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A a ‘ i 2 I v': : 5. " s’ . E . I I" x 5 1 :- . . . ‘ ' f r . I HIGAN ST NIVERSITY LIBRARIES Illlllllllllllllll 1 ill l H l I 31293 009041207 n This is to certify that the dissertation entitled Transformation by Polyomavirus: (1) Dependence on Cell Cycle and (ii) Associated Interviral Recombination presented by Hong-Hwa Chen has been accepted towards fulfillment of the requirements for _£h_._D_ degree in Mi crob i0 logy fln‘ (Li L lfl W Major professor Date September 3Ll99 l MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 .., . :0. L niversr {3/ PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE _ * ‘ * MSU Is An Afiirmetive Action/Equal Opportunity Institution “drowns-9.1 _s l l i _ l i l Transformation by Polyomavirus: (i) Dependence on Cell Cycle and (ii) Associated Interviral Recombination By Hong-Hwa Chen 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 1991 ABSTRACT Transformation by Polyomavirus: (i) Dependence on Cell Cycle and (ii) Associated Interviral Recombination By Hong-Hwa Chen The cell cycle dependence of polyomavirus transformation was analyzed in infections of nonpermissive Fischer rat cells released from CD. A 20 to 50----fold difference in relative rate of transformation was found for cells infected in early (,11 phase compared to cells infected in G2 phase. The transformation differential was reflected in an equally large differential in viral gene expression and was accounted only in part by a cell cycle dependence of viral adsorption (‘2—10 fold). My results suggest the existence of another timed step in early processing of the viral genome. Another major step in cell cycle dependence was observed, which does contribute to differences in transformation potential of cells infected at different phases of the cell cycle. Viral transcription (i.e. the early promoter) showed a strong cell cycle dependence with a large induction of viral transcripts (30—40 fold). Interestingly, viral gene expression was delayed until the G1 phase of the next cell cycle following infection. Previous work has demonstrated the existence of high frequency of homologous interviral recombination coincidental with the integration of the viral genome in the process of neoplastic transformation. I studied the role of large "ll—antigen in this process, using temperature sensitive mutants. My results do not support a. role for large T—antigen, since high levels of recombination were still ol;)ser\.'ed in conditions in which viral DNA synthesis was abolished. However, the €X[)(i?l‘llll€llts do not rule out the possibility that the dosage or the domain of large T—antigen required for recombination is different from that required for viral DNA replication. In the course of the recombination studies, l observed a gradient of recombina- fion fiequencyzflong Unarxflyonutgenonn; whlia,40—fifld dflhwenfiallxnwwwnlthe minimum in the enhancer region and the maximum between nucleotide 1245 and 1387. To my parents iv Acknowledgement I am grateful to my advisor Dr. M. M. Fluck, for her guidance. patience, financial support, many other ways of sharing, and as my ”Scientific Mother”. I also thank my committee members, Dr. J. M. Kaguni, Dr. J. B. Dodgson, and Dr. L. R. Snyder, who have given me many valuable suggestions throughout my research. I also would like to thank Dr. E. Bossi, Dr. E. Vantassell, Dr. G. Lew, and Dr. J. Wang, for their encouragement and friendship throughout the years; and Dr. P. Gerhardt and Ms. R. Solo for their advice and assistance in improving my spoken English. I also thank Dr. K. Friderici and Dr. D. Hacker for their assistance in developing my technical skills; Dr. J. Edara for initiating the projects described in the Chapter 4; Dr. J. Wirth and S. Kavanji, for their kind and careful help in reading the manuscripts; Shu—Mei Chen and Dr. Shang—Rou Hsieh, for their expert assistance in preparing the manuscript for typesetting; all my ex- and current lab- mates, who have made the atmosphere in the lab very pleasant and comfortable; and all my friends who have made the stay more colorful and fruitful. Finally, I would like to thank Dr. Chai—Liang Haung and Li—Wen Liao, from whom Ihave learned the Buddha’s teaching. My great gratitude goes to Monk Master Sheng-Yen and Master Ming-Hui, for their spiritual support. Table of Contents List of Tables ix List of Figures x Literature Survey 1 1.1 Infection Cycle of Polyomavirus ..................... 1 1.1.1 Adsorption, penetration and dempsidation ........... 1 1.1.2 Transcription, replication and tren'islation ............ 4 1.2 Neoplastic Transformation by Polyomavirus .............. 10 1.2.1 Definition of transformed cells .................. 10 1.2.2 Abortive and stable transformation ............... 11 1.2.3 Initiation and maintenance of transformation .......... 12 1.2.4 The role of large T—antigen in polyoma integration /transfor- mation ............................... 13 1.2.5 The role of middle T—antigen in the maintenance of transfor- mation phenotype ......................... 14 1.3 Cell Cycle Regulation of Gene Expression ............... 15 1.3.1 Cell cycle ............................. 15 1.3.2 Effect of cell cycle on polyomavirus infection .......... 21 vi 1.4 Recombination .............................. ‘23 1.4.1 Mechanisms of recombination .................. 24 1.4.2 Enzymology for recombination .................. 26 1.4.3 The possible role of large T—antigen in homologous recombination 30 Bibliography ................................ 34 2 Neoplastic Transformation by Polyomavirus during the Cell Cycle 52 2.1 Introduction ................................ 53 2.2 Materials and Methods .......................... 54 2.3 Results ................................... 58 2.4 Discussion ................................. 79 Bibliography ................................ 82 3 The Role of Polyomavirus Large T-antigen in Interviral Recombina- tion 84 3.1 Introduction ................................ 85 3.2 Materials and Methods .......................... 87 3.3 Results ................................... 91 3.4 Discussion ................................. 105 Bibliography ................................ 1 16 4 High Frequency of Homologous Recombination between Integrated Endogenous Polyomavirus Sequences and Exogenous Viral Genomes Introduced by Infection 118 vii 4.1 Introduction ................................ 119 4.2 Materials and Methods .......................... 120 4.3 Results ................................... 123 4.4 Discussion ................................. 137 Bibliography ................................ 145 A Gradient of Recombination on the Polyomavirus Genome 148 5.1 Introduction ................................ 149 5.2 Materials and Methods .......................... 150 5.3 Results ................................... 154 5.4 Discussion ................................. 161 Bibliography ................................ 169 viii 3.1 3.2 3.3 4.1 4.2 5.1 5.2 List of Tables Phenotypes of tsa mutant and double n‘lutants, Ma and 3a ....... Results of transformation and recombination from 3 imlependent ex- periments .................................. Comparison of transformants derived in the cross between Ma and 3a at either 33°C or 39°C .......................... Transformation of FRIJT and FR3T3 by wild type A2 and transforma- tion defective mutants 1387T and (11 23. ................ Transformation frequency of FRUI‘ cells infected by wild type A2 and Recombination occurred between Ma and 3a in the regions of Aval—- Aval, the AvaI—Bgll interval and the BglleamHl interval. ..... Recombination frequency between the endogenous polyoma sequences and the exogenous transformation—defective polyomavirus. ...... ix 9‘2 96 160 2.1 2.2 2.3 2.4 2.5 2.6 2.7 3.1 3.2 3.3 3.4 3.5 3.6 List of Figures The phases of the cell cycle and determination of transli'u‘mation fre- quencies during the cell cycle. ...................... Analysis of virus uptake in intact cells as well as in nuclei. ...... Analysis of viral transcripts. ....................... Detection of anti—late message. ..................... Western blot analysis of large T-antigeu ................. Analysis of viral DNA replication ..................... Kinetics of viral gene expression and viral DNA replication in FR3T3 cells infected 3 hours post release from (10 ................ Restriction endonuclease map of polyomavirus .............. Lack of DNA synthesis in the double mutants, Ma and 3a, at nonper- missive temperature. ........................... Test for interviral recombination. .................... Integration pattern of polyoma genome. in the transformed cell lines kept at 39°C . ............................... 61 67 78 90 95 101 Confirmation of the identity of the 1.7 and 1.3 Kb recombinant fragments. 104 Lack of viral DNA synthesis in transformed cells containing the recom- binant viral genomes at 39°C. ...................... 107 3.7 4.1 4.2 4.3 4.4 4.5 5.1 5.2 5.3 5.4 Analysis of the occurrence of recombination in the transformed cells derived in the cross between Ma and 3a. at 33°C ............. The map of restriction endonuclease sites in plasmid pMSG/LT and polyomavirus d1 23 and recombinant wild type virus, and the expected sizes of resulting fragments. ....................... The presence of wild type sequences in the twelve (.1123 recoml.)inant transformants ................................ Restriction endonuclease analysis of recoml'nnation events in two (11 23 recombinant trasnformants. ....................... Analyses of integration patterns of the polyoma sequences in both 1387TLT and d123LT cells ......................... Possible recombination events occurring in the cross between the en- dogenous large T—antigen cDNA and the exogenous polyoma genome. Restriction endonuclease map of polyonmvirus .............. Analysis of the occurrence of recombination in the regimrs of Aval AvaI, AvaI—Bgll, and BglI—BamHI. ................... Recombination frequency derived in the crosses between the endoge- nous polyoma sequences and the exogenous viral genomes. ...... A gradient of recombination frequency was detected in the regions between nucleotide 1387 and 4634 (the Hamill site).. . . . . . . . . . xi 109 132 139 144 153 159 165 Chapter 1 Literature Survey 1.1 Infection Cycle of Polyomavirus 1.1.1 Adsorption, penetration and decapsidation Polyomavirus is a double stranded DNA tumor virus discovered in 1950. The early events of polyomavirus infection, from the time of viral attachment to the cellular receptor site to the time of viral DNA replication, can be broadly divided into two stages. The first stage involves those events related to the interaction of the viral par- ticles with the host cell, i.e., attachment, penetration, and uncoating [33, 83, 143]. In the second stage, events related to the expression of the viral genetic information, i.e., transcription, translation, replication, and possibly derepression of certain host reg- ulatory systems resulting in subsequent modification of cellular or viral constituents occur[222l The course of polyomavirus infection can proceed in two distinct ways. One type of infection is productive in nature, which is described by the replication of virus in the cell nucleus. The virus then forms progeny virus particles, and subsequently kills the host cell [74, 167]. The other type of infection is nonproductive, in which the host 1 2 cells survives and acquires new properties of malignant cell [139, 168]. Whether a cell undergoes a productive or nonproductive infection largely depends on the species of cells being infected. It is well known that the cellular receptors are important determinants of virus tropism and pathogenesis. However, the receptors of few viruses have been identi- fied [229]. For examples, the receptor of poliovirus has been cloned and shown to be a member of the immunoglobulin superfamily [146], while the receptor of rhinovirus has been shown to be ICAM—l [92, 188]. ICAM——1 is a member of the integrin family for the leukocyte [72]. In the case of SV40, it has been shown that the class 1 major histocompatibility proteins act as cell surface receptors [9]. The importance of sialic acid in the adsorption of polyomavirus to cells was first demonstrated by the abolition of viral mediated hemagglutination upon treat- ment of erythrocytes with neuraminidase [73]. Treatment of 3T6 cells with l/ibrio cholerae neuraminidase to remove cell surface sialic acid prevents infection by poly- omavirus. Susceptibility of 3T6 cells to infection can be fully restored by treating the cells with ,B—galactosidea2,3—sialytransferase and CMP—NeuAc which forms the sequence NeuAca2,3Gal,81,3Ga1NAc common to oligosacharides of cell surface gly- coproteins and glycolipids. These results suggest that the oligosarcharide sequence NeuAca2, 3Galfll, 3GalNAc serves as a specific cell surface receptor involved in both polyomavirus—mediated hemagglutination and polyomavirus infection of host cells [84]. The receptor of polyomavirus could be a complex formed by more than one cellu- lar protein. The nonionic detergent octyl—D—glucopyranoside (OG) has been used to 3 stabilize peripheral cell surface proteins and lipids from a variety of isolated cell mem— branes as well as intact cells [129]. The extraction of mouse kidney cells in the pres- ence of 0G resulted in the isolation of polyomavirus receptor moieties with molecular weights of 95 kilodalton (Kd), 5O Kd, and 25 to 30 Kd [141]. The moieties were sug- gested to be subunits of a larger receptor complex, as seen in the adenovirus—receptor interaction which appears to involve more than one plasma membrane protein [99]. After binding to its receptors, the virus particles penetrate into the cells. Penetra- tion of virus particles across the cell membrane was observed to occur in two forms. In the first form, virus particles containing the viral genome can enter the cytoplasm in monopinocytotic vesicles. Thus, the virus particle is first tightly surroumled by the cell membrane, engulfed by the cell membrane which then pinches off and forms a monopinocytotic vesicle which specifically migrates to the nuclear membrane. Virus particles are now in the nucleus devoid of a membrane, and are rapidly uncoated. 1n the second form of penetration, virus particles without DNA are observed to enter the cytoplasm in large membrane—enclosed phagocytotic vesicles [137]. The. results from the direct electron microscopy visualization of the virus suggest that the virus uncoats between the nuclear membranes [143], whereas biochemical fraction studies [32] show that the virus reaches the nucleus intact, implying that uncoating occurs within the nucleus. Recently, it has been suggested by Kasamatsu and her colleagues (1991) that SV40 enters its host cell through the nuclear pore and then decapsidates in the nucleus [235]. 4 1.1.2 Transcription, replication and translation In the productive infection of polyomavirus in mouse cells, virus coded early proteins, large T—antigen, middle T—antigen, and small Taantigen , start to be expressed by 10 to 12 hours after the uptake of virus into cells. Synthesis of a variety of cellular enzymes is induced, beginning 12 to 15 hours after infection, to carry out both cel- lular and viral DNA replication. After that, late viral mRNA is then made in large quantities, and viral capsid proteins are synthesized. Infectious viral progeny begins to be observed 20 to 25 hours after infection and assembly of the viral capsids with the virions continues until 60 to 70 hours. Transcription Polyoma virus specific RNA in the cytoplasm of permissive cells, is observed im- mediately after decapsidation of virus particles. Before viral DNA replication, the cytoplasm of infected cells contains a single viral early RNA sedimenting at 19S [222, 225, 226]. The early RNA is then alternatively spliced into 3 messages encoding for the early viral proteins large T—antigen, middle T—antigen and small 'I‘—a.ntigen. Viral cytoplasmic RNA is polyadenylated [166, 224]. After the initiation of viral DNA replication, two relatively abundant species of cytoplasmic viral RNA are present. The more almndant sediments at 16 S. which is the late gene product while the less abundant sediments at 19 S [38. 208, 225]. The late SV40-specific 16 S and 19 S RNA molecules share common nucleotides sequences [225]. The 19 S RNA is the precursor of 16 S RNA, and it can be alternatively spliced into 3 messages encoding for viral capsid proteins Vl’l, VP2 and VP3 [1]. The late viral 5 RNA transcripts are far more abundant than the early viral RNA transcripts in permissive cells. The 5’ ends of the early and late strand transcripts are proximal to the point on the genome where bidirectional viral DNA replication initiates [110, 111, 114, 169]. The sequence between the early and late transcription start sites is the enhancer region and is about 380 base pairs. The enhancer region of polyomavirus can facilitate viral transcription as well as viral DNA synthesis. There is more than one transcription initiation site for polyoma early transcripts. During the late phase of productive infections, there are late—early transcripts syn- thesized from upstream transcription start sites and the RNA is not capped [55]. The 5’ proximal region of the late—early transcripts encodes an additional protein of 23 amino acids [37]. The down stream initiation site is used during the early phase of infection, leading to the synthesis of large T——antigen. The binding of large T——antigen to the origin derepresses the transcription from down stream initiation sites as well as induces the replication of DNA [37, 87, 95]. The onset of DNA replication then activates transcription from upstream sites. The early promoter of polyomavirus is autoregulated by large T—antigm [115, 159, 201] Via the direct interaction of large T—antigen with viral DNA [203, 205]. Among the three large T—antigen binding sites (I, II, and III), the autoregulation of large T—antigen involves the interaction of large T—antigen with binding sites I and II [160]. Transcription of polyoma DNA during the late phase of productive infections in mouse cells gives rise to ”giant” RNA molecules. The giant RNA molecules contain up 6 to 3—4 times the size of the viral genome [2, 25, 131, 163], and no detectable nonviral sequences [1]. A small proportion of these hybrid molecules contain single—stranded branches or deletion loops in characteristic positions, indicating that RNA splicing may occur on these high molecular weight nuclear transcripts [1]. Hyde—DeRuyscher and CarMichael (1990) showed that the nurlti—genome length transcripts can be spliced leader—to—leader, and produce a high level of polyomavirus late RNA containing multiple leaders [105]. Cytoplasmic RNA from polyomavirus— infected cells contains between 1 and 12 tandem leader units at the 5’ ends of all three late mRNA types for VPl, VP2, and VP3. It is possible that before viral DNA replication, transcription is efficient and allows the production of half~genome length primary transcripts. However, these transcripts are inefficiently spliced, and most are degraded in the nucleus. The transcript with multiple leaders is a prerequisite for the efficient accumulation of polyomavirus late mRN A. It has been suggested that the cellular factors required for polyadenylation and termination in polyomavirus—infected mouse cells are present in limited amoun ts. Less than 20 % of the giant RNA molecules are polyadenylated. Efficient polyadenylation and termination of late polyomavirus transcripts are the results of these limiting factors. The termination site of polyoma late transcription is weak so that run through transcription occurs. To test this, the 94 nucleotides of the rabbit fl—globin polyadeny- lation signal was inserted upstream of the late strand polyadenylation signal. Results showed that efficient termination of late transcription by polymerase II can occur and result in a 1.4 to 2.5 fold increase in polyadenylated virus RNA [125]. Replication DNA replication of polyomavirus requires the interaction of large T—antigen with sequence elements within the virus origin. The origin region contains two primary elements: a core element and auxiliary components. The core component is required for viral DNA replication under all conditions. The auxiliary components, containing promoter and enhancer elements, are involved in transcription as well as viral DNA replication. The core component per se is sufficient to constitute a functional repli- cation origin, but the presence of auxiliary domains increases its activity 5 to 100 fold [66, 100, 128, 132]. The polyomavirus origin core can initiate replication only in the presence of large T—antigen and the DNA polymerase o-primase complex from permissive cells. Large T—antigen can initiate DNA replication by binding to its recognition sequence within the origin and then unwinding the DNA in its vicinity via its helicase activity [63. 