 33°C. 79 Figure 7. Presence of integrated tandem repeats in A2-transformed F-lll cells. Foci were picked from monolayers of F-lll cells which had been infected at an MOI of 10 pfu/Cell with A2 at either 33°C or 37°C. Picked foci were agar cloned at the temperature at which they were isolated. For each lane, 10 ug of total cellular DNA from 33°C (lane l-3) and 37°C (lanes 4-6) cell lines was digested with EcoRI. Electrophoresis and transfer to nitrocellulose was the same as in Fig. l. Hybridization was carried out for 72 hours at 65°C using (32P)pPy-1 (2 x 1a6 cpm/ m1 of hybridization solution). 80 81 Table 3. Summary of integration analysis Temperature Single sitea Tandemrepeatsb 33°C 2/10 8/10 37°C 2/9 8/9 3The number of integration sites per cell line was determined as described in Fig. 8. bThe presence of tandem repeats was determined as described in Fig. 7. 82 Figure 8. Analysis of the number of integration sites in A2-transformed cells. The DNAs in lanes 1-3 are from cell lines isolated from a 33°C infection, and the DNAs in lanes 4-6 are from cell lines isolated from a 37°C infection. For each lane 10 pg of total cellular DNA was digested with 89111 and electrophoresed on a 0.4% agarose gel. Transfer to nitrocellulose and hybridization were conducted as described in Fig. 7. The position of Form.I DNA is marked SC. 83 C S 84 _I_r_1 fl hybridization, however, demonstrates that a fraction of the infected cells are actively producing viral genotes. In contrast to the results with the 37°C infection, a large amount of viral DNA is observed at 33°C in F-111 and FR-3T3 cells. This increase is achieved by recruiting a larger number of cells to synthesize DNA and by increasing the yield of DNA per producing cell. A substantial fraction of this DNA is genome-size form I. Similar to the requirements for viral DNA synthesis in permissive cells, synthesis in nonpermissive cells is under large T-antigen control. This is demonstrated by the existence of a non-lethal mutation in large T-antigen which greatly decreases the ability of strains carrying it to synthesize DNA at low texperature in F-lll cells (Hacker et al., submitted). Furthermore, the level of viral genotes decreases rapidly when large T-antigen is reroved by shifting cells infected with a ts-a mutant to the nonpermissive temperature. This experiment indicated a half-life of about 12 hours for polyora DNA in the absence of large T-antigen at 39°C. Finally, mutations which eliminate a large T-antigen binding site, as in Py 1-12, also decrease the viral DNA synthesis in F-lll cells. The pole g viral DNA smthesis i_n transformation by Elyoma. The arguments for and against a role for viral DNA synthesis in neoplastic transformation have been reviewed in the Introduction. Easentially, a requirerent for DNA replication has been postulated because of the integration pattern of the viral genomes and the requiretent for large T—antigen in the initial steps of transformation. Although 85 transformation is somewhat elevated under the conditions in which more viral DNA synthesis is observed, the overall results presented here argue that viral DNA replication (i.e., de novo synthesis of viral genomes) is irrelevant to neoplastic transformation. This is supported by the fact that DNA positive cells are found in nontransformed areas of the monolayer, that transformation defective mutants also induce viral DNA synthesis, that the increase in viral DNA at low tetperature is higher than the increase in transformation frequency, that transformation frequencies continue to decrease in a temperature range in which no further decrease of viral DNA synthesis is observed, and that an increase in transformation frequency is also observed at 33°C for strain N659RA although very little increase in viral DNA, if any, is observed with this strain at that temperature. Furthermore, the requirerent for large T-antigen function in polyora transformation occurs during the first 2 days post-infection (24) , a time at which no net viral DNA synthesis can be detected. An additional argurent comes from the analysis of the integration patterns. The number of viral tetplates present at later times postinfection apparently does not affect the frequency of tandem integration of viral genomes, the number of sites of integration in the host chrorosotes, or the number of genomes integrated per site. Thus, the increase in viral DNA replication does not overtly affect the integration pattern. Overall, the integration patterns observed in these experiments and others from our lab appear bimodal: either integration occurs at multiple sites in the host with tandem repeats of 86 the genomes or integration of less than a single copy at a single site is observed. These two types of integration may represent different kinds of events. The absence of change in integration patterns with increased viral DNA synthesis at late times postinfection, the requirements for large Teantigen at early times postinfection, and other unpublished data from our lab all suggest that an important step in the integration/transformation event is fixed at an early time postinfection. A final argument against an important role for net synthesis of viral genores in transformation comes from the following observation. Viral sequences integrated in transformants derived from mixed infections between two physically marked parental genomes have undergone a high level of interviral recombination (even when recombination appears to have no selective advantage for transformation). In contrast, no recombination is observed in the population of replicated unintegrated viral molecules (Hacker and Fluck, submitted). This suggests that the population of cells which is actively replicating viral DNA is not the same as the population of cells which becores transformed. Furthermore, these and other results from our lab suggest that a significant fraction of the tandem structures seen in transformed cells can be accounted for by recombination events. This result weakens one of the major rationales for the implication of viral DNA replication in transfonmation. Altogether, the results presented in this report suggest that the cells which do undergo net synthesis of viral DNA are not the precursors of the transformed cells . 