70, 187]. The unwinding of double stranded DNA at the origin core may permit DNA polymerase a—primed—DNA synthesis within the origin core exclusively on the template strand at one of several possible initiation sites [98, 78, 197]. Transition sites on each strand of the origin where RNA—primed initiation events stop and continuous DNA synthesis begins occur in the origin core. Subsequent initiation of RNA- primed nascent DNA chains (Okazaki fragments) occurs only on the retrograde arm of the replication forks [98]. An enhancer element is required for polyomavirus origin—dependent replication in developing two—cell mouse embryos, in mouse embryonic and differentiated cells, and 8 in the animal [41, 162, 199, 212, 232]. The enhancer function is normally provided either by the a or 5 element, corresponding to enhancer domains A and B, respec— tively. It has been shown that the non-polyomavirus enhancer can substitute for a and fl. Cis—acting mutations in these sequences can allow polyoma core to initiate replication in mouse cell types that are normally nonpermissive. Normally, wild type polyomavirus can replicate only in differentiated cells. There are polyomavirus mutants that can replicate in mouse embryonic carcinoma. cells. These mutants have mutations in the enhancer B domain of wild type polyomavirus DNA resulting in sequence rearranged merits [5]. For example, fl’yF9 can replicate in undifferentiated F9 cells. nyF9 has three exogenous sequences inserted into the B domain [8]. The sequences are homologous to each other, and the consensus se- quence of the inserts, GCATTCCATTGTTGTCAAAAG is termed box DNA. The box DNA can decrease activity of the SV40 promoter and enhancer in undifferen- tiated F9 cells. Thus, this sequence appears to be a negative regulatory sequence specific to undifferentiated cells. The enhancer element determines cell type specificity for the activation of the polyoma origin—core. It has been suggested that species—specific permissive factors do not interact with the auxiliary domains but rather with either the origin-core or large T—antigen or both to effect DNA replication [21]. The permissivity is most likely caused by large T—antigen induced modification of cellular proteins required to replicate the polyomavirus origin. A possible target for the large T—antigen induced modifications is DNA polymerase a—DNA primase [200]. Enhancer and promoter elements can function in either orientation, but they must 9 be in close proximity to the AT-rich side of the polyoma originvcore [46, 128, 106]. The AT-rich region nearby which is not protected by large T—antigen in footprinting studies, is nevertheless essential for replication [190]. A role for the AT-rich sequences is to open the duplex at the replication origin. Thus, the polyomavirus origin—core components are affected by transcriptional elements [68], since RNA transcript can assist the melting of the origin. The transcriptional activation of the origin has also been observed in prokaryotic systems [12]. This mechanism for altering the topological state of the origin probably accounts for the transcriptional activation first observed in the initiation of phage lambda [86]. In in vitro systems, the main components from the host cell necessary to repli— cate the double stranded circular genome of polyon'iavirus are polymerast—‘o~DNA primase, polymerase 6, proliferating cell nuclear antigen (PCNA), single-strand bind- ing protein (533) which is a helix-destabilizing protein and an important polymerase accessory protein (RF—C) [210]. DNA polymerase 6 synthesizes DNA on the lead- ing (continuous) strand, whereas DNA polymerase 0 makes the DNA on the. lagging (discontinuous) strand [210]. ATP is required for both binding of large T--antigen to the DNA and for separating the double strands by the helicase activity of large T—antigen. ATP is hydrolyzed in a DNA—dependent manner for the latter case, and the large T—antigen molecules split in half and move outward in both directions from its initial site on the DNA [3]. 10 1.2 Neoplastic Transformation by Polyomavirus In nonpermissive cells, such as rat and hamster cells, infection of polyomavirus is nonproductive and neoplastic transformation of the host cells is the major event. 1.2.1 Definition of transformed cells Polyomavirus was first observed to transform cultured cells by l)awe and Law in 1959 [62]. Ordinarily cells grow to a limited extent in culture then enter crisis and die. A proportion of polyoma—infected nonpermissive cells are found to grow at an increased rate and acquire a transformed phenotype [214]. The differences between transformed and normal cells involve changes in the reg- ulation of cell growth as well as in structural morphology [19]. The growth of normal cells is restricted by cell density, availability of growth factors, and the need for anchor- age. However, transformed cells can escape the above three limits to various extents. Transformed cells can be selected by these properties: loss of the. contact inhibition which gives rise to foci of cells in the monolayer, and loss of ancliorage—--de[wndence gives rise to the growth in soft agar [20, 138]. An important feature that distinguishes normal cells from transformed cells is the ability of the cells to regulate the entry into and out of the cell cycle in response to factors such as population density and serum [15]. In the growth of normal cells, cells will cease their growth due to cell—cell contact inhibition when they reach 100 % confluency. The density regulation of growth can thus limit the maximal number of cells growing in a particular size of culture plate. Transformed cells, on the other 11 hand, fail to show cell—cell contact inhibition when they reach 100 % confluency. Multilayered growth of transformed cells can be observed even when the cells are still subconfluent. There are several mitogenic factors present in serum to support cell growth, such as platelet derived growth factor (PDGF), epidermal growth factor (EGF), insulin, and insulin—like growth factor. PDGF can induce fibroblasts into the competent state, which can be progress to S phase in the presence of plasma [189]. Serum also contains components that promote attachment and spreading of cells on a solid substratum and that are needed for anchoragewdependent growth. Other components such as nonpeptide hormones, vitamins, Ca2+ and H+ ions can affect the density of cell growth. Addition of serum or growth factors to normal resting cell cultures can induce cells to a competent state and further to DNA synthesis and mitosis. However, transformed cells lose serum—dependent growth and grow to high density with low serum. 1.2.2 Abortive and stable transformation When nonpermissive cells (hamster or rat) are exposed to high dosage of poly- omavirus, a large proportion of the cells develop a transformed pl'ienotype, divide several times in the agar and then stop dividing. This phenomenon is referred to as abortive transformation [191, 192]. Only a small proportion of infected cells retain the transformed characters indefinitely and this occurs only after stable integration of the viral genome into host DNA. This is referred as stable transformation. Lines of stably transformed hamster cells occur at a maximum rate of about 5 (27¢... after 12 polyoma infection. In Fischer rat cells, the transformation rate is less than 1 ‘70. Stoker showed that infection of baby hamster kidney (BHK21) cells with tsa mu- tant at the non—permissive temperature could initiate a temporary change in BHK21 cells, but stable transformation did not occur [191, 192]. The defect of large T— antigen in the tsa mutant resulted in the failure of stable integration of the viral genome, so that stable transformation could not occur. These results with the tsa. mutants sug- gested that the defect of large T—antigen is not in the expression of the transformed phenotype but rather in the events leading to stable perpetuation of the viral genome. 1.2.3 Initiation and maintenance of transformation There are at least two steps in process of transforming a normal cell to a transformed cell. The first step is initiation and the second step is maintenance. Large 'l‘---—ant.igen is required for the initiation of transformation in nonpern'iissive cells. Fried (1965) showed in hamster cells infected by a tsa mutant of polyomavirus that once the effect of one temperature on transformation is produced, the reversion of transforma- tion cannot be obtained by shifting the infected cells to the other temperature [85]. For example, the cells transformed at 31.50C' retained their transformed state upon cultivation at 385°C. Thus large T—antigen is important in the initiation of the transformed state, but not for the maintenance of transformation state. Middle T—antigen is required for the maintenance of the transformed state of the cells [81]. Both F111 and normal rat kidney cells were coinfected with hr—--t mutants and ts-a mutants of polyoma. virus. The majority of clones selected from 1’1 11 cells expressed both middle T—antigen and small T—antigen, whereas the expression of the 13 large T—antigen was only detected in some of the clones. This result suggested that middle T—antigen, rather than large T—antigen, is required for the maintenance of the transformed state of the cells. 1.2.4 The role of large T—antigen in polyoma integration / transformation In cells transformed by polyomavirus in tissue culture system, one finds viral genomes stably integrate into host chromosomes [170]. Thus the integration of the polyoma genome is correlated to transformation. The integrated DNA exists as head- to-tail tandem repeats of unit—length polyoma DNA attached to cellular DNA [24]. Large T—antigen is important for the integration pattern of lnrad—to—tail tandem repeats of viral DNA. Della Valle et al. (1981) showed that in the absence of a functional large T—antigen, transformants contained multiple nontandem insertions of viral DNA segments shorter than the infecting polyoma molecule [65]. The efficiency of transformation was about 20 fold lower in the absence of large T—antigen. The integration sites of the polyomavirus DNA in different transformed cell lines are different [107, 124]. Sequence rearrangements or alterations occur inn’nediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA [97]. In addition to integrated viral genomes, there are 20 to 50 copies of free viral DNA per cell [156, 238]. Most of these molecules exist in the supercoiled form in the nuclei of the transformed cells. Basilico and his colleagues suggested that the origin of these 14 free viral DNA was due to the high rate of excision and amplification of integrated viral genomes [50]. The presence of homologous regions in the integrated viral sequences r is required for viral amplification and excision [50]. The excision events occur via intramolecular homologous recombination which is promoted directly or indirectly by the large T—antigen [16, 18, 29, 30, 31, 50, 148, 169]. 1.2.5 The role of middle T—antigen in the maintenance of transformation phenotype Middle T-antigen is the transforming protein of polyomavirus, and is located on the cytoplasmic membrane. Expression of middle T~antigen leads to profound changes in the cells and causes cell transformation. For example, the transforn'ied state of cells can be induced by the expression of middle T—antigen cDNA in normal rat fibroblasts [209]. Different levels of transformation can be observed by using the dexamethasone—inducible promoter of the mouse mammary tumor virus LT R- At low levels of expression, rat fibroblast cells showed loss of actin cables and decreased adhesion. At a higher level of induction, the cells are able to form foci; and at the maximal expression, the cells are able to grow in the soft agar [157]. Middle T-antigen is one of the important elements in the cascade of signal trans- duction events required to induce cell transformation. Middle T-antigen exerts its function by binding to the Src tyrosine protein kinase [54], and pllosphatidylinositol 3—kinase [112, 230]. The formation of complexes between middle T—antigen and Src can lead to a 10 to 50-fold increase of Src kinase activity [28, 53]. Before middle 15 T—antigen forms complexes with Src, phosphorylation of middle T—antigen by ser- ine kinases at two to three sites is required [172]. Then the middle T—antigen—Src complex binds to a phosphatidylinositol kinase [112, 230]. Middle T-antigen possesses associated protein kinase activity of pp60c‘5'“: and p62c‘y“, which are in the src—family of protein kinases [119]. The amino—terminal portion of middle T—antigen between residues 78 and 191 have been shown to asso- ciate with pp600‘3”. Recently, another member of the src protein kinase, p60fy" was shown to associate with in murine polyomavirus-transformed rat cells [104]. Cook and Hassell (1990) showed that the amino terminus of polyomavirus middle T~antigen is required for transformation [51]. The first four amino acids of constitute part of a do- main required for activation of the pp60c'3” tyrosyl kinase activity and for consequent cellular transformation. Middle T-antigen also activates cellular gene exl')re.ssion by enhancing transcrip- tion via the cellular transcription factor PEAl [221], which is similar to the mouse Jun protein (API) [7, 127, 155]. PEAl binds to polyoma enhancer A [155], and over- laps in its target specifically with TPA—inducible genes. Middle T--~-antigen, activated ras, v-src, and rafall stimulate PEAl—mediated transcription [221]. 1.3 Cell Cycle Regulation of Gene EXpression 1.3.1 Cell cycle A functional cell cycle is divided into G1, S, G2, and M phases. The G1 phase of the . . . , . ._ ke CC“ CYCIe is a period during which cells prepare for S phase. The S phase is 111‘“ 16 by beginning of DNA, histone, and some enzyme synthesis. G1 was originally defined as a time interval, a gap between the readily observed events of mitosis and DNA synthesis. The G1 events can begin during the previous cycle, at the same time as other events such as DNA synthesis or preparation for mitosis. Thus the observed Gl interval between M and S phase depends on how much progress has been made in the previous cycle [76]. Cells in viva, for example hepatocytes and neurons, remain healthy for very long periods in the nonproliferating or quiescent state called G0. Cells in culture can also be arrested in CO by contact inhibition or serum starvation. Gt) arrested cells have an unduplicated DNA content, smaller size because their protein and RNA molecules are degraded and are not rapidly synthesized. Synthesis of macronmlecules are about one-third as rapid in G0 as in proliferating cells [1.5, 58, 67]. G1 has been divided into subphases [164, 189. 202], depending on the effects of the limiting growth factors, nutrients, or inhibitors, as measured by time to reach S phase after the block in G0 is removed. These subphases are referred to as competence, entry, progression and assembly [153]. Treatment of cells blocked in G0 with PDGF or plasma (which lacks PDGF) alone cannot stimulate normal BALB/c 3T3 cells to enter S. If first treated with PDGF and then with plasma, the cells can progress to S phase, but not vice versa. The. PDGF‘ - a ‘ 6‘) . treated cells are then competent to progress to S phase [15, 58, 67, 164, 189s 20"] me For competent BALB/c 3T3 cells, it takes about 12 hours to reach S phase, the 52‘ amount of time it takes cells in G0 phase. 