87 The increased level of viral DNA synthesis at 33°C is intriguing, but this phenomenon may be trivial. Mammalian cells are quite sensitive to elevated temperatures. Mouse cells also produce higher yields of viral DNA and virus progeny at 33°C (unpublished results). Transformation frequencies in rat F-lll cells drop steadily as a function of temperature (Kalvonjian and Fluck, unpublished data), and at least in the range between 35°C and 49°C, this decrease is not paralleled by a decrease in viral DNA synthesis. The mechanism g mlyoma DNA synthesis E nonpermissive cells. It has often been suggested that viral DNA synthesis in nonpermissive cells might involve a rolling circle mechanism of replication (ll) . This has been proposed because of tandem integration of the polyoma viral genome and because of the requirements for large T-antigen (11) and a functional origin of replication in tandem formation (9). This model is appealing since it allows for the production of large yields of DNA with a single initiation event and for the production of concatameric molecules. Such a model would be consistent with a role for large T-antigen limited to the initiation of viral DNA synthesis, as suggested by experiments involving permissive cells infected with ts-a/A mutants (20,41) . _lg M experiments, however, have also suggested a role for SV40 large T—antigen in chain elongation (38). The recent discovery of a helicase activity associated with the protein supports this finding (10,39) . 0.11: experiments do not help resolve this question. The absence of high molecular weight intermediates cannot be used as an argument against rolling circle replication 89 genome are altered (11). Since ts-a mutants are not deficient in abortive transformation (13,40), it had been proposed that the defect of ts-a mutants is an "initiation" defect such as integration. Exactly what that role is remains to be elucidated. Large T-antigen appears to be required for the formation of integrated tandem viral genome structures (11), yet at least a fraction of these appear to be created by recombination (Hacker and Pluck, submitted). mat is clear from the present experiments is that the role of large T—antigen is not simply to amplify the viral genome to enhance the frequency of integration. In no way do the results exclude the possibility that a function of large T-antigen essential for DNA replication (such as the nicking or unwinding of DNA) is also essential for the integration of the viral DNA into the host genome. 10. 11. 12. 90 REFERENCES Basilico, C. 1971. The multiplication of polyoma virus in mouse- hamster somatic hybrids, pp. 12-21. In: L.G. Silvestri (ed.), The biology of oncogenic viruses. North Holland Press, Amsterdam. Bendig, M.M., and W.R. Folk. 1979. 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Knippers. 1985. A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichramosomes in an in vitro elongation system. J. Virol. 54:473-482. Stoker, M., and R. Dulbecco. 1969. Abortive transformation by the TsA mutant of polyoma virus. Nature 223:397-398. Tegtmeyer, P. 1972. Simian virus 40 DNA synthesis: the viral replicon. J. Virol. 10:591-598. Tooze, J., ed. 1981. DNA tumor viruses, 2nd Edition. Cold Spring Harbor laboratory, Cold Spring Harbor, New York. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application. Proc. Natl. Acad. Sci. U.S.A. 76:4350-4354. Vogt, M., and R. Dulbecco. 1965. Studies on cells rendered neoplastic by polyoma virus: the problem of the presence of virus-related materials. Virology 16:41-51. Watkins, J.F., and R. Dulbecco. 1967. Production of SV40 virus in heterokaryons of transformed and susceptible cells. Proc. Natl. Acad. Sci. U.S.A. 58:1396-1403. Wettstein, F.O., and J.G. Stevens. 1982. Variable-sized free episomes of Shope papilloma virus DNA are present in all non- virus producing neoplasms and integrated episames are detected in some. Proc. Natl. Acad. Sci. U.S.A. 79:790-794. Zouzias, D., I. Prasad, and C. Basilico. 1977. State of the viral DNA in rat cells transformed by polyoma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations. J. Virol. 24:142-150. Chapter 3 . A Nonlethal Mutation in Large T-Antigen of Polyoma Virus which Affects Viral DNA Synthesis]- ABSTRACT A mutation in polyoma virus large T-antigen which affects viral DNA synthesis is described. In nonpermissive Fisher rat F-lll cells, the mutation causes a 10-20 fold decrease in the yield of viral DNA at 33°C compared to wild-type A2 infections. Differences in the integration patterns of the mutant and wild-type genomes are also observed in transformed F-lll cells. The mutation maps to a region between the HindIII and NsiI restriction sites (nucleotides 1656-1910). Sequence analysis of this region revealed a C to G transition at nucleotide 1791 which causes a proline to alanine change in the amino acid sequence of large T-antigen. 1 David L. Hacker, Karen Friderici, Ming Chu Chen, Sonya Michaud, and Michele M. Fluck, Department of Microbiology, Interdepartmental Program in Molecular Biology, Michigan State University, East Lansing, MI 48824-1101. 94 95 INTRODUCTION The role of the large T-antigens of polyoma virus and simian virus 40 (SV40) in viral DNA replication is well established (12,33). Large T-antigen is required for initiation of viral DNA synthesis (9,12,33) and may also be involved in elongation (5,31) . It is the only viral protein required for viral DNA synthesis i_n _v_i£r_g (24,35) as most of the proteins necessary for this process, including DNA polymerase and DNA primase, are provided by the host cell (25). Much of our knowledge of the function(s) of the polyoma and SV40 large T-antigens has come from studies of conditional lethal mutants. These have revealed the multifunctional nature of these proteins and have led to the mapping of several domains. For polyoma large T-antigen, regions important in DNA replication (7,8,16), DNA binding (14), and cellular immortalization (26) have been defined. In addition, this viral protein has ATPase (17) and nucleotide-binding activities (3) that are important in viral DNA replication. In the course of a study on polyoma viral DNA replication in nonpermissive rat cells, we discovered a defect in a viral strain previously assumed to be wild-type. The present report concerns the characterization and mapping of this mutation. Interestingly, the mutation maps outside of the domains of large T-antigen that were previously defined by mutations. MATERIALS AND METHODS Cells and viruses. Mouse NIH-3T3 (21) and two Fischer rat cell lines, F-lll (13) and FR-3T3 cells (28) , were grown in Dulbecco's modidfied Eagle's medium (DMEM) with 5% heat-inactivated calf serum. 