17 The addition of plasma, which provides factors such as EGI’ and insulin, for competent cells, can lead these cells to progress to S phase [130, 236]. Competent cells incubated with plasma in a medium without essential amino acids reach a. point called V. It requires 6 hours for cells at V to reach S phase after the essential amino acids are provided. This length of time is very similar to the duration of GI phase for the cycling cells. After 3T3 cells have reached V point, the only growth factor required to progress to S phase is insulin—like growth factor—1 (lGlf—l) [130, 236]. Rapid synthesis is required during this middle part of G1, and enzymes required for DNA synthesis are made during progression. In the assembly subphase, movement of enzymes into the nucleus and organization of these enzymes into a complex to catalyze DNA synthesis may require considerable time. Induction of cellular genes after external stimulation Competent cells produce immediate—early mRN A including c-fos, which appears in a few minutes after treatment with mitogenic factors and c-myc, which appears several hours later. Some of these mRNA turn over rapidly; their levels peak for only a short time. The presence of these mRNA are observed even when synthesis of new protein is inhibited, suggesting that transcriptions of early mRNA do not require production of new proteins. Progression through the cell cycle is regulated primarily during the G1 phase [154]. Synthesis of new mRN A is required for progress through G1. Inhibitors and mutations affecting G1 are effective in blocking proliferation. For examples, a-—amanitin, which 18 is an inhibitor of RNA polymerase 11, blocks cells in G1 [216] , and a cell line that is arrested in G1 at the non-permissive temperature is defective in RNA polynn-rase 11 Protein synthesis in early G1, possibly directed by newly transcribed messages, is also required for cell growth [154, 177]. It has been estimated in mouse fibroblast cells by subtractive or differential hybridization that 3 ‘70 of the mRNA species in exponentially growing cells are absent in non—growing cells [231]. Most molecules are made continuously to serve house—keeping functions. A number of normal cellular genes have been postulated to have roles in the control of cell proliferation. Included among these are those characterized as proto— oncogenes, those whose expression is cell cycle dependent, and those whose protein products are components of the cytoskeletal framework. Cellular proto—oncogenes encode proteins with three major sites of action: the cell surface membrane, cyto- plasm, and nucleus [26, 223]. The nuclear acting group of proto—oncogenes includes c-fos, c-myc, c-myb, c—erbA, p53, and c-jun etc. [215]. Because of their nuclear loca- tion, it has been suggested that products of these genes function as transcriptional regulators [117]. The c-erbA gene codes for a thyroid hormone receptor [171, 227]. The c-Myc protein acts in conjunction with MAX protein [27], and functions as a transcriptional activator. c-jun codes the transcription factor A111, and binds to a DNA sequence in the upstream promoter regions of many genes. c-fos codes for a trans—activator which does not operate via direct binding to DNA but by interacting with c-jun/AP-l [47]. The wild type p53 is a tumor suppressor gene, and regulates the cell growth. If both alleles of p53 are mutated, p53 becomes an oncogene resulting in loss of the control of cell growth [75, 80, 101]. The c-ras gene can be activated 19 in mid—G1. The Ras protein is a guanosine triphosphate (GTP)—binding G protein, and it is membrane localized. The early gene responses can also be induced by viral infection. When quiescent cells are infected by polyomavirus, cellular DNA synthesis and cell division are induced to allow viral replication. Zullo et al. showed that infection of quiescent BALB/c 3T3 cells by polyomavirus resulted in the biphasic accumulation of RNA from the early response genes c-fos, c-myc, and JE [239]. They found that the first peak was due to the interaction of VPl, the major virus capsid protein, with its receptors. The second peak was due to the expression of polyomavirus early messages. Studies with virus mutants indicated that large T—antigen alone was not sufficient to induce the second peak. Middle T—antigen was dispensable, and small T—antigen either alone or together with large T—antigen, may be the regulator for the second wave of induction [90]. Furthermore, Glenn and Eckhart (1990) showed that infection of both BALB/c 3T3 and NIH 3T3 cells with polyomavirus lead to the expression of earva response genes,c- fos, c-myc, and c-jun, biphasically. Large T—antigen was not suflicient, and middle T—antigen was dispensable for the induction of the early—response genes [90]. In mid-G1, several enzymes activities increase. These enzymes includes transin (which is a protease), ornithine decarboxylase (which catalyzes polyamine synthesis), hydroxymethylglutaryl coenzyme A reductase for isoprenoid synthesis, and a 68 Kd nuclear protein that is a RNA helicase. Both p53 and p68 proteins are increased [61, 102,113] In late G1 phase, several gene products involved indirectly in DNA synthesis are increased. These include thymidine kinase [52, 181], dihydrofolate reductase [791, 20 thymidylate synthetase [11], DNA polymerase a, and proliferating cell nuclear antigen (PCNA). Induction of DNA polymerase 01 activity during the S phase of the cell cycle [14, 48] suggests that expression of this gene is closely coupled to the onset of DNA replication. At the end of the G1 phase, the migration of the enzymes, which are produced on ribosomes in the cytoplasm, to the nucleus to catalyze DNA syntl'iesis is observed. Enzymes in the nucleus then form a multienzyrne complex called replitase [153]. The replitase includes enzymes for DNA replication, such as DNA polymerase (1 [‘12, 217], and enzymes that catalyze precursor synthesis, such as ribonucleotide reductase, dihydrofolate reductase, and thymidine kinase (TN) [69, 94, 96. 120, 1119, 158, 211, 213]. After G1 phase, DNA synthesis and histone synthesis occur. and the cell cycle is completed by mitosis and cytokinesis. The results above were obtained from proliferative cells stimulated from serum- deprived, metabolically blocked, or spatially restricted quiescent cultures. These methods perturb normal cell growth by artificially bringing cells into a nomycling or G0 phase from which, up on stimulation, cells must re—enter the cell cycle. The G0 phase is usually not present in the exponentially growing cells. For example, Wahl et a1. (1988) have studied the expression of human DNA polymerase a in both quiescent cells stimulated to proliferate as well as in actively growing cells separated into progressive phases by counterflow centrifugal elutriation. Results showed that the regulation of human DNA polymerase a was positively correlated with cellular transformation and activation of proliferation. However, results using expont-sntially growing cells separated into different phases by elutriation showed that DNA poly— 21 merase a was constitutively expressed throughout the cell cycle, with only a moderate elevation prior to the S phase and a slight decrease late in the G2 phase [217]. These results suggested that what is observed in the serum—released cells may not occur in the exponential growing cells. 1.3.2 Effect of cell cycle on polyomavirus infection It has been shown that the process of viral infection is a function of the physiological state of the host cell. The infection of polyomavirus can be initiated during the cell cycle while the integration of viral DNA into the host cell DNA occurs during the S phase. Integration Eremenko and Volpe (1984) showed that the integration of the viral genome into host cell DNA was not observed until the next S phase. These cells were synchronized by the double thymidine block, infected by SV‘IO occurred middle G2 phase in the mouse 3T3 cells. In the other experiment, cells were synchronized by mitotic shake off, where cells were infected in early G1 phase, the integration of the viral DNA was seen in late S phase of the same cell cycle. These results suggested that the integration of SV40 viral DNA occurred preferentially during S phase, and that the G1 phase was necessary for the viral integration [77]. Transformation The neoplastic transformation induced by polyon‘iavirus in nonpermissive cells is very low [193]. It has been shown that the physiological state of the cells can affect its competence for neoplastic transformation. Basilico and 1\«'larin (1966) showed that the susceptibility of baby hamster kidney cells (BHK21) to transformation by polyomavirus varied in different stages of the mitotic cycle [17]. They found that the transformation rate was about 2 fold higher in cells infected in G2 cells than in G1. The 2—fold difference observed was suggested to be directly related to the 2—fold increase of DNA content in G2 cells. The duplicated cellular DNA thus offers twice as many targets for viral integration which can then lead to 2-—fold higher of transformation frequency. In mouse 3Y1 cells infected by SV40, Tamura (1983) found that the highest trans- formation frequency was observed when the growth of resting 3Y1 cells was stimulated by sparse replating after virus inoculation, and the lowest frequency was in resting stage [198]. A 8 to 30—fold increase in transformation frequency was observed in rest- ing cells infected with SV40 compared to infection of growing cells. In his eXpm‘iment, similar results were also observed after infection of 3Y1 cells with polyomavirus. The reduction of transformation in proliferating cells infected with SV40 was also observed in BALB/c 3T3 mouse cells [142]. Viral DNA replication The relation between replication of SV40 DNA and the various periods of the host— cell cycle was investigated in synchronized CV1 cells by Pages et al. (1973). CV1 cells 23 synchronized by a double thymidine block were infected with SV40 at the beginning of S, middle of S or in G2 phase. Infection with SV40 was also [1)erformed on cells obtained in early G1 through mitotic shake off. Their results showed that as long as cells were infected in G1 phase (either early, middle or late G1), the viral progeny DNA molecules were detected during the S phase of same cell cycle. However, if infection took place once the cells had entered the S phase, no progeny DNA molecules were detected until the S phase of the next cell cycle [152]. The explanation for these results were that the infected cells has to pass through a critical stage situated near the end of G1 or the very beginning of S in order to gain competence for the eventual initiation of viral DNA synthesis. Adsorption The influence of the various phases of the cell cycle on adsorption of SV40 was stud- ied by measuring the binding of tritiated thymidine—labeled virus to cells in various phases. The results showed that a 2 to 3—fold increase of viral adsorption was obtained in GI—infected cells compared to cells infected at G2 or M phase [152]. 1 .4 Recombination Mammalian cells readily integrate foreign DNA into their chromosomes. In the inte- gration of the polyoma viral genome, there is little or no nucleotide sequence homology at the joint between viral DNA and chromosomal DNA. The absence of homology indicates that polyoma integration is nonhomologous recombination. 24 Homologous recombination are present in the polyomavirus such as in gene ampli- fications [50, 16, 195], excision [29, 30, 31], and interviral recon‘ibination [93]. Hacker and Fluck (1989) showed that high levels of interviral recombination were obtained among the integrated viral genomes in transformed Fischer rat (19111) cells [93]. When duplicated regions of the polyoma genomes are present, homologous se- quences can recombine and produce complete viral molecules that can be excised and circularized as free DNA [31, 50]. The newly replicated n'iolecules can recombine with homologous sequences of the parental strands. This type of recombination can lead to an increase in the copy number of the integrated viral genomes [50, 195]. 1.4.1 Mechanisms of recombination Nonhomologous recombination The nonhomologous recombination is a two—step process: Free ends of double strand DNA are generated in the first step and joined in the second. DNA ends arise from er- rors of DNA metabolism, and are subsequently eliminated by sticking them together. The end joining is the general defense mechanism in mammalian cells for dealing with broken chromosomes. Homologous recombination Two working models have been proposed to explain the results of llOD’IOIOgOLlS re- combination of DNA introduced into mammalian cells. One is the double-strand break repair (DSBR) model [10, 34, 35, 82, 108, 121, .185, 186], and the other is the 25 single—strand annealing (SSA) model [6, 36, 43, 133, 134, 178, 218]. Both models are based on the observation that double—strand breaks in DNA at certain locations can stimulate recombination. In the DSBR model [196], recombination is initiated by a double---straml break in one of the DNA molecules (the recipient) that is enlarged to a double strand gap. That gap is then repaired by using the second molecule (the donor) that shares homology with the region flanking the double-strand break in the first molecule. In this model, only the recipient molecule needs to contain a doi11,)le-strand cut in the homologous region. In contrast, both SUDSIII'EIIPS must be linearized to generate recombinants by the SSA model. According to SSA model, the DNA ends act as entry sites for a strand-specific exonuclease. Degradation of DNA by such a nuclease generates complementary single-stranded DNA for pairing [133, 1331]. Alternatively, the single—stranded DNA can be generated by helicase unwinding of the linearized DNA duplex without extensive exonuclease degradation. The essential featin‘e of the SSA model is that double—strand breaks made close to the region of homology shared by each parental DNA molecule are required to initiate recombination. Although both the DSBR and SSA models emphasize the importance of generating single-strand DNA for pairing steps in the reactions, the outcomes of recombination predicted by the two models are very different. In the SSA mmlel [43, 178. 2181» recombination is nonconservative and produces crossover products exclusively. The flanking sequences of the recombinant DNA produced by SSA model are usually . - _ . N A rearranged. However, in the DSBR model, only about 50 % of (hp recombinam I) . . . . . . . . . .,. “Lion 18 assocrated With crossover of the flanking markers if there IS no bias in the usol step of the reaction. 1.4.2 Enzymology for recombination RecA and auxiliary proteins In prokaryotic systems, two types of homologous recombination are observed. One type of recombination utilizes the RecA protein, does not require extensive DNA synthesis, and is found in phage, bacterial, and fungal systems. The. other type is RecA—independent and replicatiorrdependerit,such as in phage lambda. 'l‘T and T4 [56]. The ReCA protein is an ATP—dependent, single-strand DNA binding protein [150]. It is also, a protease and is required in the regulation of its own synthesis [233]. The protease activity of RecA can cleave the lexA repressor [.161], which regularly binds to the RecA promoter, and allows the expression of RecA. The principal function of the RecA protein in recombination is to catalyze strand invasion [71]. RecA is able to promote the annealing of a single st rand to a recipient molecule of double helix and form a D—loop by hydrolyzing ATP in the process [182, 144]. After binding to the single—strand DNA, RecA is able to partially denature the recipient duplex DNA molecule to accept the invading single strand DNA [59]. Then a reciprocal exchange of DNA strands between the two double helixes can then occur. That is, as a strand from a donor double helix invades a recipient double helix, the displaced strand in the recipient molecule can invade the first double helix [228]. This is the same as the initial step in recombination in the Holiday model [103]. 27 Although the RecA protein can catalyze a variety of DNA strarid—exchange reac- tions, three other E. coli proteins play roles in conjunction with the RecA protein [71]. These proteins are E. coli single-strand DNA binding protein (SSB), topoisomerases, and the RecBC enzymes. The E. 0012? SSB protein has an even greater affinity for single-strand DNA than does the RecA protein [184]. When the SSH protein is added to the various reactions with RecA, it dramatically improves the ellicient use of RecA and ATP, so that less RecA protein and less ATP are required to generate an equiv- alent level of strand exchange [145, 183]. When a. topcfisomerase is added to a, basic reaction mixture containing RecA, ATP, single—strand circles, and supercoiled duplex rings, the single-stranded circles become more stably interwrapped with the duplex ring at a region of homology [60]. The RecBC enzyme is a multisubunit protein product of the RecB and RecC pro- teins in E. coli [207]. The RecBC enzyme was first identified as a. potent exonuclease as well as an endonuclease driven by the hydrolysis of ATP [13. 151, 91]. llecBC can initiate genetic recombination by traveling internally down a DNA double helix and creating a region of local denaturation. In the denatured region, several hundred base pairs of positive and negative strand DNA are held apart so that their hydrogen bonding surfaces are exposed. In this reaction the RecBC works with the SSB protein, whose role is to stabilize the separated strands. This is the DNA molecule that will initiate recombination by serving as the donor. ()ne of its exposed singlevst randed regions will attack a second DNA molecules [71]. Topoisomerase The DNA topoisomerases are involved in nearly all biological t rausaction of DNA. These include the relaxation of negatively and positively supercoiled domains which are generated in a DNA template during replication and transcription [136, 231, 89]. The participation of bacterial gyrase or eukaryotic DNA topoisomt-irase in nonhomol- ogous recombination has been pr0posed based on in vitro and in vivo studies. For example, sequencing the integration sites of SV40 indicated that eukaryotic DNA topoisomerase I might be doing the strand transfer during viral integration [10, 39]. The eukaryotic topoisomerase l—mediated nonhomologous recombination may initiate in regions that contain single—strand gaps which can be converted to cl()t.ible-strand breaks by topoisomerase I [45]. Although both eukaryotic topoisomerase and lambda integrase play roles in the integration of viral or phage DNA into the host chromoson’ie, the nwchanisms are clif- ferent [57, 122]. The major difference between topoisomerase and lambda integrase is that in the t0poisomerase—catalyzed reactions, the same internucleotide bond broken in the first transesterification step is re—formed in the second transesterificatiou step. However, in a lambda—type recombination catalyzed by integrase. strand switching occurs between the two steps: the 3’ side of a transiently strand is joined to the 5’ side of another transiently broken strand. DNA topoisomerase might be required for the formation of recombination interme- diates in which two DNA strands are wound plectonemically around each other [44, 118, 60] as well as for supercoiling of intracellular DNA. DNA topoisomerases can 29 also act as suppressors of recombination. In yeast, mutants with a. null mutation in the topl gene or a temperature sensitive mutation in the top? gene, the frequency of mitotic recombination in the rDNA gene cluster at a semipermissive temperature is 50—200 times higher than it is in the wild type TO P+ controls [419]. Thus the topoiso- merases I and II appears to suppress mitotic recombination within the rDNA cluster. Mutations in yeast gene t0p3 increase recon‘ibination between the long terminal re- peats of the Drosophila retrotransposon Ty [219]. TOP3 is a protein homologous to bacterial topoisomerase I. The possible mechanism invoked to explain the suppression of recombination by a topoisomerase is that the enzyme relaxes supercoiled regions of intracellular DNA and thus suppress DNA supercoiling— stimulated recombination [320]. For example, for the heavily transcribed rDNA cluster, intramolecular recombination is elevated and excision of extrachromosomal rings occurred when topoisonmrase activity was insufficient to relax the supercoiled domains in a. topoisomerase double mutant. [116]. In conclusion, it is possible that the topoisomerase can function either positively or negatively in recombinational synapses of two complementary DNA strands from two different molecules or different regions of the same molecules. That. is. recombination might normally be minimized by processes that would dissociate two inadvertently paired DNA strands, recombination would form only from structures that have es- caped dissolution—by helicase [204]. 