96 The polyoma viral strains A2 (19), N659RA [RA] (l0), and BZ (32) were grown on baby mouse kidney cultures from plaque purified viruses as described (34) . The pseudo-wild-type strain RA was obtained by marker rescue of the middle T-antigen defect of the hr-t mutant NG59 (10). N659 was derived from the Pasadena small plaque strain by nitrosoguanidine mutagenesis (l) and has been partially sequenced (2) . The hr-t mutant B2 was derived from the Pasadena small plaque strain by mutagenesis with ICR-l9l (32) . 82 contains a deletion of nucleotides 491 through 730 (numbered as in [29]). Viral reconstruction. Viral DNA for the construction of mutant viruses was prepared in one of two ways. Both the A2 and RA genomes were cloned at their BamHI sites into pAT153. Plasmid DNA was isolated as previously described (22) . Alternatively, viral DNA from A2- and RA-infected NIH-3T3 cells was prepared by the method of Hirt (20). The restriction fragments shown in Fig. 1 were exchanged between the two parental viral genomes. The names of the resulting viruses consist of the name of the parental virus contributing the largest DNA fragment followed in parentheses by letters designating the restriction enzymes used and the name of the virus contributing the smaller fragment. For instance, A2 (E/N RA) is an A2 virus in which the 350 base pair EcoRI to NsiI fragment has been replaced with the corresponding fragment from the RA virus. Viral DNA (500 ng) was digested with 20 units of restriction enzyme for four hours. The digested DNA was resolved by agarose gel electrophoresis, and the fragments were recovered from the gel by 97 Figure 1. Map of polyoma virus. (A.) Linear map of the polyoma virus genare showing the muddle (mT) and large T-antigen (LT) coding regions and the locations of several ts-a mutations. The genare is numbered as in Soeda gt & (29) . (B.) The restriction fragments from A2 which confer competence for DNA replication at 330C in F-lll cells for the RA/A2 hybrid viruses. 98 3.. m— ‘— 2.23.25 5 .95: 2.3. 32:15am :unzxsua 33w: .3 952: h a 9:13 hE 99 electrophoresis onto 3MM paper backed by a dialysis membrane (18) . A total of 50 ng of viral DNA fragments were ligated in 10 pl of ligation buffer (66 mM Tris, pH 7.5; 10 mM MgC12; l mu DTT; and l mM ATP) for 16 hours at 16°C using T4 DNA ligase (New England Biolabs) . For transfections, 10 ng of DNA was applied to 1.5 x 105 NIH-3T3 cells /35 mm dish using 500 ug/ml DEAE dextran at 33°C for 1 hour. The dishes were washed and overlayed with DMEM containing 0.9% agar and 5% calf serum. Plaques were picked 10 days later, and viral stocks were grown on NIH-3T3 cells. Viral DNA replication assaj. Cells were infected at a density of l x 105 cells/60 mm culture dish at a multiplicity of infection (MOI) of 10 plaque forming units (pfu)/cell. At the times indicated, viral DNA was isolated by the procedure of Hirt (20). For each time point, 10% of the extracted DNA was digested with EcoRI to linearize the viral DNA. The digested DNA was electrophoresed on a 0.7% agarose gel and then transferred to nitrocellulose (30). The blots were hybridized in 2X SSC/lx Denhardt's solution (0.1 ml/emz) for 18-24 hours at 65°C using a polyoma probe (1-2 x 109 cpthJg; 5 x 105 chl of hybridization solution) . Transformation. F-lll cells were seeded at a density of 1 x 105 cells/60 mm culture dish and infected with either A2 or RA at an MOI of 10 pfu/cell. For the competition experiment, the cells were infected with B2 and either A2 or RA at the MOIs given in Fig. 3. Infected cells were grown at either 33°C or 37°C in DMEM containing 5% heat-inactivated calf-serum. Cells which overgrew the monolayers of 100 A2- and RA-infected cells were transferred to 35 mm culture dishes and grown in DMEM containing 5% heat-inactivated calf serum. The transformants were then cloned in agar. Integration analysis. Total cellular DNA was isolated from A2- or RA-transformed cell lines as described (15). For each cell line, 10 pg . of DNA was digested either with EcoRI, which cuts polyoma DNA once, or with BglII, which does not cut the viral DNA. Digested DNA was electrophoresed on 0.7% agarose gels if digested with EcoRI or on 0.4% gels if digested with BglII. After transfer to nitrocellulose (30), the blots were hybridized to a polyoma probe (1-2 x 109 cpm/pg) . Hybridization was at 65°C for 40-48 hours in 2X SSC/lX Denhardt's solution (0.1 ml/cmz) using 1 x 106 cpn of the labeled probe per ml of hybridization solution. Sequencing. Sequencing was carried out according to the procedure of Maxam and Gilbert (23) . The nucleotide numbering system is that of Soeda fl al. (28). RESULTS Comparison g viral DNA synthesis between .A_2_ and RA. Previous results from our laboratory (Hacker and Fluck, submitted) have shown that some strains of polyama virus, including wild-type A2, undergo elevated levels of viral DNA synthesis in nonpermissive F-lll cells at 33°C, while only minimal levels are observed at 37°C (Fig. 2A). In contrast, little replication occurs at 33°C with the RA strain, so that the difference in viral DNA synthesis between the two temperatures is minimal (Fig. 2B). A similar reduction in DNA synthesis with RA was observed in Fisher rat FR-3T3 cells at 33°C (not shown). 101 Figure 2. comparison of viral DNA synthesis between A2 and RA in F-lll cells. F-lll cells were infected with A2 or RA, and viral DNA was extracted and analyzed as described in Materials and Methods. The infections were carried out at 33°C and 37°C as indicated. (A.) A-2 infected F-lll cells. (B.) RA-infected F-lll cells. 