30 1.4.3 The possible role of large T—antigen in homologous recombination Structure and function of large T—antigen Large T—antigen of polyomavirus is a 100 kilodalton protein with 785 amino acids. The homology between large T—antigen of polyoma and SV40 is about 90 ‘X... The structure of SV40 large T—antigen is studied more thoroughly. S\7-10 large T» antigen contains an origin binding region (131—371 amino acids) [66, 109]. a ZnH—finger do- main (302—320 amino acids) [‘22, 23], a. Leucine zipper domain (3415-370 amino acids), a helicase activity domain (131—680 amino acids) [63, 187, 116], an ATP binding re- gion (418—528 amino acids) [88, 206], a DNA polymerase a ~binding domain('.372—5l7 amino acids), a retinoblastoma binding region (105—1 14 amino acids) [64], and a p53 binding domain [123, 135], which is not present in large T—antigen of polyoma. Also, there is homology of SV40 large T—antigen and E. coli RecA protein in the 372 to 648 mino acids of SV40 large T—antigen ‘ and 36 to 352 amino acids of RecA [1.79]. The multifunctions of large T—antigen make it possible for it to be involvetil in the molecular events which are essential for both viral productive infection and for cellular neoplastic transformation. Large T—antigen plays an important role in the initiation of viral DNA replication by exerting its function of binding to the origin region, association with polymerase a—primase, helicase activity, and A’Tl’ase activity. Once the initiation of DNA replication occurs, large T~antigen is required for elongation of replication forks using its helicase and ATPase activities. In viral transcription, large T—antigen can feedback control its own expression by 31 a negative regulation mechanism of blocking RNA polymerase II elongation. Large T—antigen can also act as a positive regulator of late gene expression via modification of a cellular protein rather than binding directly to the late promoter. Regulation of large T—antigen Large T—antigen is regulated in a very complex way such that its many functions in vitro can be carried out by one protein. One mode of regulation at the posttransla- tionallevel is phosphorylation since large T—antigen is known to be a phosphol)rotein. For example, the the phosphorylation of large T-antigen at serine residue is faster than the turnover of the protein itself, suggesting that regulation by phosphoryla- tion is important. Two clusters of phosphorylation sites on large T~antigen have been mapped, one near the N terminus and one near the C terminus. Each cluster contains one phosphothreonine and four phosphoserine residues [173, 1,65]. Dephosphorylation of serine groups on SV40 large T—antigen has been shown to activate its specific DNA binding to site If within the origin of S\t'-10 DNA replication in vitro. It has been shown that the incubatimr of large T—antigen with alkaline phosphatase can remove all serine—bound phosphates [180], increase the binding of large T—antigen to site II at the origin of replication and stimulate its ability to support in vitro DNA replication [147]. Recently, it has been shown that phosphatase 2A (PP‘2A) can potentiate SV40 DNA replication in vitro [126]. Lawson et al. showed that the I’P'ZA can dephos- phorylate large T—antigen and lead to activation of DNA replication. Formation of stable complexes between both polyoma small T——antigen and middle T—antigen with 32 protein PP2A was observed. It is possible that small T—antigen plays a role in DNA replication by activating the ability of PP2A to (l€].)l108pl101‘yla10 large T antigen, thereby activating large T—antigen [175, 174, 237]. Besides phosphorylation, large T—antigen undergoes other posttranslatior1al mod- ifications, such as oligomerization, acetylation, and glycosylation. It. has been shown by Hurwitz (1990) that large T-antigen oligomerizes as two IIPXEHIH‘I'S on the replica- tion origin in the presence of ATP. Results of DNase protection and DNase protection studies showed that large T—antigen was organized into a two lobed structure at the origin . The largest complex was found by scanning transmission electron microscopy to contain 12 monomers of 12 large T~antigen. As a higher level of regulation, large T—antigen is con'ipartmentalized in different structural systems of the nucleus. The interaction of large "ll—antigen with the chro- matin and the nuclear matrix is suggested to be another level of regulation. Deppert and his colleagues (1989) showed that the associations of the tsA large T antigen with both the cellular chromatin and the nuclear matrix were temperature-sensitive, while that of the SV40 wild—type large T—antigen was not [176]. l-‘urtherrncn'e. Mann found that large T—antigen was still associated with the nuclear matrix after cells were shifted to the nonpermissive temperature for 1 hour. His results suggested that the association of SV40 large T—antigen with the nuclear matrix was DNA replication independent and origin—binding independent [110]. 33 The role of large T—antigen in recombination Recently, the role of large T—antigen in intracl’m)mosomal recombination was studied by Bastin and his colleagues. They showed that the recombination between two copies of defective middle T—antigen located side~by~side was promoted from 10"~ to 10'“2 per cell generation when large T—antigen was present. Recombinatitm between the two copies of defective middle T—antigen was promoted ('rven by the g\,' 10 large '1‘. antigen which can not activate polyoma DNA replication. The promotion of recon‘ibination by large T—antigen was not DNA replication-dependent. They suggested that the possible role of large T—antigen in homologous recombination was to melt. and unwind the DNA at the viral replication origin so as to create a fax-"orable substrate for homologous recombination [194] . [11 [21 [31 [41 [71 [81 [91 [101 [111 Bibliography N. H. Acheson. Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome. Proc. Natl. .-‘1cml. Set, 7324754— 4758, 1978. N. H. Acheson, E. Buetti, K. 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Synthesis ol‘ double—strande‘ 550-- ‘c’ g 25» 0' £3 20.. c .3 15-- E g 10-- .2 2 5~- 2 . ’— 0 v ' 1 L 1 1 L n o 3 5 9121518 2124 2'7 30 61 Time of infection (hours post GO) Figure 2.1: The phases of the cell cycle and determination of transformation frequen- cies during the cell cycle. 62 was also observed in cells infected in G1 phase for both infection of cells in suspension, and cells synchronized at low cell density by starvation only without the treatment of trypsin. Similar results were obtained in multiple experiments with small variations in the length of the cell cycle observed (from 24 hours to 30 hours). However, a peak of transformation at 3 hours post infection was always observed and followed a continuous decrease until 24 hours . Thus, in all subsequent experiments, time points chosen were 3, 6, 9 and 24 hours post G0. As follows below many parameters of infection were followed. In Figures 2.3, 2.5 and 2.6 which follow all parameters were assayed in the same experiments. The cell cycle dependence of each 1*)henotype was tested in duplicated samples in at least 3 individual experin‘ients. Viral adsorption: To assay whether different viral uptake at different phases of the cell cycle accounts for the differences in transformation, total DNA was extracted from infected cells 20 hours post infection, prior to any de novo DNA synthesis. As shown in Figure 2.2.A, a peak in viral uptake was observed in cells infected early in the G1 phase, with a 2 to lO—fold ratio between cells infected at 3 hours and those infected at 24 hours post release from GO. We also analyzed nuclear uptake in the same experiment at 20 hours post infection. Total DNA was thus extracted from nuclei, as described in Materials and Method. As seen in Figure 2.2.8, a peak of virus signal in the nuclei was also observed in cells infected in early GI, with a 22--fold ratio between cells infected 3 hours and those infected as 24 hours post release from G0. The difference between adsorption and nuclear uptake suggests that there may be a cell cycle—dependent step between viral adsorption and entry into the nucleus. 63 This is also suggested by the difference between the adsorption differential and the transcription differential (see below). However, this difference relies on an accurate measure of the adsorption differential. This may be problematic for two reasons which would both lead to an underestimation: (1) only the 5.3 Kb signal was used for the quantification measurement ignoring other bands in the 3 hours sample, (2) the signal in the 24 hours sample may contain viral genomes which were associated to the cell membrane but would never enter into the cells. However, comparing the range of differences in adsorption and nuclear uptake transcription/transformation in every experiment suggests that adsorption alone truly does not account for the difference in the other parameters. Viral gene expression: Viral gene expression was monitored in cells infected at different phases. For this purpose, FR3T3 cells were synchronized and infected at various times as described above. Total RNA was extracted 48 hours post infection, blotted and hybridized as described in Materials and Methods, and the results are shown in Figure 2.3. A 33—fold difference in viral RNA was observed in cells infected 3 hours compared to those infected 24 hours post release from G0. .-\ probe containing glyceraldehydephosplate dehydrogenase (Galel) sequence was used to hybridize to the same blot and shows that equal amounts of RNA were loaded in each lane. The size of the transcripts was heterogeneous (Figure 2.3). Some of the RNA represents species of 4—5 times the unit length of polyoma genome. .\lost of the mature viral transcripts in infected rat cells would be expected to be early transcripts. whose sizes are 183 and 198 [8, 9]. The large size of RNA transcripts in the Northern blot 64 Figure 2.2: Analysis of virus uptake in intact cells as well as in nuclei. (A). Cells were released from GO and duplicated plates were infected at 3, (i, 9, and 24 hours post release from G0. Total DNA was extracted 20 hours post infection, digested with EcoRI, electrophoresed on a 0.7 ‘kf agarose gel. .»\ “P—labeled probe containing the entire polyoma DNA was used for hybridization. (B). Cells were infected as described and nuclei were isolated from cells 20 hours post infection. Total DNA was extracted and analyzed as described above. 65 A. Total DNA 'fetin s t 3. Nuclear DNA time of infection (hrs ggstQO) 3 6 9 a .5.3>.... - Figure 2.2: Analysis of virus uptake in intact cells as well as in nuclei. 66 Figure 2.3: Analysis of viral transcripts. 20 pg of total RNA, prepared from cells 48 hours post infection, was ele(,'t.roplioresed, blotted and hybridized with a 32P-labeled probe containing either the <—.>ntir<-—> polyoma DNA or the GapDH DNA, as described in l\fa,teria,ls and methods. 67 time of infection (hrs post GO) if 3 6 9 24 E I." " 2ss> , ’ 18%; . ’ GapDH> GapDH> ....~.-“ Figure 2.3: Analysis of viral transcripts. 68 suggests that the RNA was not degraded. The compact band GapDH 2.4 Kb message in each lane, further rules out the possibility of R NA degradation. The heterogeneity in the size of the polyoma signal was not caused by contamination with DNA, since treatment with DNase did not alter the RNA pattern while an added plasmid DNA control was completely digested (data not shown). Another possible explanation for the high molecular weight RNA is that run through transcription of the polyoma early messages obviating the poly A addition signal, generates heterogeneous transcripts which are larger than the viral gt‘IIOITHPS. Run through transcription has been described in the case of late transcripts produced in polyoma-infected mouse cells [17]. Run through transcription of early message would require the production of anti—late message. To test this, hybridization of the same Northern blot to a single—strand riboprolw of appropriate imlarity was carried out. The RNA signals detected by a riboprobe for anti—late message observed in cells infected at 9 and 24 hours post infection in figure 2.4 are somewhat strong; whereas, there was little or no RNA signal detected by a. probe containing the entire polyoma DNA (Figure 2.3). The signal seen in cells infected at 9 and 24 hours in Figure 2.3 could be due to the nonspecific binding of the riboprobe, since it. has been shown that riboprobe usually gives rise to high background. The higli molecular weight RNA could be the precursor of rRNA. The unchanged hybridization pattern with the riboprobe (Figure 2.4) support this hypothesis. We also assayed for the presence of true late messages by hybridizing a Northern blot to the appropriate riboprobe. The results showed that little or no late message was present in the infected FR3T3 cells (data not shown). From the above results, we conclude that the heterogeneous size of 69 polyoma RNA transcripts represent early as well as anti—late messages derived from run-through transcription. Viral early protein expression: As a further analysis of viral gene exi'n‘ession, the expression of viral proteins was analyzed 48 hours post infection by Western blotting analysis as described in Materials and Methods. For detection of large T—ant igen, a polyclonal rabbit anti-polyoma small T—antigen which detects both large T antigen and small T—antigen was used. As shown in Figure 2.5, the expression of large T— antigen followed the same time course as did RNA expression, i.e., a peak of large T—antigen expression was observed in cells infected early in G1 phase, with a lO-fold increase in cells infected 3 hours compared to 2.1 hours post release from GO. Viral DNA replication: To determine whether viral DNA synthesis parallels viral gene expression, total DNA was extracted from cells 48 hours post. infection. As shown in Figure 2.6, the maximal viral DNA signal was seen in cells infected in early G1 phase, with a 5—fold and a 2.6—fold increase in cells infected 3 hours compared to 9 hours and 24 hours post release from G0 respectively. There was an increase of DNA signals in cells infected 24 hours post release from G0, and this has been observed in 2 to 3 different experiments. As discussed below. it is less likely that either the. viral DNA synthesis occurs prior to early message transcription, or viral DNA synthesis is mediated by cellular protein(s) which replaces the requirement of large rl‘~antigen. The kinetics of viral gene expression and viral DNA replication: The ki- netics of both viral DNA and RNA syntheses in cells infected 3 hours post release Figure 2.4: Detection of anti—late message. RNA was isolated from cells 48 hours post infection as described in Materials and Methods, and hybridized to a 32P-labeled strand-specific riboprobe specific for the detection of anti-late message. 71 time of infection (hrs post GO) 3 6 28$> 185> Figure 2.4: Detection of anti—late message. K1 [\3 Figure 2.5: Western blot analysis of large T antigen. Cell lysates were harvested from infected cells 18 hours post infection. Aliquots of the cell lysates were analyzed in 10 % polyacrylamide gel, and electroblotted onto nitrocellulose filter paper. The filter paper was blocked with 2 9f» BSA, and reacted with the primary antibody: rabbit anti—small T antigen, and then reacted with the secondary antibody: goat anti—rabbit lgG. 73 time of infection (hrs post GO ) '35 3 6 9 24 E LT-Ag > a an: 4.-- - ., A...- will... W ‘bu \— ’ ‘ 4 ~ ~ W wan-iv V‘ Figure 2.5: Western blot analysis of large T—antigen. Figure 2.6: Analysis of viral DNA replication. Cells were infected at various times, and total DNA was prepared from the infected cells 48 hours post infection. 10 ,ug of total DNA was digested with licoRl and electrophoresed on a 0.7 % agarose gel, and probml to a 32 P— labeled probe containing the entire polyoma sequence. 75 time of infection (hrs post GO) Figure 2.6: Analysis of viral DNA replication. n-.— SIA..A.A&.- i 76 from GO were studied. The timing of cell cycle in the same infection was determined by incorporation of 3HsTdR and following cell multiplication. The first cell cycle was within 0 and 26 hours post release from GO, and the second cell cycle was within 27 and 40 hours post release from GO. Both total DNA and total RNA were extracted at 12, 24, 36, 48, and 60 hours post infection. As seen in Figure 2.7, both signifi- cant amount of viral transcripts and viral DNA were not ol‘)served until 36 hour post infection (Figure 3.7.A). These results showed that, viral gene replication and gene expression occurred in the cell cycle subsequent. to the time of infection. though virus entered the cells in early G1 of the first cell cycle. The RNA signal remained at a high level at least through 60 hours post infection. A more detailed kinetic study of viral gene expression in cells infected 3 hours post release from G0, was carried in the intervals of every 3 hours. In the same experiment, incorporation of 3H-TdR and cell number counting were carried out to determine the phases of cell cycle, and showed that cells had a cell cycle shortened to 23 hours. Results showed that viral gene expression was not, detected until cells entered the GI phase of next cell cycle (24 hours post infection). when a large synchronous burst of expression was observed (Figure 2.7.8). Result of kinetics of viral DNA synthesis is shown in Figure 2.7.(3. We found that the viral DNA synthesis occurred at 24 hours post infection, i.e., in the same cell cycle as cells were infected. Similar to what observed in Figure 2.6, cells infected at 24 hours had high level of viral DNA replication, but little or no viral DNA was synthesized in these cells (Figure 2.3). One explanation for this result is that the viral DNA synthesis can occur before the early gene tra,nscri1’)tion. However, this is least Figure 2.7: Kinetics of viral gene expression and viral DNA replication in l"R.3T3 cells infected 3 hours post release from GO. (A). Cells were infected 3 hours post release from Ct). Total RNA was prepared from the infected cells 12, 24, 36, 48, and 60 hours post infection. 20 fig total RNA was analyzed in 1 % formaldehyde agarose gel, hybridized to a 32P-labeled probe containing the entire polyoma DNA and GapDH, as described in Materials and Methods. (B). Cells were infected 3 hours post release from C0. Total RNA was prepared from the infected cells 12, 18. 2'1 and 27 hours post infection. 5 ,ug total RNA was analyzed on data blot, hybridized to 32P—labeled probe containing the entire polyoma DNA. (C). In the same experiment, total DNA was prepared from cells 12, 24, 36, 48, and 60 hours post infection. 10 ,ug DNA was digested with EcoRI and analyzed as described in Materials and l\~'lethods. For hybridization, a 32P-labeled probe containing the polyoma DNA was used. 78 A. 12 24 36 48 60 Imock 28$> 18$> 6mm. .0‘..‘..D B 12 18 24 27 O O O O C. 12 24 36 48 60 5.3 Kb > Figure 2.7: Kinetics of viral gene expression and viral DNA replication in FR3T3 cells infected 3 hours post release from G0. 79 likely, since it is known that large T—antigen is required for viral DNA synthesis. The other possible explanation is that cellular proteins can carry out the synthesis of viral DNA, and replace the function of large T—antigen. However, there is no evidence for the latter. 