33° 102 33° 37° 1 4 7 1 4 7 103 Transformation by E _i_n_ the gesence g; a Transformation frequencies of RA and A2 were compared in rat F-lll cells at 339C and 37°C, and the frequency of transformation by RA was two-fold less than that of A2 regardless of the temperature (not shown). Differences in the transfonming behavior of these two strains were also apparent when maxed infections were conducted with a transfonmation defectivewmutant. As reported elsewhere (11) , mixed infections with wild-type strains and nontransfonming mutants lead to strongly depressed transformation frequencies as compared to infections with wild-type virus alone. This is evident in mixed infections with A2 and B2 (Fig. 3) . No dominant lethal effect was seen, however, in mixed infections with RA as the transforming parent. As shown in Fig. 3, the yield of transformants in the mixed infection was higher than in the infection with RA.alone. This observation suggests that the transformation potential of RA is slightly deficient and can be complemented in trans by a functional large T-antigen. Analysis gf_integrated viral sequences in_transformed cells. To analyze the potential effect of the RA.mutation on integration patterns, A2- and RA-transformed cells were analyzed for the number of viral integration sites and for the presence of integrated tandem repeats of the viral genome. For this purpose total cellular DNA from Veach transformant was digested with BglII and EcoRI. Since BglII does not out within the polyoma genome, the number of polyoma-specific restriction fragments observed on blots of BglII-digested DNA corresponds to the number of distinct integration 104 Figure 3. Transformation in mixed infections of F-lll cells. F5111 cells were infected with mixtures of transforming (A2 or RA) and nontransforming (B2) strains and polyoma. The ratios ofoOIs of A2 or RA to B2 are given. Transformation was scored by the appearance of foci over the monolayer. The transfonmation efficiency was determined by dividing the number of foci obtained in thelmixed infection with the number of foci obtained in the infection with the transforming strain alone. 105 RAIBZ b b I A2I32 A V i V A T— 1/10 1/20 1/5 1/1 MOI 23+- 17" 10¢. a q q u 4 2 1 Km >uco.o_:u catactozca; 1/0 (WT/B2) 106 sites. Digestion with EcoRI, which cuts the polyoma genote once, provides a method to determine the presence of tandem repeats within a cell line. A summary of the analysis of 19 A2- and 16 RA-transformed cell lines is shown in Table l. A distinct difference in integration patterns was observed between A2- and RA-transformed cells. This difference was reflected in both the number of integration sites and the presence of tandem repeats. The temperature at which the infections were carried out, however, had no apparent effect on integration by either of these strains. Single integration sites were observed in 10 of the 16 RA-transformed cell lines but in only 4 out of the 19 A2-transformants. Similarly, tandem repeats are present in only 50% of the RA-transformants but in 84% of the A2-transformants. These results suggest the presence of a defect in RA which affects integration of the viral genore during neoplastic transformation of nonpermissive cells. Viral DNA synthesis with reconstructed viruses. To locate the region of the polyoma virus genome coding for the defect in viral DNA synthesis, we constructed hybrid viruses between the RA and A2 strains. A number of reconstructions were made either by using restriction enzymes which cut at two sites within the polyoma genore or by using combinations of two enzymes, each of which cuts at a single site in the viral genole. The restriction fragments used the construction are shown in Fig. 1. Each reciprocal pair of hybrid viruses was tested for replication at 33°C in F-lll cells. men the fragments shown in Fig. l originated from the A2 genome, the resulting hybrid genomes 107 Table 1. Integration analysis.a Strain Temp.b Single sitec Tandem repeatsd A2 33°C 2/10 8/ 9 A2 37°C 2/ 9 8/ 9 RA 33°C 4/ 8 3/ 8 RA 37°C 6/ 3 5/ 8 aInfections with A2 and RA were carried out at either 33°C or 37°C. Transformants from.these infections were agar cloned and maintained at the same temperature as the infection. bTemperature. CAs determined by digestion of total cellular DNA from the transformants with BglII, an enzyme which does not cleave polyoma DNA. dAs determined by digestion of total cellular DNA from the transformants with EcoRI, an enzyme which cuts polyoma DNA once. 108 replicated like A2 at 33°C. When the restriction fragments were taken from RA, the resulting hybrid genotes replicated like the RA virus at 33°C. The EcoRI/NsiI fragment from A2 (nucleotides 1560-1910) was the smallest fragment tested which conferred A2-1ike DNA replication on RA (Fig. 4) . Since the HindIII fragment from A2 also rescued the replication defect in RA (Fig. 4), the mutation is likely to be between the HindIII site at nucleotide 1656 and the N511 site at nucleotide 1910. Only large T-antigen is encoded by this region of the genome (Fig. l). Seglence analysis o_f_ RA. A plasmid containing the RA virus was used to determine the sequence of the large T-antigen coding region downstream of the HindIII restriction site (nucleotide 1656). (he sequence difference was found at nucleotide 1791, where a C to G transition has occurred in RA. This results in an amino acid change from proline to alanine at amino acide position 412 of large T-antigen. Other sequence differences in the middle T-antigen coding region between NG59, the parent strain of RA, and wild-type A2 have been observed previously (3) . A valine codon which is not part of the published sequence for wild-type A2 (29), but is present in wild-type A3 (27), was discoverd near the HindIII site in both the A2 and RA strains from our laboratory. DISCUSSION The experiments presented in this report describe a novel mutation in large T—antigen of polyota virus strain RA. The effect of this mutation on viral DNA replication in permissive mouse cells is not 109 Figure 4. Comparison of viral DNA synthesis using hybrid viruses. F-lll cells were infected with RA, A2, or hybrid virus constructions. Infections were performed at 33°C, and viral DNA was extracted at l, 4, and 10 days postinfection as described in Materials and Methods. The viral fragments contributed by eadh parent are represented in cartoon form at the bottom of the figure. 110 =1£I :. we“: :35... 515... o. v _ o. c . .N(==.:on_ .oth :_o..m 129 The MOP1033 stock contains a defective viral genome as evidenced by the presence of an AvaI/BamHI restriction fragment which migrates at 4.3 kbp (Fig. 2). This fragment does not interfere with the detection of recombination since it does not migrate close to the 1.3, 1.7, and 3.6 kbp AvaI/BamHI restriction fragments. Another prominent restriction fragment of about 1.0 kbp appears at 10 days postinfection. Longer exposures show that this fragrant is present in the tsB stock. High level gf recombination .13 integrated genomes. Transformed foci arising from the infections described above were visible by 10 and 17 days postinfection for the 37°C and the 33°C cultures, respectively. These were isolated, and total DNA was extracted from the transformants and analyzed for integrated recombinant viral genores as described in Materials and Methods. a.) Recombination i_n the 3.6 kbp AvaI/BamHI interval. Recombination in this interval was analyzed by hybridizing blots of the AvaI/BamHI digestions with the polyoma FcoRI/XbaI probe (Fig. 3). In 23 of the 65 (35%) cell lines analyzed, the 3.6 kbp AvaI/BamHI restriction fragment is present, indicating that recombination between the two parents has occurred (Table l) . In addition to the recombinant fragments, the 5.0 and 5.3 kbp fragtents from the two parental viral genotes are also visible in many cases. Both parental genomes are present in 14 out of 23 cell lines, while a single parental genome is present in 6 out of 23 cells lines. b.) Recombination i_n the 1.3 kbp and the 1.7 kbp AvaI/BamHI intervals. The blots from the analysis above were washed 130 Figure 3. Recombination in the 3.6 kbp AvaI/BamHI interval. Total cellular DNA (10 pg) was digested with a combination of AvaI and BamHI. Hybridication was to the polyoma EcoRI/XbaI probe. The cell lines represented here were established from the 37°C coinfections in Exp. Nos. 6 and 7 (Table 1). The locations of the 3.6 kpb AvaI/BamHI fragrent, the MOP1033 genome—size fragment (5.3 kbp), and the large AvaI fragment fromlt53 (5.0 kbp) are shown. 131 132 .mfimuoo now muocuw: pom mameumum: mow .coflowmce now wmmmmmo on nowum c395 heamwucocomxm mums won—330 Zoo on» .o com o .N 3352 ucmefiummxu mom .cofloomcfi wow uncommon ou uoflo ucmcamcoo mum: non—.1350 duo on» .5 can m .m J mama—HZ ucgummonm mono .mucmEomuw onus» wo oceumowmflcwow may How H .53 cu momma .ooon Héeoopm on... cu Esophagus»: ho pouooumo mos ucmemmum H333 one. o.m on» no mocwmouo one .woouo mIHHmo: on» on cowumufiofiuoao an empowumo mos mucmpeomum .5303»me HERE m9. 54 can mtm on» no mocwnmum ween SEN Sea 3me H33 om} em} 3% EA Hobos oxm oxm o\m o\m m\~ m) mxm m\s s o} es o\o o\m mi m) m} .3. o «\m in {N «\m «\m is {a «\m m {m «\m in {M is S. S. is 1. SA SR 3} EA Qs m\s mxs mxs m m} m} mxs m\s I I u- I N we on so on -- .. -- I H ooz .es ofifinoo enema moss; seem; ofifimoo moses. ones; seem; mongoose H332 1* H38. * H38 comm oomm 838% mos: Zoo pwauommcmuu 5 mogumcflgomu mo hump—5m .H manna 133 and then hybridized with the HpaII-S probe to detect evidence of recombination within the small AvaI/BamHI interval or between the AvaI sites (Fig. 4). In many of the cell lines, both the 1.3 and the 1.7 kbp restriction fragIents are present. As before, the 5.0 and 5.3 kbp fragments from the two parental genores are also visible. The presence of either the 1.3 or the 1.7 kbp fragment is evidence that recombination between the MOP1033 and ts3 genomes has occurred during the integration/transformation process. Either the 1.3 or the 1.7 kbp fragment is present in 24 of the 65 (37%) cell lines analyzed. In all, 25 of the 65 (38%) contain at least one of the three fraglents that is diagnostic of recombination between MOP1033 and tsB. In most cases, more than one of the recombinant restriction fragments is present. Fourteen of the recombination-positive transformants have all three of the fragments, five have the 1.3 kbp and the 3.6 kbp fragments, three have the 1.7 kbp and the 3.6 kbp fragtents, and three have only one of the fragtents. The structure 3f the integrated viral genomes. The transformants in which the parental genotes have undergone recombination have the following hallmarks of normal polyoma transformants: head-to-tail tandem integration of the recombinant genotes as evidenced by the presence of 5.3 kbp genote-size restriction fragtents in digestions with a single-cutter such as EcoRI (not shown), multiple restriction fragtents in digests with a zero-cutter such as BglII, and free viral DNA as shown by the presence of form I polyoma 134 Figure 4. Recombination in the 1.3 and 1.7 kbp AvaI/BamHI intervals. Total cellular DNA (10 pg) from transformants established from coinfections with MOP1033 and ts3 was digested with a combination of AvaI and BamHI. Hybridization was to the HpaII-S probe. The DNAs in lanes 1-9 are from transformants established from the 37°C coinfections in Exp. Nos. 6 and 7. The locations of the 1.3 and the 1.7 kbp AvaI/BamHI fragments are shown. 135 5.3 — .- 136 DNA in digests with the zero-cutter BglII (not shown). Along with recombinant viral genotes, parental genomes are also present in most of the transformants. The parental genores are integrated as head-to-tail tandems as evidence by the presence of genome-size restriction fragments (5.0 and 5.3 kbp) in the products of AvaI/BamHI codigestions (see Figs. 3 and 4) . The presence of free viral DNA does not affect the interpretation concerning the presence of integrated recombinant viral genomes since the free DNA has been shown to be excised from integrated copies (1) . The existence of integrated recombinant genomes is based on the following facts. First, three of the transformants contain only a recombinant genole with no obvious sign of the colplete parental genores (not shown). Second, the intensity of the hybridization signal from the recombinant fragments colpared to the genore-size fragtents suggests that they exist as integrated copies rather than being generated post-excision. Third, the recombinant restriction fragments are generally absent in cell lines generated from the 33°C infections. Furthermore, recombination has also been detected at similar high frequency under circumstances in which an integrated recombinant genore is required for transformation (Kalvonjian gt _a_l_._L submitted). Finally, the DNA from one cell line containing all three recombinant fragments was extracted by the Hirt procedure and isolated as low and high molecular weight fractions. AvaI/BamHI digestion of this high molecular weight DNA revealed that the recombinant fragments are present in this fraction (not shown). 