2.4 Discussion In this paper, we present evidence for multiple levels of cell cycle control in the infectious cycle of polyomavirus in FR3T3 cells released from Ct). A peak of trans- formation frequency was observed in cells infected early in CI phase with a 20 to 50—fold differential between the highest and lowest transformatitm frequencies. To understand the factors which are responsible for the variations in transformation, we analyzed Viral adsorption, viral gene expression. and viral DNA replication in cells infected at different phases, and found that (RNA, DNA, or proteins) track with transformation with maximal levels obtained in (ll-infected cells. Cell cycle regulation of transformation frequency induced by polyomavirus was first investigated by Basilico and Marin (1966) [2]. They found a 2-—fold increase in neoplastic transformation for cells infected at the S/(J‘Z boundary compared with cells infected in other phases. They suggested that the. two fold increase was proportional to the number of cellular chromosomes, which doubled at the S/G‘Z boundary, so that there might be twice as many targets available for integration by the polyoma genomes. However, the difference in transformat ion frequency in our experiments was much higher (20 to 50—fold), and maximal snscept ibility was observed in C1 infected 80 cells. Whether the results we obtained are specific for cells released from (if) or for F R3T3 cells is not clear yet. Tamura (1983) and Matsuzaki(1989) have also studied transfm'mation of Fisher rat 3Y1 cells by SV40 under various growth conditions [10, 14]. 'l‘hey showed that the frequency of transformation by SV40 and polyon'iavirus was reduced when cells were in the proliferating state at the time of virus inoculation as cmnpared to cells in the quiescent state. The 2 to 10—fold differential in viral adsorpticm (assayed as the total viral DNA entering into host cells) between cells infected at 3 hours compared to those infected at 24 hours post release from G0 suggests that the armearance of the polyoma. receptors on the cell surface is cell cycle regulated and maximal on G1. The effect of cell cycle on SV40 viral DNA replication in nonpermissive cells was analyzed by Pages et al. (1973). They showed that the CV1 cells infected with SV40 during G1 have viral DNA replication occurring within the same mitotic cycle. But if the time of infection passed the end of G1, then these cells were unable to initiate SV it) DNA replication until the next mitotic cycle [11]. They suggested that virus had to pass through a critical stage situated near the end of G1 or the lwginning of S phase in order for viral DNA replication to occur. Differential rates of viral adsorption has been described for SV40 by Pages et al. (1973) [11] as measured more directly by binding of 3H— TdR—labeled virus particles to CV1 cells. Their results showed a. 2 to 3—fold increase in G1 compared to G2 or M phase infection. However, Basak and Compans (1991) found that the expression of the SV40 receptor in Vero cells is increased about 3—fold in S, and G2/M phase compared to G1 phase (personal communication). 81 The differences in adsorption (2—10 fold) liietween various phases of the cycle only partially account for the differences in viral gene expression ($50—40 fold) and /or in transformation (20—50 fold) in every experiment. This and other observations discussed below suggest the existence of another rate limiting step in the infection cycle between adsorption and gene expression which is also regulated during the cell cycle. This step (also maximal in differential rates for nuclear uptake were 3.7-fold larger than those for cellular uptake in the same experiment. The kinetics of viral gene expression showed that the polyoma early promoter is very strongly cell cycle regulated in FR3T3 cells released from GO. Interestingly, viral gene expression was only detected in the cell cycle following that in which infection had occurred. Thus, it appears that the majority of viral germmes which entered cells at 3 hours into the first G1 phase of the cell cycle did not. get processed in time to catch the transcription competence step and waited a full cycle to become expressed. Furthermore, if any genomes did enter cells in S, or Gil as is suggested by the adsorption results, these genomes did not l‘)eeome transcription competent in the next G1 phase, since very little gene expression was detected in cells infected in S or G2. As proposed above, there may be another cell cycle regulated rate limiting step in viral infection between adsorption and viral gene expression. In conclusion, our results showed that the maximal transformation frecuwncy in- duced by polyomavirus occurs in cells at G1 phase soon after release from Gt). There are at least three cell cycle regulated steps in the polyoma infectious cycle: adsorp- tion, transcription, and undefined event(s) including the processing of the polyoma genome. Bibliography [1] F. M. Ausubel, R. Brent, R. E. Kinston. D.D. Moore, .l.G. Seidman, and K. Struchl. Current Protocols in A’Ioleculur Biology, pages <1.1.4——4.1.6. Wliey Interscience, 1987. [2] C. Basilico and G. Marin. Susceptibility of cells in different stages of the mitotic cycle to trasnformation by polyoma virus. Virology, 28:429—‘137, 1966. [3] W. H. Burnette. Western blotting: Electroplroretis transfer of protein from SDA— polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radiolabeled protein A. Anal. Brooke-rm, 112:195-«203, 1981. [4] J. J. Chirgwin, A. E. Przbyla, R. J. MacDonald, and W.J. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Bio- chemistry, 18z5294—5299, 1979. [5] G. Glenn and W. Eckhart. Transcriptioal regulation of early—response genes during polyomavirus infection. J. Viral, 611:2193—2201, 1990. [6] V. Glisin, R. Crkvenjakov, and C. Byus. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry, 13:26:33—2637, 1974. [7] B. E. Griffin, M. Fried, and A. Cowie. Polyoma DNA: A physical map. Proc. Natl. Acad. Sci., 71:2077—2081, 1974. [8] R. Kamen and H. Shure. Topology of polyoma virus messenger RNA nu>l<1>cules. Cell, 7:361—371, 1976. [9] G. Khoury, B. J. Carter, F. J. Ferdinand, I’. \I. Howley, M. Brown, and M. A. Martin. Genome localization of simian virus :10 RNA species. J. ViroL, 17:832— 840, 1976. [10] A. Matsuzaki, A. Okuda, H. Tamura, M. ()htsu, and G. Kimura. Frequency of cell transformation by the small DNA tumor viruses: Infection of proliferating cells and quiescent cells. Microbiol., 33:65? 667, 1989. [11] J. Pages, S. Manteuil, D. Stehelin, M. Fiszman, M. Mark, and M. Girard. Rela- tionship between replication of simian virus 40 DNA specific events of the host cell cycle. J. Virol., 12:99—107, 1973. 82 [12] [13] [14] [15] [16] [17] [18] 83 R. Seif and F. Cuzin. Temperature-sensitive growth regulation in one type of transformed cells induced by the tsa mutant of polyomavirus. J. Virol, 2412721— 728,1977. E. M. Southern. Detection of specific sequences among DNA fragn'ients separated by gel electrophoresis. J. Mol. Biol, 98:503-517, 1975. H. Tamura. In vitro transformation of rat fibroblasts (3y1) with SV-lO: ef- fect of cellular growth state, before and after virus inoculation, on frcxiuency of transformation. Fukuoka Medicine, 74:796—812, 1983. P. S. Thomas. Hybridization of denatured R NA and small DNA fragment s trans- ferred to nitrocellulose. Proc. Natl. Acad. Sci., 77:5201—5205, 1980. J. Tooze. The molecular biology of tumor viruses, chapter rllransformation by polyoma and SV40, pages 350—402. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1973. J. Tooze (ed). R. W. Tseng and N. H. Acheson. Use of a. novel 81 nuclease RNA—mapping technique to measure efficiency of transcription termination on polyoinavirus DNA. M01. Cell. Biol, 6:1624—1632, 1986. J. Zullo, C. D. Stiles, and R. L. Garcea. Regulation of c—‘mye and c—fos mRNA levels by polyomavirus: distinct roles for the capsid protein \I’Pland the viral early proteins. Proc. Natl. Acad. Sci., 84:121041214, 1987. Chapter 3 The Role of Polyomavirus Large T-antigen in Interviral Recombination Previously our laboratory has demonstrated the existence of high levels of interviral homologous recombination among the integrated polyoma viral genomes in the trans- formed cells. To assess whether large Twantigen plays a role in the interviral recom— bination, two double mutants were constructed to have both a temperature sensitive mutation of large T—antigen and mutations in restriction endonuclease sites which were as nonselective recombination markers. Coinfections of Fischer rat (FR3TB) cells with the two double mutants were carried out at both permissive 330C and non- permissive (39°C) temperatures. Viral DNA synthesis was undetectable in cells kept at 39°C. Foci of transformed cells were selected and integrated viral genomes were analyzed for the occurrence of recombination events. Homologous recombination was detected in 33 % of cells kept at the nonpermissive temperature. These results do not support the hypothesis of a role for large T—antigen in interviral recombination. If however, large T-antigen does play a role, the dosage or the domains required for recombination must be different from that required for viral DNA synthesis and other activities of T antigen must be involved. 84 85 3. 1 Introduction The head—to—tail pattern of integration of polyomavirus and SV40 into the genome of nonpermissive cells has raised the issue of the mechanism by which these are produced. In earlier studies with SV40 by Chia, and Rigby (1981), it was suggested that viral DNA replication by a rolling circle mechanism generates the precm'sors for the integrated genomes, even though they also detected some which were formed by recombination [8]. Hacker and Fluck (1989) formed direct evidence showed that high levels of homol- ogous recombination was found coincidental with the process of viral integration and transformation of polyomavirus [17]. They showed that in Fischer rat (F111) cells coinfected with MOP1033, a transformation—defective virus, and ts3, a temperature sensitive mutant defective in decapsidation, an elevated level of interviral recombina- tion was observed after cells were allowed to decapsidate at 330C for 24 hours. This result indicates that interviral recombination plays an important role in the processes of viral integration and transformation, although it could not resolve whether the tandem structure are generated by recombination events. Interestingly, recombina- tion was observed only among those genomes which are integrated in the genome of transformed cells, while no recombination was detected among the non~integrated viral genomes present in the population of infected cells from which the transformed cells were infected. Results of recent experiments in our laboratory suggest that a further amplification of the viral genomes can occur by post—integration events (Syu and Fluck, unpublished - 4': EWETIJ 86 data). In an attempt to further our understanding of the integration process, we have started to address the issue of a potential role for large T—antigen in the process of interviral homologous recombination. The rationale for this experiment is that a tsa mutant, which has a thermolabile large T—antigen, has decreased transformation at 39°C. The transformation observed at 39°C is either due to the leakiness of the tsa mutant or a large T—antigen—independent event. The role of large T—antigen in viral integration has been shown by Stoker (1969). He showed that cells infected with a tsa mutant have the same level of abortive transformation at nonpermissive temperature as that of cells infected with wild type, while the stable transformation induced by tsa was reduced about 20—fold [21, 22]. These results indicate that large T—antigen is required for stable transformation. The inadequecy of stable perpetuation of the viral genome into host cell DNA causes the reduction of stable transformation induced by tsa. Della Valle et al. (1981) showed that in the absence of large T-antigen, polyoma integrated into the host chromosomes with a pattern of non—tandem repeat and with incomplete viral genomes integrated [10]. It has been shown that large T—antigen is inyolved in the process of viral excision in transformed cells [1, 2, 9, 19]. When duplicated regions of polyoma genomes are present after in situ replication by the ”onion skin” model, homologous regions can recombine, producing complete viral molecules that can be removed and circularized as free DNA [3, 4, 5, 14, 15, 18]. Furthermore, a more direct examination of the role for large Ill-antigen in recom- bination has been addressed in a different system by Stonge et al. (1990). They 87 showed that the recombination between two defective copies of middle T~antigen located side—by—side in FR3T3 cells was promoted from 10‘7 to 10‘2 per cell gener- ation when large T—antigen was present [23]. The promotion of recon'ibination was also observed by SV40 large T-antigen which cannot initiate viral DNA replication of polyomavirus, suggesting that the role of large T—antigen in recombination is DNA replication—independent [23]. In the present experiments, we attempt to determine whether large T~antigen plays a role in interviral recombination which is important in the process of viral integration and transformation. To do this, we asked whether temperature sensitive mutants with a defect in large T—antigen can affect the high levels of recombination associated with the integration of the viral genome in FR3T3 cells. Our results do not support a role for large T—antigen since we observed high levels of recombination even with a temperature labile large T—antigen. However, the experiments cannot exclude the possibility of a role for large T—antigen in interviral recombination, for example, if either the dosage or the domain required for recombination is different from that for viral DNA replication. 3.2 Materials and Methods Virus: The polyoma mutants tsa, MOP 1033, and ts3 were used to construct dou- ble mutants. The tsa mutant [13] bears a point mutation at nucleotide 2193, located within the carboxy—terminal coding sequence of large T—antigen. MOP 1033 was derived from a wild—type strain by site directed mutagenesis eliminating the AvaI 88 sites of nucleotides 659 and 1018 [25]. The mutation at nucleotide 659 does not have a phenotype, and the mutation at nucleotide 1018 introduced alterations into the middle T—antigen reading frame producing a transformation-defective virus. The ts3 mutant [11] harbors a a mutation within the VP2 protein which prevents decapsida- tion at the nonpermissive temperature. Since ts3 lacks the Bamlll site (nucleotide 4634) also located within the VP2 coding region, it is assumed that the BamHI site is the site of the decapsidation mutation, although this has not been proven. ts3 can transform nonpermissive cells normally after a short decapsidation period at 33°C. Wild type A2 virus was used in the experiments as positive controls. Double mutants carrying either MOP 1033 or ts3 mutations with the tsa mutation were constructed by ligating the appropriate I'lindIII fragments from the appropriate parental strains. The phenotype of the double mutants were verified. MOP 1033 tsa (Ma) lacking the Aval sites grows only at 33°C in permissive cells and does not transform nonpermissive rat FR3T3 cells at any temperature. ts3- tsa (3a) lacks the BamHI site, grows at 33°C. Similarly to tsa, 3a shows a decrease in transformation at 39°C, even after incubation at 33°C for 24 hours to bypass the decapsidation defect. Infection: Fischer rat (FR3T3) [16] cells were cultured and maintained in Dul- becco’s Modified Eagle medium (DMEM, Gibco) supplemented with 10 % calf serum (Hazleton). To maximize transformation, cells were grown to con fluency and starved with 0.2 % gamma globulin (GG) free serum [7]. Cells were then released from G0 by trypsinization and addition of 10 % calf serum (Gibco). Coinfection of cells with Ma and 3a at a multiplicity of infection (MOI) 50 and 50 was carried out 3 hours post 89 release from G0, the earliest time for cell reattachment. Infections with the single parents or wild-type A2 virus served as controls. To allow decapsidation of ts3 and 3a to occur, infected cells were kept at 33°C for 24 hrs after infection and then half of the cultures were shifted to 39°C. Foci of transformed cells were obtained two to three weeks post infection, and the clonal transformed cell lines were grown in DMEM supplemented with 10 % calf serum at the same temperature at which the foci were derived (at either 33°C or 39°C). Preparation and analysis of DNA: Total DNA was extracted from infected or transformed clonal cell lines by lysing the cells in a solution of 0.2 “fl SDS, 10 mM Tris, pH 7.5, 10 mM EDTA, and 50 ,ug/ml proteinase K (Sigma) [12]. For analysis of the occurrence of recombination, 10 ug DNA was digested with endomrclease restriction enzymes (AvaI+BamHI+Bglll, PvnIl+AvaI+l3glll, HindlII+Aval+BamIll, l'lcoRI, BclI, BglI, HaeII, EcoRV, and Mspl) (Figure 3.1), electrophoresed on agarose gels and transferred onto Hybond—N filter paper [20]. Preliybridization was carried out in a buffer containing 5 X SSPE, 5 X Denhardt’s solution, 0.5 % (w/v) SDS and 50 pg/ml salmon testis DNA for one hour at 65°C. I’beridization was carried out in the same buffer at 65°C for 48 to 60 hours. For hybridization, l x 106 cpm/ml of 32P—filabelled probes containing the entire polyoma DNA, or the fragments 4 or 5 of MSpI—digested polyoma. DNA were used for different hybridizations as mentioned in each figure legends. Probes were labeled to a specific activity of 1—2 x 10‘J cpm/iig template DNA by using a multiprime DNA labeling kit (Amersham). _ (Era-E a“. 90 Or‘l ?~ :2 " 'x 0 o) (o (D -» NWQG s s s was V. b—d N... (3992.133? Eris ,; (659) ”591 1 “L6" AvaI BamHI (4634) MspI (4409) (1318) AvaI EcoRV (4108) (1103)MS I (1144) Pvu I - (1213) Mspx (1488) MspI (2032) Pqu A M50 (2615) 2991 Figure 3.1: Restriction endonuclease map of polyomavirus. Origin is located at the nucleotide 0/ 5292. BglI, EcoRI, EcoRV , BamHI, BclI cleave polyoma once, HaeII and AvaI cleave polyoma genome twice, PvuI I cleaves polyoma genome four times, and MspI cleaves 8 times. 91 3.3 Results Analysis of recombination in the cross between Ma and 3a at 39°C: To assess whether large T—antigen plays a role in the interviral recombination associ- ated with transformation, we constructed double mutants with the tsa temperature sensitive mutation in large T—antigen and the imitations in restriction endonuclease markers of MOP 1033 and ts3 used to follow recombination [17]. The properties of the double mutants are described in Materials and Methods, and sun‘u'narized in Table 3.1. There was no selection pressure for recombination in the cross between Ma and 3a since 3a can transform normally after decapsidation at 33°C for 2] hours. FR3T3 cells were coinfected with Ma and 3a as described in Material and Methods. Infected cells were kept at 33°C for 24 hours , and then half of the cultures were shifted to 39°C. Total DNA was extracted at 4 hours post infection (hpi). at 48 and 60 hpi (39°C) and 60 and 90 hpi (33°C). Focus formation assay was used to assay transformation. A total of 49 foci of transformed cell lines were collected from 3 independent experiments carried out at 39°C. Clonal cell lines were amplified at 39°Cand total DNA was isolated. Viral DNA synthesis was examined to confirm that large T-antigen could not be detected at nonpermissive temperature. Total DNA was extracted 4 hours post infection (hpi) to measure the input of virus, 48 and 60 hpi at 39°F. and 60 and 90 hpi at 33°C. Digestion with a combination of AvaI and BamHI reveals t he expected bands of a 5.3 kb fragment for Ma and a 5.0 kb fragment for 3a. In these signals as shown in Figure 3.2, there was little or no increase in viral genome copy number over the 'm'ra‘nlsl _._ II 92 Table 3.1: Phenotypes of tsa mutant and double mutants. Ma and 3a. \"irus Strains tsa M a“ 3a° replication 33°C + + + 39°Cd — — transformation 33°C + ._ + 39°C° +C (reduced) — + (reduced) Restriction A val“L A val ‘ .