137 Further analysis 2:: the 1.3 and 1.7 kbp AvaI/BamHI restriction fragments. The presence of the 1.3 and 1.7 kbp fraglents in the same cell lines is especially interesting since these may represent different recombination events. Conceivably, the 1.7 kbp AvaI/BamHI fragment may arise by incomplete digestion at the first AvaI site (nucleotide 672). To further analyze the origin of the 1.3 and 1.7 kbp restriction fragtents, the DNAs from fourteen of the recombination-positive cell lines were digested with a combination of restriction enzymes: AvaI, BamHI, and either PvuII or BglI. The restriction sites in and around the small AvaI/BamHI interval for these two additional enzymes are shown in Fig. 5. Digestion with PvuII, BamHI, and AvaI generates restriction fragments of 687, 1,046, and 1,174 bp that hybridize to the HpaII-S probe (Fig. 5) . The presence of these fragments depends upon the presence or absence of the AvaI sites. The 1,174 bp fragment is generated by digestion of the MOP1033 genore, and the 687 bp fragment can originate from either the tsB genome or a recombinant genome. Digestion of the 1.3 and 1.7 kbp AvaI/BamHI fragments with BglI generates restriction fragments of 747 and 570 bp and 747 and 929 bp, respectively, that hybridize to the HpalI-S probe (Fig. 5). It should be noted, however, that the B911 site is absent in the MOP1033 genote. Therefore, the 1.3 and 1.7 kbp AvaI/BamHI fragments will be digested with BglI only when this restriction site is contributed by the tsB genore. Of the fourteen cell lines analyzed, ten contained both the 1.3 and the 1.7 kbp fragments, three contained only the 1.3 kbp fragtent, 138 Figure 5. Analysis of the 1.3 and 1.7 kbp AvaI/BamHI restriction fragments. Total cellular DNA (10 pg) from recombination-positive cell lines was digested with AvaI and BamHI (A.); AvaI, BamHI, and PvuII (B.); and AvaI, BamHI, and BglI (C.). The»locations of the restriction sites for these enzymes within and around the wild-type 1.3 AvaI/BamHI interval are shown along with the sizes of the expected restriction fragments for (B.) and (C.) above. Hybridization was to the HpaII-S probe. 139 1.7 — . 1.3 - '0 . . 1.0 — . 0.7 — .. c g 747 bp . 570 bp . I I a 747 bp I 929 bp . I r fl I HpoE-S I BamHI PvuII PvuII BglI AvaI AvaI PvuII 4647 5143 5277102 672 103] "59 687 hp P ' 1046 bp ' ; J 1174 bp 140 and one contained only the 1.7 kbp fragrent. As expected, both the 1.3 and the 1.7 kbp AvaI/BamHI fragtents (Fig. 5, lanes A) were further digested by PvuII in all of the cell lines examined (Fig. 5, lanes B). Addition of BglI to the AvaI/BamHI reactions led to digestion of the 1.3 kbp AvaI/BamHI fragment in 4 out of the 14 cell lines analyzed (Fig. 5, lanes C). The 1.7 kbp AvaI/BamflI fragment, present in eleven of the cell lines, was not digested with BglI (Fig. 5, lanes C). In the ten cell lines with both the 1.3 and the 1.7 kbp fragments, the 1.3 kbp fragment was digested with BglI in two cases. These results can be interpreted to mean that the 1.7 kbp AvaI/BamHI fragrent is generated by a recombination event in which the MOP1033 and tsB genotes recombined between nucleotides 672 and 1016 (interval C, see Fig. 7). If this is the case, then the region between the BamHI site and the AvaI site at position 672 (interval A) is contributed by MOP1033. On the other hand, the 1.3 kbp fragment is occasionally digested by BglI (4 of 14 cell lines), indicating that this restriction site can be donated by the ts3 genome if the recombination event occurs between the BamHI site and the AvaI site at nucleotide 672 (interval B). Conditions which ageit recombination. Previous results from this laboratory have shown that viral DNA replication in nonpermissive cells is higher at 33°C than at 37°C for many strains of polyora (Hacker and Fluck, submitted). The same is true for MOP1033 and ts3 (see Fig. 2) . To determine if the level of replication affects recombination, we isolated transformants from cells that had been infected with MOP1033 and ts3 and then maintained at either 33°C or 37°C following the 24 141 hour incubation at 33°C to allow for decapsidation. The presence of the 3.6 kbp AvaI/BamHI restriction fragment was detected in only 3 of 24 (12%) cell lines isolated from cultures maintained at 33°C (Table 1). On the other hand, 20 of 41 (49%) of the cell lines from cultures maintained at 37°C were positive for the presence of the 3.6 kbp fragtent (Table l) . Similar results were observed when the cell lines were analyzed for the presence of either the 1.3 or the 1.7 kbp AvaI/BamHI restriction fragment. Of the 24 cell lines isolated from 33°C infections, only 5 (21%) were positive for either one of these fragments, while at least one of these fragments was detected in 20 of 41 (49%) cell lines isolated from cultures maintained at 37°C (Table l) . To begin analyzing which cellular factor(s) may affect recombination, cells were maintained in different growth states prior to infection with MOP1033 and tsB (Table 1). For "exponential cells", the cultures were maintained in a state of active cell division prior to passage for infection. For "confluent cells," the cultures were allowed to becore confluent and were maintained this way for 24 hours prior to passage for infection. This population of cells was essentially synchronized at the time of infection. The two protocols for growing the cells did not noticeably affect the recombination frequency (Table 1). Analysis g§_ts3 and MOP1033 viral stocks. Several approaches were taken to address the possibility that the wild-type fragments observed in the transformed cell lines were not generated by recombination but 142 rather were due to a wild-type contamination of one of the viral stocks. First, genomic DNA from cell lines transformed by tsB alone were analyzed by codigestion with AvaI and BamHI. The»blots were hybridized with the HpaII-S (Fig. 6) and the EcoRI/XbaI (not shown) probes. 0f the 17 cell lines studied, none contained the 1.3, 1.7, or 3.6 kbp AvaI/BamHI restriction fragment. Second, no transformants were ever obtained from infections with MOP1033: an indication that the stock of this nontransfonming virus does not harbor a wild-type containment. Finally, analysis of the input viral DNA by AvaI/BamHI digestion (Fig. 2) did not reveal the presence of the 1.3 kbp or the 1.7 kbp restriction fragment. Transfonmation frequencies 12 mixed infections with MOP1033 and t§§;. To ascertain that recombination between the two parental genomes has no selective advantage over infection by the ts3 parent alone, transforming frequencies were measured in single and mixed infections (Table 2). The presence of the nontransforming virus decreases the transformation rate of the ts3 virus by 1.5 to l7-fold at 37°C. This effect (the dominant lethal effect) of a nontransforming parent in mixed infections has been previously noted (12) and is described in detail elsewhere (Oh 3t 314 submitted). Transformation at 33°C was 1.5 to 2-fold higher than at 37°C for the mixed infections as is typical for cell transformation by polyota (8; and Hacker and Fluck, submitted). The higher transformation rate and the lower recotbination rate at 33°C suggests that steps other than homologous recombination affect the integration/transformation pathway. 143 Figure 6. Analysis of tsB-transformed cell lines. Transformants were established from infections with tsB, and total cellular DNA (10 pg) from these cell lines was digested with a combination of AvaI and BamHI. Hybridization was to the HpaII-S probe. The locations of the 1.3 kbp AvaI/BamHI fragment and the large AvaI fragtent (5.0 kbp) from tsB are shown. 5.0 — 144 6:9 145 Table 2. Transformation rates. No . Transformantsb Exp. No.a MOP1033¢ ts3C MOP1033 x ts3 ‘ 33°C 37°C 3 a 32 26 17 4 a 19 22 14 5 o 100 10 6 6 a 56 33 15 7 o 75 15 8 aThe experiments are numbered the same as in Table l. bBased on the infection of l x 105 cells at an MOI of 10 pfu/cell. cCultures were maintained at 37°C after the decapsidation period. 146 DISCUSSION . The experiments presented above were designed to determine the level of recombination between viral genotes in the process of transformation by polyola virus. There was no selective advantage for recombination between viral genores since one of parents, MOP1033, is a nontransforming strain. Unusually high but variable levels of recombination were observed in the transformants. Of the 65 cell lines analyzed, 25 had integrated viral genomes which had been generated by recombination. The maximal level observed corresponds to a recombination frequency of sixty-five percent in a 1.3 kbp interval of the viral genore (see Table 1, Experiment 5) . These values are minimal estimates since we have not analyzed recombination events leading to the double-mutant recombinant (which would generate fragments larger than genore-size) and since recombination of two molecules of the same genotype cannot be scored. In the recombination-positive transformants (designated recombinant transformants) , recombination in the 1.3 or 1.7 kbp AvaI/BamHI interval is accorpanied in eighty-eight percent of the cases by recotbination in the large AvaI/BamHI interval. The recombination events are homologous as judged by the size of the restriction fragments. The integration patterns in recombinant transformants are typical of polyoma-transformed cells: integration of head-to-tail tandems at multiple sites of the host genore with production of sore free viral DNA. Parental genotes were also recovered in all but three of the recombinant transformants. As shown in Table l, recombination frequencies varied from experiment to experiment. It is surprising, however, that higher 147 levels of recombination were observed at 37°C than at 33°C. FOr instance, the 3.6 kbp AvaI/BamHI fragment was present in 49% of the 370C cell lines but only 12% of the 33°C cell lines. we have shown elsewhere that DNA replication is substantially increased in nonpermissive cells at 33°C compared to 37°C (Hacker and Fluck, submitted). However, the apparent antagonism between recombination and replication in our experiments may be fortuitous. First, data from other experiments show that the integration/transformation linked recombination occurs prior to the detection of increased viral DNA synthesis (15; Friderici and Fluck, unpublished data). Second, it appears that cells in the process of synthesizing viral DNA are not the precursors of transformed cells (Hacker and Fluck, submitted). Since large T-antigen is required for the formation of tandems (8) and a fraction of the tandems may be assigned to recombination events, it appears that the role of large Thantigen in the fonmation of tandems may not be viral DNA replication per se. Recently, results from an intramolecular recombination assay involving a polyoma-mouse hybrid replicon containing 1.03 copies of the polyota genore suggest that large T-antigen may have a recombinogenic activity (24). It is not known, however, if this is a direct effect of large T-antigen binding to the viral genole. The results of the analysis of the 1.3 and 1.7 kbp AvaI/BamHI fragments proved to be very interesting. For the 1.7 kbp fragment, the absence of the B911 site and the AvaI site at nucleotide 672 illustrates trat this fragrent is generated by a recombination event in 148 interval C (Fig. 7). The 1.3 kbp fragment results from a recombination event either in interval A (Fig. 7) or in interval B (Fig. 7). The number of recombination events which occurred within these three intervals in the fourteen cell lines that were analyzed is tabulated in Fig. 7. In this group of recombinant transformants the highest number of events occurred in the stallest region, the 359 bp interval C, while the lowest number of recombination events occurred in the largest region, the 747 bp interval A. This gradient of recorbination suggests the existence of a site on the viral genome near the AvaI sites that enhances recombination. Downstream from the AvaI site at nucleotide 1031 is an eight base-pair sequence 5'-GCTGGl‘CT-3' (nucleotides 1162-1155 [16]) in which six of the eight base pairs match the consensus sequence of the lambdaphage Chi site (5'-GCI‘GGTGG-3') , a nucleotide sequence which stimulates recombination by the RecA-RecBC pathways in E: .c_:_o_l_i. (23) . It is not known whether this sequence in the polyota genomes has an effect on recombination in lambdaphage. Chi sites have previously been identified in the mouse immunoglobulin genes (21) . We have attetpted to determine the level of recombination among unintegrated viral genotes present in the population of infected cells at early times after infection (0-10 days). Interestingly, we did not detect the recombinant genotes in this population. The presence of the wild-type restriction fragments should have been detectable at a level of 5% of the total viral DNA as determined by reconstruction experiments. The absence of recombinant fragments in the population of 149 Figure 7. Gradient of recombination in the 1.3 and 1.7 kbp AvaI/BamHI intervals. Partial restriction maps of the small AvaI/Baml intervals from MOP1033 and ts3 are shown. The open and closed symbols represent restriction endonuclease sites whiCh are absent or present, respectively. The number of recombination events which occurred in each of the intervals (A, B, and C) in the fourteen recombination-positive cell lines are tabulated. 150 on s : $3 «a; soeosooru o m < .1 r n . Woo one . no o5 . no A: l .33.... 