-\ val + endonuclease pattern Bamlll+ Baml-Il+ BamHI- a. Ma is constructed from both MOP1033 and tsa. b. 3a is constructed from both ts3 and tsa. c. Transformation induced by tsa mutant observed at nonpermissive temperature is about 20—fold decreased compared to that observed at permissive l_(:‘lll])(?l“ctllll‘(‘. d. The first 24 hours was at 33°C. 93 input signal (extraction at 4 hpi) in cells kept at 39°C, whereas, a. significant amount of synthesis was observed for cells kept at 33°C. The viral DNAs detected were all parental viral DNA, and no recombination was evident. These results suggest that the expression of the large T-antigen defect causes the absence of DNA replication function at high temperature. The results for the number of transformation were shown in Table 3.2. To analyze recombination, total DNA from transformants was digested with a con'ilnnation of AvaI, BamHI and BglII (Figure 3.1). A 1.3 Kb fragment will be generated if recom- bination has occurred between the small AvaI—BamHI interval; a 1.7 Kb fragment will be generated if the recombination occurred between the two Aval sites; and a. 3.6 Kb fragment will be generated if the recombination occurred between the large AvaI~BamHI interval (Figure 3.3). Total 13 out of 49 foci cell lines in 3 experiments showed acquisition of recombinant viral genomes. In experiment 1, recombinant viral genomes were detected in seven out of twenty-one transformed cell lines picked at 39°C, and were analyzed in more detail. In Figure 3.3, three cell lines (# 1, 3, and 5) had only the 1.7 Kb fragment; one cell line (# 4) had both 3.6 Kb and 1.3 Kb fragments; and two cell lines (# 6 and 7) had both 3.6 Kb and 1.7 Kb fragments. One cell line transforim—id with wild type A2 was used as control. Cell line # 2 is not a recombinant, because no recombinant viral genome was detected. Cell line # 8 had only one 3.6 Kb fragment (data not shown). Thus, in 4 out of 7 transformed cell lines containing recombinant viral genomes (#1, 3, 5, 8) a single recombination event had occurred; wheree-is viral genomes in 3 cell lines (# 4, 6, 7) had undergone 2 recombination events. Among 94 Figure 3.2: Lack of DNA synthesis in the double mutants, Ma and 3a, at noupermis~ sive temperature. Total DNA was extracted from FR3T3 cells coinfected with Ma and 3a 48 hrs and 60 hrs post infection at either 33°C or 390C. Infected cells were kept at 330C for 24 hrs before half of the cultures were shifted to 39°C. DNA was isolated, digested with a combination of AvaI and BamHI, and analyzed as described in l\‘laterials and Methods. Arrows show the corresponding sizes of a 5.3 Kb fragment derived from Ma, and a 5.0 Kb fragment derived from 3a. 95 39°C 33°C mu: ow vv N I Figure 3.2: Lack of DNA synthesis in the double mutants, Ma and 3a, at nonpermis- sive temperature. Table 3.2: Results of transformation and recombination from 3 independent experi- ments. 96 tem pera t ure 330C 39"C5’ A ‘2 50" l 00" Transform at ion M a. 0“ 0“ Experiment 1 3a 50“ 9-1.7" .\la. x 3a 17.8b 18.5‘ Recombination .\la x 3a 7/16 7/‘21 Experiment 20’ Recombination .\la x 3a ND6 4/12 A2 50“ 13” 'l‘ransforn‘iation Ma 0“ 0“ Experiment 3 3a 40” 5“ .\la x 3a ‘20f 5f Recombination Ma. x 3a ND6 2/16 a. Foci number averaged in 2 parallel infections. b. Foci number averaged in 9 parallel infections. c. Foci number averaged in 10 parallel infections. CL. . Undetected. (D :‘H g. The first ‘24 hours was at 330C. . The same experiment as described in Figure 3.2 Foci number averaged in 5 parallel infections. 97 Figure 3.3: Test for interviral recombination. Total DNA of8 transformed cell lines derived at 39"C was digested with a combination of Aval, BamHI, and Bglll, and analyzed as described in Materials and Methods. Transformant isolated from type A2 virus infection is shown as control. ("ell lines number 1, 37 contain recombinant viral genomes. Arrows show the, sizes of 3.6 Kb, 1.7 Kb and 1.3 Kb fragments. Figure 3.3: Test for interviral recombination. 99 these seven recombinant transformants, except # 3, complete viral genomes were detected as determined by digestion with Mspl which cuts the polyoma genome eight times (data not shown). In cell line # 3, Mspl fragment 3 is missing. Among the 21 foci cell lines analyzed, 13 out of 21 had a tandem repeat. of viral genome integrated, whereas 8 out of 21 had nontandem of viral genome integrated. The ratio of recombination occurred in the transformed cells with a tandem repeat of the viral genome compared to nontandem of the integrated viral genome was 4 : 1 (6/13: 1/8) In the six cell lines which have the complete viral genomes, the extent of of the viral repeats was further determined . Four restriction endonucleases which cut the polyoma DNA once or twice were used: BglI (cleavage site: nucleotide 87), EcoRI (nucleotide 1560), EcoRV (nucleotide 4108), and BclI (nucleotide 50233), and HaeII (nucleotides 87 and 97). Results showed that all the six cell lines possess partial head-to-tail tandem repeat (data not shown). In .5 cell lines the repeated region did not include the EcoRI site in the middle of early region, but the repeats extended through the BclI, BglI, and HaeII sites in. the origin-enhancer region. The number of viral integration sites was (l(,,‘t()l‘n'1in€(l by digestion with Bglll, which does not cut the polyoma genome. The results showed that 9 out of 21 cell lines have a single integration site, 6 out of 21 cell lines have 2 to 3 integration sites, 6 out of 21 have a single integration site with a ladder-like pattern of bands on top of a basal band. Examples of 6 cell lines are shown in Figure 3.4. 100 Figure 3.4: Integration pattern of polyoma genome in the transformed cell lines kept at 39°C. Total DNA of was extracted from transformation cells, and digested with Bgll ,elec- trophoresed in a 0.4 % agarose gel, and analyzed as (.lescribed in Materials and Meth- ods. Cell lines # 5, 6, and 7 are recombinants and labeled as same as shown in Figure 3.3, and cell line # 9, 10, 11 were foci picked in the same experiment but did not contain recombinant viral genome. 101 .1 I .fifie m. Integration pattern of polyoma genome in the transformed cell lines kept Figure 3.4 at 39°C . 102 Proof that the 1.3, 1.7 and 3.6 Kb fragments are recombinant: To confirm that the 1.3 Kb and 1.7 Kb fragments observed in Figure 3.2 were true recombinant fragments, DNA of cell lines with these fragment was digested with a. combination of Aval, BamHI and Pqu (Figure 3.1), and probed with fragment 5 of Mspl—digested polyoma DNA. In this analysis, a 1.0 K1) fragment. will be generated from a 1.7 Kb recombinant fragment; a 0.7 Kb fragment from a. 1.3 Kb fragment (either from a recombinant or from the 3a parental type), and a 1.1 Kb fragment. from the Ma parental genome(Figure 3.5.A). The results showed that. cell lines # l, 3, and 5 have a 1.0 fragment; cell lines # 1 and 4 has a 0.7 Kb fragment, and cell lines # 6 and 7 have both 1.0 and 1.1 Kb fragments (Figure 3.5.13). These results support the conclusion that the 1.3 Kb and 1.7 Kb fragments in the recombinant cell lines are the recombinant products. To confirm the identity of the 3.6 K1) recombinant fragment. cell lines with the 3.6 Kb recombinant fragment were further analyzed by digestion with a. combination of HindIII, AvaI, and BamHI, and hybridized to a. probe containing the Ecolt 1-——Xbal fragment of polyoma DNA (Figure 3.1) The expected 2.2 kb fragment was generated in all the cell line with a 3.6 kb fragment (data not shown). A defective large T—antigen present in the transformants containing re- combinant viral genomes: To further confirm the lack of viral DNA synthesis in cells with recombinant viral genomes, three cell lines (# 3, 6, and 7) were chosen and grown in parallel 33°C or 39°C. Cell lines # 6 and 7 contain a complete integrated viral genome, whereas cell line 3 does not. Total DNA was extracted after 5 days ifimi .‘l‘ 103 Figure 3.5: Confirmation of the identity of the 1.7 and 1.3 Kb recmnbinant fragments. (A). A 1.1 Kb, a 1.0 Kb, or a 0.7 Kb fragment will be generated after digestion with a combination of Pqu, Aval, and Bamlll, and hybridized to a probe containing the fragment 4 of Mspl digested polyoma genome. (I3). 10 fig DNA was digested with a combination of Pvull, Aval, and Bamlll, and amilyzed as describt-wil in l\"laterials and Methods. 104 A. On ‘ l t l O O 1.1 Kb 0 1.0 Kb 0.7 Kb Figure 3.5: Confirmation of the identity of the 1.7 and 1.3 Kb recombinant fragments. 105 incubation at 39°C, or 10 days at 33°C, and analyzed by digestion with a combination of AvaI and BamHI. The results showed that a IT to 27—fold increase of replicat ion at 33°C in cell lines # 6 and 7 (Figure 3.6). In cell line # 3, no increase of viral signal was found at 33°C compared to at 39°C due to its i1’1complete viral genome. This result indicates that the transformed cells with recombinant viral genon‘ies derived at 390C are not able to amplify viral DNA at 30"’('. (as expected for a tsa mutant). and the ability of DNA replication of large T—antigen can be restored by shifting cells to 330C. Analysis of the recombinants derived at 33“C: Sixteen foci were picked and amplified from cells coinfected with both Ma and 3a at 33"C. From the digest ion with Aval, BamHI and Bglll restriction enzymes, seven out of sixteen cell lines contained recombinant fragments of 3.6 Kb, 1.3 Kb or 1.7 Kb. Among the cell lines grown at. 33°C, ten cell lines showed the presence of high levels of free viral DNA (Figure 3.7). The integration site of the polyoma genome was determined by digestion with Bglll. Results showed that 9 cell lines had single integration site and (5 cell lines had ‘2—3 integration sites (data not shown). 3.4 Discussion We have investigated the effect of large T—antigen on viral DN\ synthesis. viral integration, transformation and interviral recoml)ination. Results of these assays in the transformed cells derived from coinfection with Ma and 3a are summarized in Table 3.3. 106 Figure 3.6: Lack of viral DNA synthesis in transformed cells containing the recombi- nant viral genomes at 39°C. Total DNA was extracted from 4 cell lines kept. at either 39"C or after shifted to 33°C for 10 days, and digested with a combination of Aval and Bamlll and analyzed as described in Materials and Methods. Cell lines f}: 3, 7, and 6 were the same cell lines as in Figure 3.3, and a cell line which was transforiméd by Al? was used as a control. Arrows show the sizes of the 3.6 Kb, 1.7 Kb .and 1.3 Kb fragments. 107 39°C 33°C 3.6> qua-— “a 1.7> .. ].3> Figure 3.6: Lack of viral DNA synthesis in transformed cells containing the recombi- nant viral genomes at 39°C. Figure 3.7: Analysis of the occurrence of recombination in the transformed cells derived in the cross between Ma and 3a. at 33"( '. 10 [1g of DNA was digested with a combination of Aval, Bamlll. and Bglll, and analyzed as described in Materials and Methods. 109 Figure 3.7: Analysis of the occurrence of recombination in the transformed cells derived in the cross between Ma and 3a at 33°C. Table 3.3: Comparison of transformants derived in the cross betwwn Ma and 33. at either 33°C or 39°C“ 110 temperature 3:1“(9' 39%,?! Viral DNA synthesis in transformed cells 17 X 27 X l X Integration pattern?) complete genome XI) 1.3/21 incomplete genome N l) 8/‘21 Viral sequence flanking to Mspl 3 XI) 7/8 celluar DNA in cell lines with incomplete viral genome Mspl -l Kl) '1/8 single site 8/10 9/21 # of integration siteC ‘2—3 sites 132/10 6/‘31 single site w/ a. ladder 0/l0 6/‘21 Free viral DNA of Yes 10/16 ()/'.’1 both parental strains No (i/ 10 ‘21/‘21 Transformation 17.8’7 18.5')‘ Incidence of Recombination 7/16 7/2 1 l / 7 3 i / 7 # of events/cell 2 12/7 4/7 3 1/7 0/7 # of events occurred tandem N1) 6/13 in viral genome nontandem N l) l/S Ratio of tandem/nontandem 3.7 a. Data obtained from cells coinfected with Na and 3a. in EXperiment 1. I). Results obtained from Mspl—digestion. c. Results obtained from BglII—digestion. d. Number averaged from 9 parallel infections. e. Number averaged from 10 parallel infections. f. The first 24 hours was at 33°C. 111 For viral DNA synthesis, little or no viral DNA synthesis in the infected cells was kept at 39°C. However, it is possible if the viral DNA synthesis occurs only in 5 % of the population of infected cells, the increase of viral signal might be underdetectable. The lack of viral DNA synthesis was also observed in transformed cells kept at. 390C. The ability of viral DNA synthesis can be restored by shifting transformed cells to permissive temperature. For example, a 17 to 27 fold increase of viral DNA synthesis was observed in cell line # 6 after shifting from 300C to 33"(.'. Integration of a complete viral genome is required for the viral DNA synthesis to occur. In cell line # 3, no increase of viral DNA synthesis was observed after shifted to 33"’(..' owing to its possession of incomplete viral genome. The integration pattern of polyoma genome was analyzed in trzmsformant s derived from both 33°C and 39°C. The viral integration pattern observed at the nonpermis- sive temperature is the original integration event without further viral amplification and rearrangement. Thus, the integration pattern by tsa observed at nonpermis- sive temperature is the original integration event. 8 out of ‘21 of the viral genome transformed cell lines picked at 390C were found with nontandem repeat integrated; whereas the other 13 cell lines had tandem repeat of polyoma genome integrated. lntriguingly, among the 8 cell lines with nontandem repeat. of the viral genome , we found 7 cell lines had lost the fragment 3 of Mspl --digested viral DNA, and only 1 cell line lost the fragment 1 of Mspl—digested viral DNA. This result indicated that the junction of viral and host DNA resides in the Mail—fragment 3 in these 8 cell lines, suggesting that there might be a hot spot in Mspl—fragment 3 for nonl‘iomologous recombination between viral genomes and host chromosomes. 112 The number of integration sites was analyzed, and results showed that 9 out of 21 cell lines had a single integration site, 6 had 2-—3 integration sites, and 6 cell lines had a single site integration with a ladder~~~like pattern of bands with unit length of 5 Kb distance observed on top of a basal band. Syu and Fluck (1991) showed that the ladder—like pattern was generated via a post—integration event which required functional large T—antigen such as in situ amplification of viral DNA [‘24]. As we discussed above, the large T—antigen of tsa was impaired in viral DNA synthesis of newly introduced viral genomes in infected cells as well as of integrated viral DNA in transformed cell. However, the presence of ladder—like pattern of bands suggested that the amplification of viral genome occurred and there were some levels of large T—antigen present in these cells. Thus, the leakiness of tsa mutant can facilitate the post—integration amplification of viral gentmm. although there is a severe defect in viral DNA synthesis with newly introduced viral genomes. Transformation induced by tsa at nonpermissive temperature has been shown to be 20—fold decreased compared to that at permissive temperature. In our experi- ments, variations in transformation were observed. For example, in experiment 1, no difference of transformation frequency was observed between cells coinfected with Ma and 3a at 33°C and 39°C. However, in the other CXPCI‘IYUCIHS, the termierature effect in transformation frequency was observed. The susceptibility of transformation varied from experiment to experiment. The high susceptibility of transformation in this experiment could be due to the usage of high MOI of Ma and 3a (50 and 50). It has been known that tsa mutant is leaky in transformation when high MOI of tsa. mutant is used [22]. 113 The assessment of recombination occurred between Ma and 3a. showed that there was no difference of recombination frequency either at permissive or nonpermissive temperature. The crossover events were determined in the 7 transformed cell lines containing recombinant viral genomes; 4 cell lines had a single crossover event as evidenced by the fact that they have only one recombinant viral l'ragn‘ient; whereas the other 3 have two crossover events with two different recombinant viral fragments. Interestingly, we found that among these 7 cell lines, 6 have tandem repeat of the polyoma genome integrated, and only 1 has nontandem viral genome integrated. As far as we know, there was no selection pressure for recombination in the cross between Ma and 3a. The wild type polyoma sequence was the recombinant products; whereas no reciprocal recombination product was detected. The reciprocal recoml‘)i- nation product carrying both mutations of Ma and 3a could be lost during the cell proliferation by exonuclease digestion or simply because it cannot replicate. The interviral recombination was independent of viral DNA synthesis because high levels of interviral recombination occurred in the absence of viral DNA synthesis. The evidences of lack of viral DNA synthesis in cells coinfected with .