9 J? IT Hue m3 OF 0 D L... moot—Os. o>< _o>< :3 .153 151 unintegrated viral genores is retarkable considering the percentage of cell lines which were found positive for recotbination. These results can be interpreted in two ways. The first is that the cells destined to becote transformed are selected from a small pool of cells (possibly in a restricted window of the cell cycle) in which recorbination is occurring at a high level. In these cells integration of the viral genores and fortuitous recombination between viral genomes are both enhanced. The second is that only the viral molecules which are involved in the integration pathway (i.e., which interact with the host chromosore) undergo interviral recombination. These alternatives are not mutually exclusive. The present experiments do not exclude the possibility (which we consider unlikely at this time) that one genore integrates and then the second parent recombines with it at a high frequency. The experiments described in this report were initiated as an attetpt to identify the pathway that leads to formation of head-to-tail tandem integrants of the viral genote in polyoma transformants. Although interviral recombination was not selected for in these experiments, a significant portion of the integrated viral genotes in the transformants studied have undergone interviral recombination. The recombinant transformants have the typical integration pattern of polyota transformants. Thus, the present experiments suggest that homologous recombination may account for the formation of a large fraction of the integrated head-to-tail tandems. We cannot exclude the possibility that viral DNA replication plays a role in tandem 152 formation, but it appears that the bulk of the cell population in which extensive viral DNA replication is occurring does not contribute to the population of transformants (Hacker and Fluck, submitted). To our knowledge, the recorbination frequencies observed in these crosses represent the highest recombination rates reported in a mitotic mammalian system, including recombination studies involving plasmid DNA and transfection procedures (9,17,18,25,30). The mechanisms underlying such elevated recombination rates remain to be elucidated. 3. 5. 10. 11. 153 REFERENCES Basilico, C., S. Gattoni, D. Zouzias, and G. Della Valle. 1979. Loss of integrated viral DNA sequences in polona-transformed cells is associated with an active viral A function. Cell 17: 645-659. Birg, F., Dulbecco, M. Fried, and R. Kamen. 1979. State and organization of polyota virus DNA sequences in transformed rat cell lines. J. Virol. 29:633-648. Bjursell, G. 1978. Effects of 2'-deoxy-2'-azidocytidine on polyora virus DNA replication: evidence for rolling circle- type mechanism. J. Virol. 26:136-142. Botchan, M., W. Topp, and J. Sambrook. 1976. The arrangetent of simian virus 40 sequences in the DNA of transformed cells. Cell 9:269-287. Bullock, P., W. Forrester, and M. Botchan. 1984. DNA sequence studies of simian virus 40 chromosomal excision and integration in rat cells. J. Mol. Biol. 174:55-84. Chla, W., and P.W.J. Rigby. 1981. 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Virology 19:158-168. 156 SUIVMARY AND CObCLUSIONS The roles of viral DNA replication and recombination in the integration of the polyoma virus genome into the host chromosome of nonpermissive cells was investigated. The experiments described in Chapter 4 establish that interviral recombination is involved in integration. Although recombinant genomes are undetectable in the population of unintegrated genotes during the early steps of nonpermissive infection, the level of recombinant genomes in transformants is high and can account for a large fraction of the integrated viral genomes. The role of viral DNA replication in integration is not as clear. The experiments described in Chapter 2 indicate that a high level of viral DNA synthesis occurs in a subpopulation of the infected nonpermissive cells at 33°C. This increase in the number of viral genomes does not dramatically affect the transformation frequency at 33°C nor the integration pattern in transformants obtained from.330C infections. The experiments in Chapters 2 and 3 show that large T-antigen has a role in viral DNA synthesis in nonpenmissive cells. Experiments from another laboratory demonstrated a requirement for large Thantigen in the integration of tandem viral genores. It is not known if this requirerent for large T-antigen involves viral DNA replication. Unintegrated high molecular weight species of viral DNA have been hypothesized to be the precursors to the integrated tandem copies of the viral genome. The failure to detect these can be explained in several ways. It is possible that such intermediates do not exist. If 157 they do, they may either be present in low abundance or be short-lived due to resolution to genome-size by intramolecular recombination. The results presented here suggest other areas of research to pursue. One of these concerns the nature of permissivity of cells to infection by polyora. Only a stall percentage of nonpermissive cells can support synthesis of viral DNA. In these cells, the block in virus production appears to be in late gene expression. Therefore, at least two stages of inhibition of viral replication occur after the virus enters the nonpermissive cell. Experiments from other laboratories have implicated the cellular DNA polymerase o/DNA primase as a major factor in permissivity with regards to viral DNA replication, but other host factors are probably involved as well. It is likely that the block in late gene expression involves host factors important in positive or negative regulation of gene transcription. The second major area of interest developed from these experiments involves the mechanims of DNA recombination in mammalian cells. The results in Chapter 4 suggest that a high level of recombination occurs in a small population of cells. It may be that the viral genotes in these cells interact in a specific way with the host chromosotes to allow for both a high level of interviral homologous recombination and nonhomologous recombination between cellular and viral DNA. It is possible that a specific interaction with the nuclear matrix makes the viral DNA more accessible to the cellular recombination machinery. The recombination system described here may be useful in elucidating the viral-host cell interactions involved in the recombination events described in Chapter 4. 14.11115 [[11]] R]! E” V” 0307 N I” U " m WI. '3 A I'llllg T "2 III/[[1]]