\Ia and 3a in the transformed cells at nonpermissive temperature was obtained and discussed above. These results with the tsa mutant indicated that. the mutation in large T—antigen did not affect interviral recombination. Two possible interpretations of this are : (1) large T—antigen is not involved in the recombination process. (‘2) 'l‘here are low——levels of large T—antigen present in the cells grown at 3906' either due to the leakiness of the tsa mutation or due to the incubation period at 330C. (3) The domain of large T— antigen essential for recombination resides in (*lifl'erent region of large ',I‘-~ant.igen from 114 that required for viral DNA replication. A ladder—like pattern of bands was observed in 6 out 21 transformed cell lines, suggesting there was some large T~antigen present for DNA synthesis of the integrated viral genome. The leakiness of tsa. mutants may give rise to the low level of large T—antigen to carry out some functions of large T—antigen. Syu and Fluck (1991) observed that rearrangements of integrated viral sequences resulted from homologous recombination in the tsa transformed cells at 39°C [24]. This result also suggests that recon‘ibination can still occur with little or no large T—antigen. Stonge et al. (1990) showed that the rate of homologous recombination was pro- moted from 2 x 10‘7 to 10"2 per cell generation before and after the large T-antigen was introduced [23]. They studied the recombination between two defective copies of middle T-antigen integrated side by side in FR3T3 cells, which is very similar to the viral excision process. A clear role for large T—antigen in homologous recombination has been demon- strated in viral excision [2, 3, 5, 9] and amplification [3, 4, 5, 21]. These processes may be linked to requirement for viral DNA replication. The present type of recom- bination we studied is different , i.e., the occurrence of recombination between two de novo introduced viral genomes rather than between two integrated viral sequences. The evidences of the occurrence of recombination independent of viral DNA syn- thesis were also obtained from other groups. Stonge et al. (199(1) showed that re- combination between two defective middle T-anti gen can be promoted by SV40 large T-antigen [23]. They suggested that the role of large T-antigen in recombinatimi is by binding and unwinding the origin region, since SV40 large T-antigmi cannot initiate 115 DNA synthesis from a template containing polyoma origin. Bom'gaux et al. (1990) showed that viral DNA synthesis is not required for the homologous recombination, but the region near the origin is required [6]. In conclusion, the different effects of the tsa large T——antigen in different aspects of polyoma infection were obtained. On one hand, the large T—antigen of tsa mutants are defective in viral DNA replication although that our results do not exclude small levels of replication from integrated viral genomes. On the other hand. little or no effect of tsa was observed on: (1) transforn'iation; no rmluction of transl'orn’iation observed, (‘2) integration pattern; tandem repeat of the polyoma genome found integrated in 62 % transformed cells, suggesting the presence of large T-antigen during integration, (3) appearance in ”ladder pattern”; a post—integration replication event occurred, (4) recombination; high levels of interviral recombination occurred at nonpermissive temperature. [11 [101 [111 Bibliography C. Basilico, S. Gattoni, D. Zouzias, and G. Della Vale. Loss of integreued viral DNA sequences in polyoma transformed cells is associated with an active viral A function. Cell, 17:645—659, 1978. C. Basilico, D. Zouzias, G. Della Vale, S. (lattoni, V. Colantuoni, It. l"enton, and L. Dailey. Integration and excision of polyon'ia virus genomes. ("o/(l Spring Harbor Symp. Quant. Biol, 44:611—620. Ifls‘f). M. Botchan, J. Stringer, T. Mitchison, and .l. Sambrook. Integration and excision of SV40 DNA from the chromosome of a transformed cell. Cc/l. 2011-13-1 .32. 1980. M. Botchan, W. Topp, and J. Sambrook. The arrangement of simian virus 40 sequences in the DNA of tramnsformed cells. Cell, 9:269—287. 1976. M. Botchan, W. Topp, and J. Sambrook. Studies on simian virus 40 excision from cellular chromosomes. Cold Spring ”(Ir/H)!" ngp. Quu'nl. Biol. > ’i! D in" {"43 26> ' 21>..- l.8> 1.7» l.6> l.5>. Figure 4.3: Restriction endonuclease analysis of recombination events in two (11 23 recombinant trasnformants. 135 they were derived from a recombination event in which the exogenous virus acquired intron sequences from the endogenous sequences. Thus, as we have observed in many other crosses, reciprocal recombination is not observed for any particular event. The absence of class 2 recombinants might. reflect the failure of generating these events or selecting them. An easy case could be found for the latter one, i.e., selecting these events. Since multiple copies of the plasmid are integrated in I’ll LT, it. is possible that the generation of a wild type middle T-antigen copy from a. single plasmid copy might not lead to sufficient middle T-antigen production even in the presence of dexamethasone for the expression of the transformed phenotype. The recovery of wild type virus from transformed cells: As the recombi- nation with 1387 T which has a point mutation cannot be studied by restriction endonuclease mapping, an alternative method was devised. Low molecular weight DNAS were extracted (as described in h’laterials and Methods) from two liibTTlfl‘ cell lines which have high levels of free viral DNA and were used to transect NIH 3T3 cells. These viruses were plaque purified and amplified to a titer of '108 pfu/ml. A transformation assay was performed in FR3T3 cells with the recovered virus stocks. Both viruses induced neoplastic transformation in FR3T3 cells very efficiently (data not shown). Since the original virus, 1387 T cannot. transform Flt3T3 cells (see Ta- ble 4.1), the transforming ability must resulted from the presence of recombinant. wild type genomes in the 1387 T recombinant. transforinants. 136 Analysis of the integration patterns of both endogenous pMSG/LT poly- oma and intact viral DNA in 1387TLT and d123LT recombinant transfor- mants: The integration patterns of the pMSCl/LT plasmid in FRLT cells and the viral genomes in 1387TLT and d123LT recombinant trasnformants were determined. Three restriction endonucleases were used to digest the total DNA extracted: (1) Bcll does not cut the pMSG/LT plasmid, but cuts polyoma DNA once at ntusleotide 5023; (2) Bglll cuts pMSG/LT once, but does not cut polyoma DNA; (3) Bst ICII cuts neither DNA. The digested DNAs were resolved in a 0.4 % agarose gel and hybridized with a 32P—labeled probe containing the entire polyoma DNA. The results are shown in Figure 4.3 and Figure 4.4. As alluded above, the integration pattern of ph’lSG/LT in FltlfI‘ cells is compli- cated. When analyzed with enzymes which do cut the plasmid (Bcll and Bglll), two bands of very high molecular weight were seen. In Bcll digestions, one band was sharp and another broad, while two broad band were obtained with BstEII. In Bglll digestions, which cuts pMSG/LT once, 4 distinct strong band and 2 weaker bands were detected, and no 10 Kb fragment (the size of the plasmid). This result suggests that the pMSG/LT plasmid is integrated at 2 major sites containing multiple copies of the plasmid, and is not a simple head to tail complete tandem repeats of the plasmid. In the fifteen 1387TLT recombinant transformants analyzed, clear alterations of the endogenous sequences were observed in both Bcll and BstElI digestion. The re- sults with Bcll digestion are shown in Figure =1.3.A. In twelve dl23l.T recombinant transformations, 5 cell lines showed alterations of endogenous sequences, 6 cell lines had alternations in Bell digestion, and 8 cell lines had alternations in Bglll digestion. 137 Examples of digestions of 4 dl23LT recombinant transforn'iants with Bell, llgll and BstEII are shown in Figure 4.3.8. The high incidence of alterations of the endoge- nous sequences suggested that the viral genomes had integrated into the endogenous pM SG / LT sequences. 4.4 Discussion Our results showed that high levels of recombination frequency was observed bet ween the endogenous large T—antigen cDNA and the. exogenous polyoma viral genoi’nes. Recombination frequency in the cross between integrated and unintegrated polyoma sequences was measured by the transformation frequency of the infected cells, since all the transformants are recombinants. In infection with 1387 T. the reconurination frequency was 1/34 of that observed in cells infected with wild type A2 virus at the same multiplicity of infection. Comparing the recombination frequency obtained from the present experiments to other systems, we found that the recombination between polyouurvirus genomes in infection of cells containing pre—integrated viral sequences occurred at a high rate. For example, the frequencies of gene targeting in embryonic stem cells are: 1/30t) for the Err—2 gene [9], 1/1000 for the hprt gene [31], ”400,000 for the 2T-nl~2 gene [32], 1/117 for the fig — microglobulin gene [20], 1/250 for the T—cell receptor fi—subunit. [24], 1/40 for the homeobox gene Hort—3.1 [25]. In a. hybrid murine— human cell line. the frequency of correction of a human fls-globin gene was 3/126 by gene targeting [28]. Possible explanations for the high levels of recombination frequency observed in Figure 4.4: Analysis of integration patterns of the polyoma sequences in both 1387TLT and d123LT cells. (A). 10 pg of DNA extracted from FRLT and fifteen 1387TLT cell lines was digested with BclI, and analyzed as described in l\/Iaterials and h‘lethods. (B). 10 pg total DNA extracted from FRLT and 4 d123LT cell lines was digested with Bell, Bglll and BstEII, and analyzed as described in Materials and Methods. 139 .— —l 5123456789101112131415 B. BstEll Bglll Bell ’3 5 ’3 E I 2 3 4 E 1 .2 3 4 E 1 2 3 4 - v ‘5‘. Figure 4.4: Analyses of integration patterns of the polyoma sequences in both 1387TLT and d123LT cells. 140 the cross between the integrated and unintegrated viral genomes are (1) the presence of high levels of large T—antigen, (‘2) high copy numbers of the endogenous integrated plasmid DNA, or (3) a hot spot for recombination in the polyoma genome. The first possibility is that large T—antigen plays an important role in homologous recombi- nation. However this can be ruled out by the fact that no significant difference in recombination frequency was observed in FRI.T cells with or without, dexamet hasone— induction. In previous experiments, we found that high levels of int(_.‘r\»'iral recombi- nation occurred in the absence of functional large T: antigen [7]. The second possibility is that high copy numbers of the endogenous plasmid pMSG/LT sequences can cause recombination to occur with a higher frecpiency. The pMSG/LT integrates at 2 sites of cellular chromosomes with a complicated integration pattern in FRLT cells, so that there are more. templates available for recoml)ination to occur. However, this possibility can also be ruled out by the obser\-'a.t;ion that no difference of recombination frequency was observed with increasing copy number of the integrated plasmid DNA in embryonic stem cells [2]. The third possibility is that a tentative hot spot for recombination is located in the polyoma genome. The hot spot may cause high levels of recombination obserwxl in the cross l_)(-_=tween uninte- grated and pre—integrated viral genomes. A gradient. of recombination along the 40 % of polyoma viral genome was observed [5]. This might indicate the presence of a hot spot which could play a role in the high frequency of the homologous recombination. From the analysis of the integration pattern of polyoma genome in the recom- binant transformants, the acquisition of the wild type polyoma sequences resulted from the recombination between the integrated polyoma sequence and exogenous vi- 141 ral genome. Two possible explanations for these recombination events were raised. One is that a double crossover occurs and the crossover sites are located between the intron (nucleotide 795) and the deletion mutation (nucleotides 1121 to 121:3 for (ll ‘23). The other is that a double crossover occurs downstream of nucleotide l2-l7), and which is then followed by a gene conversion event during DNA replication or repair (Figure 4.5). However, the regions of ClaI—ICcoRI, BclI—EcoRl. and Bgll—llindlll from 1the endogenous plasmid pMSG/LT sequences remain unchanged. suggesting that the crossover must have occurred downstream of the EcoRl (nucleotide L362) or I’lindIII (nucleotide 1658). The alterations of the pre—intcgrated polyoma. sequences occurred at a high rate. These alterations arose from the integration of newly introduced viral genomes in the same sites of the integrated endogenous plasmid pMSG/LT. The fact that most (50 ‘70) transformants, 2 viral genomes recombined at the site of integration. and in contrast, integration at more than one site with a single transformant is rare (ll) 70). This fact opens the possibility that the viral genomes may borne to special sites on the host chromatin or scaffold; such targeting might be related to the path of entry into the nucleus. The very high or high rates of interviral recombination occurred among the inte- grated genomes arising in mixed infection or in infected cells containing pie—integrated viral sequences respectively. However, on the other hand, the levels of recombination were not detected among unintegrated genomes in mixed infection. The contrast between the recombination occurred predominantly in the integrated viral genomes and not in the unintegrated viral genomes suggests that interaction with the host 142 chromosome or scaffold plays a major role in the recombination process. Thus, in contacting the host chromatin, viral genomes encounter a major host recombination pathway. A comparison of the recombination frequency of the cross between unintegratr—rd and integrated viral genomes to that of the cross between 2 parental mutant genomes in FR3T3 cells, suggests an answer to the question concerning the timing of recombi- nation in the latter crosses. In these crosses, the recombinant viral genomes are inte- grated into host DNA and serve as the transforming genomes of stably transformed cells. Three major classes of events might lead to interviral recoinbination associ- ated with integration. Interviral recombination may occur prior to, simultaneously with, or post integration. The first possibility appears unlikely since recombination was not observed among unintegrated viral genomes derived from the same infected cell population from which the transformants with integrated recombinant genomes were recovered. The third case was that one genome was integrated first followed by integration of the second genome into the first one by homologous recombination. However, results in the present experiments showed that the recombination fl‘t-‘(lllf‘lle between unintegrated and integrated viral genomes is 10—fold lower than that ob- served in crosses between 2 co—infecting viral geiumies. Thus, the recombination of 2 viral genomes occurs simultaneously as they integrate. Interestingly, illegitin‘iate recombination between the viral and the host genome might occur concurrently to homologous interviral recombination. 113 Fi 'ure 4.5: Possible recon'ibination events occurring in the cross bet ween the endo e- g s nous large T-antigen cDNA and the exogenous polyoma genome. The open triangles represent for the deletion of intron in large T-antigen cDNA, and the solid triangles are the good copy of intron in the d] ‘23. The open squares are the deletion of nucleotide 1140 to nucleotide 1213 in the d] ‘23. and the solid squares are the good copy of corresponding sequence of mutation in (H 23. Two possible events could occur: (A). A double crossover occurs between the intron and deletion mutations. (B). A double crossover occurs downstream from the deletion mutation, and followed by a gene conversion event. 144 rat pMSC LT-Ag cDNA pMSC rat Polyoma vtrus A. A double crossover occurred between Intron and mutation. 5- A double crossover occurred downstream from mutation site, and followed by a gene conversion. gene conversion Figure 4.5: Possible recombination events occurring in the cross lwtween the endoge- nous large T—antigen cDNA and the exogenous polyoma genon'ie. Bibliography [1] A. L. M. Ten Asbroek, M. Quellete, and P. Borst. Targeted insertion of the neomycin phosphotransferase gene into the tubulin gene cluster of trypanosoma brucei. Nature, 348:174—175, 1990. [2] R. L. Brinster, R. E. Braun, D. Lo, M. R. Avarbock, and F. Oram. Targeted correction of a major histocompatibility class 11 ea gene by DNA microinjected into mouse eggs. Proc. Natl. Acad. Sci., 86:7087——7091, 1989. [3] M. R. Capecchi. 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Disruption of the proto— oncogene int—2 in mouse embryo-derived stem cells: A general strategy for tar- getting mutations to non—selectable genes. Nature, 336:348-352, 1988. [23] D. A. Melton, P. A. Krieg, M. R. Rebaglia, T. Maniatis, K. Zinn, and M. R. Green. Efficient in vitro synmthesis of biologically active RNA and RNA hy- bridization probes from plasmids containing a bacteriophage SP6 promoter. Nu- cleic Acids Res, 12:7035-7056, 1984. [24] P. Mombaerts, A. R. Clarke, M. L. Hopper, and S. Tonegawa. Creation of a large genomic deletion at the T—cell antigen receptor fl—subuuit locus in mouse embryonic stem cells by gene targeting. Proc. Natl. Acad. Sci., 88:308-1-73087, 1991. [25] H. L. Mouellic, Y. Lallemand, and P. Brulet. Targeted replacement of the home- obox gene hour—3.1 by the escherichia coli lacz in mouse chimeric embryos. Proc. Natl. Acad. Sci., 87:4712—4716, 1990. 147 [26] D. Roth and J. Wilson. Genetic recombination, pages 621—653. Am. Soc. Micro- biol., Washinton, D. C., 1988. R. Kucheriapati and G. R. Smith ed. [27] R. Seif and F. Cuzin. Temperature-sensitive growth regulation in one type of transformed cells induced by the tsa mutant of polyomavirus. J. Virol, 24:721~ 728,1977. [28] E. G. Shesely, S Kim H, W. R. Shehee, and T. Papayannopoulou. Correction of a human fl‘—gene by gene targeting. Proc. ."Vall. Acad. Sci., 882429441298. May 1991. [29] O. Smithies, R. G. Gregg, S. S. Boggs, M.A. Koralewski, and R. S. Kucherlapati. Insertion of DNA sequences into the human chromosomal ,fi—globin locus by homologous recombination. Nature, 317:230—234, 1985. [30] L. Sompatrac and K. Danna. Efficient infection of monkey cells with DNA of simian virus 40. Proc. Natl. Acad. Sci., 78:7575—7578, 1981. [31] K. R. Thomas and M. R. Capecchi. Introduction of homologous DNA sequences into mammalian cells induces mytations in the cognate gene. :‘Vatuxrc, 321:34—38, 1986. [32] K. R. Thomas and M. R. Capecchi. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell, pages 503—512, 1987. [33] K. R. Thomas, K. R. Folger, and M. R. Capecchi. High frequency targeting of genes to specific sites in mammalin genome. Cell, 44:419—428, 1986. [34] A. Zimmer and P. Gruss. Production of chimaeric mice containing embryonic stem (ES) cells carring a homoeobox how [.1 allele mutated by homologous re- combination. Nature, 338:150——153, 1989. Chapter 5 A Gradient of Recombination on the Polyomavirus Genome We have analyzed the transformation frequency along the polyoma genome in 2 types of crosses in which we have previously described high levels of homologous recombi- nation were obtained: (1) in infections of normal FR3T3 cells, recombinant genome are recovered integrated at the cellular genome in 33 % of stably transformed cells and, (2) in infections of FRLT cells, integrated polyoma sequences were found in 3 ”/6 on the infected cells. Our results demonstrate that the frequency of recombination is nonuniform along a segment of the polyoma genome analyzed so far between nu~ cleotide 4634 in the late region and nucleotide 1387 in the middle of the early region, encompassing the enhancer and the origin. A gradient of recon’ibination was seen and, by overlapping results from different crosses, a minimum was observed in the en— hancer region with a continuous increase when moving towards the early region. The maximal recombination mapped so far in the early coding region between nucleotides 1245 and 1387 Show a 60—fold increase over the frequency observed in the enhancer region 2 Kb away. 148 149 5. 1 Introduction Our laboratory has previously demonstrated the occurrence of high levels of ho- mologous interviral recombination in infection of nonpermissive rat cells by poly- omavirus [8]. Recombination is not detected between unintegrated viral genomes, but among the integrated genomes which serve as the transforming sequences of stably transformed cells. Recombination is also detected albeit at lO—fold lower lev- els between unintegrated infection viral chromatin and preintegrated polyoma se— quences [5, 4]. The results suggest that viral genomes engaged in the integration process interact with a major host recombination pathway. Interestingly, the 2 phe— nomena appear to happen at the same time. Recombination with the host chro- mosome which is thought to be the mostly illegitimate recombination event occurs concommittant with homologous interviral recombination. In the present study, we have analyzed the transformation frequency along the polyoma genome in the 2 types of crosses described above. Our results demonstrate that the frequency of recombination is nonuniform along the part of the polyoma genome analyzed so far. A gradient of recombination was seen and, by overlapping results from different crosses, a minimum was observed in the enhancer region with a continuous increase when moving towards the early region. The maximal recombi- nation mapped so far, in the early coding region between nucleotide 1245 and 1387. show a 60—fold increase over the frequency observed in the enhancer region. 150 5.2 Materials and Methods Cells and virus: Fischer rat (FR3T3) [7] and FRLT [4] cells were used in the experiments. FRLT cells are FR3T3 which contains a stably integrated plasmid with the large T—antigen cDNA sequences in multiple copies as described in chapter 4 [4]. Cells were maintained in Dulbeco Modified Eagle medium (DR/IBM. Gibco) and supplemented with 10 % calf serum (Hazleton). Wild type polyoma A2 [11] was used as a control in the experiments. Two double mutants, Ma and 3a, harbor both the tsa temperature sensitive mutation in large T—antigen as well as mutations in the Aval and BamHI restriction endonuclease sites, respectively. Ma contains the MOP 1033 point mutations in the AvaI sites at nucleotides 659 and 1018, and the latter abolished the transforming ability of the virus due to a termination mutation in the middle T—antigen reading frame. 3a is temperature sensitive in decapsidation and has a point mutation at nucleotide 4634. 3a can transform normally if allowed to decapsidate at 33°C for 24 hrs [5]. D1 23 and d1 1015 are nontransforming mutants with deletion in the middle T—antigen/large T—antigen frame. D1 23 has a deletion between nucleotide 1121—1245 [6]; dl 1015 has a deletion between nucleotide 1246- 1275 [10]; 1387 T has a point mutation at nucleotide 1387 which caused a termination in the middle T—antigen [3]. Isolation of transformed cells: To maximize transformation, syncl‘ironized cells were used [5]. For this purpose, cells were grown to confluency. The following day, cells were transferred to low serum medium supplemented with 0.2 (70 garma-globulin free serum (Gibco) for 1 day. To release cells from G0, both trypsinization and adding 151 fresh DMEM supplemented with 10 ‘70 calf serum were used. The reattachment of the cells occurred within 3 hours. Infections were carried out at a multiplicity of infection (MOI) of 10 plaque forming units (pfu)/cell. Both focus formation in monolayer cell culture and colony formation in soft agar were used to identify the transformed cells as described previously. Estimation of the recombination frequency: Recombination was analyzed in the viral genome found integrated in the genome of stably transformed cells. In the cross between Ma and 3a in FR3T3 cells, no selection pressure for recombination associated with transformation was applied since the parent 3a can transform nor— mally after decapsidation at 33° for 24 hours. The occurrence of recombination was determined by digestion with a combination of AvaI, BamHI and Bglll. The cell lines containing recombinant viral genomes were further analyzed for the occurrence of recombination between the AvaleAval interval (nucleotide 1(l'18~7659), the small AvaI—BamHI interval (nucleotide 659—4634), and the long AvaI——l3amIII interval (nu- cleotide 1018—4634) (Figure 5.1). In the cross between the integrated polyoma sequences and the exogenous uninte- grated viral genomes, both viral genomes are noutransforming. In these experiments, individual transformants were derived from recombinants. W’e took the. transfor- mation frequency as a measurement for recombination frequency. The occurrence of recombination events in DNA intervals was analyzed, and the recon'ibination fre- quency was normalized by distances between the intron and the mutations of the exogenous mutants. Figure 5.1: Restriction endonuclease map of polyomavirus. Aval cleaves polyoma DNA twice at nucleotides 659 and 1018, Bgll cleaves polyon‘ia DNA once at nucleotide 89, and BamHI cleaves polyoma DNA once at nucleotide 4634. 153 Orl (89) 8911 (659) Aval BamHI (4634) (1818) AvaI ~ Figure 5.1: Restriction endonuclease map of polyomavirus. 154 Preparation and analysis of total DNA: Total DNA was extracted from trans- formed cells for the analysis of the occurrence of recombination. 10 lag of DNA was digested with a combination of AvaI, BamHI, and Bglll, electrophoresed on a 1 (70 agarose gel, transferred onto Hybond—N filter paper, and hybridized to a 32PHlabeled probe containing the entire polyoma DNA. Probes were prepared by multiprime la- beling kit (Amersham) to a specific activity of l—2 x 109 cpm/irg template DNA. 5.3 Results A gradient of recombination frequency in the cross of Ma and 3a: In chap- ter 3, we have described the recombination between Ma and 3a. in FR3T3 at 390C. There was no selection for recombination in the cross between Ma and 3a, because 3a can transform after decapsidation at 33°C for 24 hours. 65 transformants were picked from cells coinfected with Ma and 3a. 20 out of 65 transformed cell lines were found containing recombinant viral genomes. The occurrence of recombination events among these recombinant transformants was further studied among three DNA in- tervals: from the first to the second AvaI site (nucleotide 659—1018), the first Aval to the BglI site (nucleotide 659—89), the small AvaI—BamHI interval (nucleotide 659— 4634), and the long interval of the AvaI to the BamHI site (nucleotide 1018-4638). Digestions with a combination of AvaI, BamHI, and BglII were carried out for this analysis. If the recombination occurs within the AvaI—Aval interval, a 1.7 Kb frag- ment would be detected; if the recombination occurs within the small AvaI—BamHI interval, a 1.3 Kb fragment would be detected; or if the recombination occurs with 155 the long AvaI—BamHI interval, a 3.6 Kb fragment would be detected. In the digestion with a combination of AvaI, BamHI and Bglll, results showed that 7 out of 20 cell lines had the 1.7 Kb fragment, 15 had the 3.6 Kb fragment, and only 6 had the 1.3 Kb fragment. After normalization with size of DNA interval, a ratio of 4.7 : 1.1 : 1 was found in intervals of the 2 AvaI sites, the small AvaI—Baml'll interval, the large AvaI—BamHI interval. If the recombination occurs between the AvaI—Aval interval. a 1.7 Kb fragment will be observed; if the recombination occurs between the first AvaI—Bgll interval, a 1.3 Kb fragment as well as a 374 bp fragu‘urnt will be observed; or if the recombination occurred between the BglI—BamHI interval, fragments of 747 bp, 570 bp, and 374 bp will be observed. In order to normalize the distance effect on recombination frequency, the number of recombinants was divided by the length of the DNA interval. In the digestion with a combination of Aval, BamHI, and Bgll, we found that recombination occurred within the interval of two Aval sites in 5 cell lines, and only 1 cell line showed occurrence of recombination between Bgll—llaml-II interval by the fact that it has 744 bp, 570 bp, 374 bp fragments (Figure 5.2). Combining the results of the cross between MOP 1033 and ts3, carried out by D. Hacker. we found that 15 events, 9 events, and 5 events occurred between the two Aval sites, the A valeBglI interval, and the BglI—BamHI interval, respectively. After normalizing the incidence of recombination occurred by the size of each DNA interval, we found a 6.7 : 2.4 : 1 ratio of recombination frequency between the two AvaI sites, the A val—Bgll interval, and the BglI—BamHI interval (Table 5.1). Taking the above results together, we estimated the recombination frequency along 156 Table 5.1: Recombination occurred between Ma and 3a in the regimis of Aval-Aval, the AvaI—BglI interval and the BglI—BamHI interval. DNA interval AvaI—A vaI“ AvaI-Bgllb Bgl [_- BamH IC Size of the DNA intervals (bp) 359 570 748 Incidence of recombinationd 16 9 5 Incidence of recombination/bp 16/359 9/570 5/748 Ratio of recombination frequency 6.7 2.4 1 a. Between nucleotide 659 and 1018, b. Between nucleotide 659 and 89, c. Between nucleotide 89 and 4634, d. Recombination events occurred among 20 recombinant transfm'mants derived from a cross between Ma and 3a, and a cross between MOP 1033 and ts3. 157 polyoma genome and found a 40 : 20 : 6.7 : 2.4 : 1 ratio of recombination frequency in the intervals between nucleotide 1387—1245, 1245—1140, 1140795, 795—89, and 89—4634 (the BglI—BamHI interval) (Figure 5.4). Recombination between endogenous large T—antigen cDNA and exoge- nous viral genome: The other type of recombination studied was recombination between an integrated polyoma plasmid containing large T-antigen cDNA and ex- ogenous sequences, i.e., viral genomes introduced by infection. In these experiments, FRLT cells, which have the large T—antigen cDNA stably integrated into the cellular chromosomes, were infected by three transformation—defective viruses. These cells can become transformed only if recombination occurs between the integrated large T—antigen cDNA and the unintegrated viral genomes. FRLT cells infected with wild type polyoma A2 were used as positive control. The homologous recombination in these crosses was determined. The number of foci were 156 in 1387 T—infected FRLT cells, 47 in (11 1015—infected cells, and 9 in (II 23—infected cells. In A2—infected FRLT cells, the foci number was 109 (Table 5.2). For negative controls, FR3T3 were infected with 1387 T, (II 1015, and (“23 individually. Our results showed no transformation detected in these infected cells (Table 5.2). We normalized the relative recombination frequency by the distances between the intron of T—antigen DNA and the site of mutations in the viral genome. 'I‘hese distances are 347 bp for (11 23 (nucleotide 795-1140), 452 bp for (II 1015 (nudeotide 7954245), and 539 bp for 1387 T (nucleotide 7954387). Our results showed a 10 : 4 : 1 ratio of recombination frequency in the infection of FRLT cells with 1387 Figure 5.2: Analysis of the occurrence of recombination in the regions of Aval ~Aval. AvaI—Bgll, and BglI—BamHI. 10 pg of DNA extracted from cell lines 1, and 3—7 (same labeling as in Figure 3.2) was digested with a combination of AvaI, BamHI, and BglI, and analyzed as described in Material and Methods. As size markers, viral DNA of A2 strain was (.ligested with the same restriction endonucleases. (1018) (659) {89) (4634) AvaI Aval Bgll BamHI l m m I 1.7 U U L l m I 9.359 + 1.3 \J [ 9.359 1 9.57 . 8.748 [ Figure 5.2: Analysis of the occurrence of recombination in the regions of Aval—Aval, AvaI—BglI, and BglI—BamHI. 160 Table 5.2: Recombination frequency between the endogenous polyoma sequences and the exogenous transformation—defective polyomavirus. virus A2 1387 T dl 1015 dl 23 MOI 1 10 10 10 # of foci“ 109 I56 47 9 distance between intron and mutation sites (bp) ND" 593 452 3-17 # of foci/hp ND 150/593 17/432 9 /:5.17 ratio of recombination frequency ND 10 4 1 # of foci in infected FR3T3 cells 65 0 0 0 a. Total number for 8 parallel infections, b. Undetected, c. Total number for 2 parallel infections. 161 T, dl 1015, and d1 23 (Table 5.2). A subtraction suggests that 6 times more recombination events/per nucleotide could be assigned to interval between 1245-1387 than in the interval 795—1 1‘11) (Fig- ure 5.3). 5.4 Discussion We have compared recombination frequencies along the polyoma genome between the BamHI site located at nucleotide 4634 and I’lUClL‘OthlC 1387, an interval representing about 40 % of the viral genome, which encompasses some late coding scqu-FIH'C, the enhancer, the origin, and approximately half of the early region. These studies were carried out in two types of crosses using nonpermissive rat FR3T3 cells, a system in which we have previously reported elevated interviral recombination. In one case, infections were carried out with 2 parental genome marked with defects in specific restriction endonuclease sites which allow the assignment of the crossover site to a specific interval between 2 restriction redonuclease sites. In these experiments, there was no selection for recombination. Mixed infections were carried out and transformed cells were selected. Moreover, the viral genome integrated in these cells were screened for recombination in a 1.7 Kb interval spanning from the BamHI site to the AvaI site at 1018. Recombination was also scored in the corre- sponding 3.6 Kb AvaI—BamHI interval region were further analyzed for the presence of restriction redoclease sites within the 1.7 Kb interval and crossover sites were thus assigned to either of 3 intervals. 162 Figure 5.3: Recombination frequency derived in the crosses between the endogenous polyoma sequences and the exogenous viral genomes. The mutations of the endogenous polyoma sequences, or in the dl 23 and dl 1015 mutation are shown. Deletions are represented by doted lines, while a point mutation is represented by a simple dot. The hatched boxes show the distances involved in the 3 crosses between the intron mutation and the mutations in dl 23, d1 101.5, and 1387 T mutations. Deductively, a ratio of l : 3 : (i of recombination frequency was obtained for the three intervals 795—1140, 11110—1245, and 1245—1387, respectively. 163 distance ratio of 3:510:25 recombination 6 foci O LT-Ag ; § (bp) frequency GUM - .- N E E d1 23 m 347 l x 9 3 § d] 1915 m *5? ‘l x ‘7 a 1337 r W 593 1° x ‘55 Q If) f\ 8 :2 E 8 X Figure 5.3: Recombination frequency derived in the crosses between the endogenous polyoma sequences and the exogenous viral genomes. 1 64 Figure 5.4: A gradient of recombination frequency was detected in the regions between nucleotides 1387 and 4634 (the BamHI site). After chromosomal walking on the polyoma genome, a 40 : 20 : 6.7 : 2.4 : 1 ratio of recombination frequency was observed in the intervals between nucleotides 1387—1245, 1245—1140, 1140—795,795—89 and 89—4634 (the ligll—Baml—ll interval). 165 F i ure 5.4: A radient of recombination fre uencv was detected in the re ‘ions between . 8 nucleotide 1387 and 4634 (the BamHI site). 166 An analysis of 20 recombinants derived from 4 crosses showed that the majority of the recombination events had occurred in a short interval of 374 nucleotides (the small AvaI interval between nucleotide 6.59 and nucleotide 1018). Compz—u‘ison of the number of events in each interval normalized as recombination events per unit length revealed a 6—10 fold ratio in the Bgll—Bamlll interval which encompasses the origin- enhancer region. This increase in recombination in the small Aval interval compared to the BamHI—Bgll interval was observed in transformants derived from interaction with wild type large T—antigen [8], or those with a. temperature sensitive large T— antigen [8] carried out at 33°C as well as those. carried out at the nonpermissive temperature. In the other case, infections were carried out with a. cell line, derived from l‘ilt3'l‘3, carrying multiple copies of a plasmid with polyoma. sequences using transforination defective mutants with either point or deletion mutations. Recombination was se- lected for by selecting for transformation. Three infections were analyzed using 3 different mutants at the same multiplicity of infection and the transforn'iation fre- quencies in the 3 infections were compared to each other, and assumed to faithfully reflect recombination frequencies in the 3 overlapping intervals defined in these infec- tions. By subtraction, a relative recombination frequency for 3 adjacent intervals was obtained and revealed a gradient of recombination as well, that is increasing when moving away from the origin towards the 3‘end of the early region. Since the intervals studied in the 2 types of infection are overlapping, a case can be made for a continuous gradient of recombination increasing from a. low value in the enhancer region to high levels in the early region. The steepness of the gradient 167 (60—fold increase over a 2 Kb interval) revealed in these studies are very elevated compared to other systems. Because of the lack of markers in the other half of the genome in the present experiments, this region was not studied. However, it appears that elevated recombi- nation is also observed in that region. in that region. In most of the recombination events studied in the crosses between the Aval“ and the Bamlll‘ mutants. double recombinants were observed with a recombination event in the small Aval interval and a second one in the 3.6 Kb large AvaI—Bamlll interval. Furtl'n-‘r experimcmts with mutants marked in multiple regions will be required to map the, whole genome in a single cross. A site in the late region (between nucleotide 3092 and 32713) has been previously suggested to represent a hot spot of recon‘ibination [1, 2]. The fact that recombination appears to occur rarely in the enhancer region is somewhat surprising since the viral chromatin in this region is relatively devoid of nucleosome and it has been previously suggested that nucleosome free l).\';'\ (as that introduced in transfection experiments) is much more recombinogenic than chro- matin [16]. The existence of recombination gradients has been associated with the presence of a hot spot of recombination. The best studied system is the E. coli Chi system [12, 14, 15] which is encoded by the sequence 5’—GCTCGTGG—3’ [13] and which mediates generalized homologous recombination in E. Coli catalyzed by the enzymes of the Rec BCD pathway [9]. Whether a similar system is active here is of course a question quite far from reach at the present time. A few points are worth noting in this context are that the rate of homologous recombination undergone by the polyoma 168 viral chromatin are unusually high; recon'ibination is seen in as high as 530 % of transformation cells derived from experiments carried out at moderate multiplicities (10—50 plaque forming units/per cell). Experiments also suggest that only those viral molecules which are interacting with the host chromosome (those actually engaged in the process of integration) are undergoing recombination. Thus it is likely that these are in contact with the host recombination machinery. Interestingly, I'lorilioxnol(.)gous recombination between the viral and the host genome appears to oeenr simultainmusly with homologous recombination of ‘2 viral chromatin. The only viral protein which might be invoked in this process is large T—antigen. However, no strong case of the requirement of large T—antigen in this process is available to date. Thus this system should be a very helpful model system to study some of the parameters of homologous recombination in mammalian cells. [1] [31 [61 [101 [111 Bibliography P. Bourgaux, D. Gendron, and D. Bourgaux-Ramoisy. Preferred crossover sites on polyoma virus. J. Virol., 64:2327—2336, 1990. P. A. Bourgaux, B. S. Sylla, and P. Chartrand. Excision of polyoma virus DNA from that of a transformed mouse cell: identification of a hybrid molecule with direct and inverted repeat sequences at the \I'iral-«cellar joints. Virology, 1222:84— 97,1982. G. Carmichael, B. Schaffhausen, D. Dorsky, 1"). ()liver, and T. Benjamin. 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