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The mitochondrial inner membrane contains a large protein complex that functions in inner membrane organization and formation of membrane contact sites. The complex was variably named the mitochondrial contact site complex, mitochondrial inner membrane organizing system, mitochondrial organizing structure, or Mitofilin/Fcj1 complex. To facilitate future studies, we propose to unify the nomenclature and term the complex “mitochondrial contact site and cristae organizing system” and its subunits Mic10 to Mic60.

Poly(ADP-ribose)polymerase-1 (PARP-1) is a predominantly nuclear enzyme that exerts numerous functions in cellular physiology and pathology, from maintenance of DNA stability to transcriptional regulation. Through a proteomic analysis of PARP-1 co-immunoprecipitation complexes, we identified Mitofilin, a mitochondrial protein, as a new PARP-1 interactor. This result prompted us to further investigate the presence and the role of the enzyme in mitochondria. Using laser confocal microscopy and Western blot analysis of purified mitochondria, we demonstrated the mitochondrial localization of a fraction of PARP-1. Further, the effects of overexpressing or down-regulating Mitofilin showed that this protein promotes and is required for PARP-1 mitochondrial localization. We also report several lines of evidence suggesting that intramitochondrial PARP-1 plays a role in mitochondrial DNA (mtDNA) damage signaling and/or repair. First, we show that PARP-1 binds to different regions throughout the mtDNA. Moreover, we demonstrated that the depletion of either PARP-1 or Mitofilin, which abrogates the mitochondrial localization of the enzyme, leads to the accumulation of mtDNA damage. Finally, we show that DNA ligase III, known to be required for mtDNA repair, participates in a PARP-1-containing complex bound to mtDNA. This work highlights a new environment for PARP-1, opening the possibility that at least some of the nuclear functions of the enzyme can be also extended to mtDNA metabolism.

Murine polyomavirus (Py) infection initiates by the recognition of cell membrane molecules containing terminal sialic acid (SA) residues through specific binding pockets formed at the major capsid protein VP1 surface. VP1 Pockets 1, 2, and 3 bind terminal SA, Gal, and second branched SA, respectively. The consequence of recognition on viral cell entry remains elusive. In this work, we show that preincubation of Py with soluble compounds within Pocket 1 (N-acetyl or N-glycolyl neuraminic acids) increases Py cell binding and infectivity in murine 3T6 fibroblasts. In contrast, Gal does not significantly alter Py binding nor infectivity, whereas sialyllactose, in Pockets 1 and 2, decreases cell binding and infectivity. Binding experiments with Py virus-like particles confirmed the direct involvement of VP1 in this effect. To determine whether such results could reflect VP1 conformational changes induced by SA binding, protease digestion assays were performed after pretreatment of Py or virus-like particles with soluble receptor fragments. Binding of SA with the VP1 Pocket 1, but not of compounds interacting with Pocket 2, was associated with a transition of this protein from a protease-sensitive to a protease-resistant state. This effect was transmitted to the minor capsid proteins VP2 and VP3 in virus particles. Attachment of Py to cell monolayers similarly led to a VP1 trypsin-resistant pattern. Taken together, these data present evidence that initial binding of Py to terminal SA induces conformational changes in the viral capsid, which may influence subsequent virus cell entry steps.

Murine polyomavirus (MPyV) infection occurs through recognition of sialic acid (SA) residues present on the host cell membrane, but the nature of the molecules involved and the exact role of this interaction in virus cell entry still need to be clarified. In this work, mutations at residues R(77) or H(298) of the MPyV VP1 protein were shown to lead to a complete loss of virus infectivity, which, however, could be restored by lipofection of virus particles into the cytoplasm of the host cells. Using virus-like particles (VLPs), it was demonstrated that the non-infectivity of these mutants was due to impaired cell entry caused by total abrogation of SA-dependent cell binding. This indicates that SA residues are essential primary cell receptors for MPyV. As the alpha4beta1 integrin has been identified recently as a cell receptor for MPyV, the relationship, if any, was investigated between SA-containing and alpha4beta1 integrin receptors. The ability of mutants R(77)Q and H(298)Q and wt VLPs to bind to cells overexpressing the alpha4beta1 integrin was studied in SA-positive (BALB/c 3T3 cells and Pro-5 cells) and SA-deficient (Pro5-derived Lec-2 cells) backgrounds. Overexpression of alpha4beta1 integrin did not restore binding of mutant VLPs in any of these cell lines or, indeed, that of wt VLPs in a SA-deficient background. Moreover, evidence is provided that overexpression of the sialylated alpha4beta1 integrin enhances wt VLP cell binding, suggesting that, in addition to its function at a post-attachment level, alpha4beta1 integrin acts also as one of the SA-containing receptors for initial cell binding.

The initial interaction of murine polyomavirus (Py) with host cells occurs through direct binding of the major capsid protein VP1 with cell membrane molecules containing terminal sialic acids; however, these Py receptor molecules have not yet been identified. Analysis of the capsid protein primary sequences of all murine strains revealed the presence of integrin ligand motifs in the DE and EF loops of VP1 (LDV and DLXXL, respectively) and at the N terminus of VP2 (DGE). We show that infectivity of the Py A2 strain in mouse Swiss 3T3 fibroblasts is significantly reduced only in the presence of natural integrin ligands carrying an LDV motif or antibodies directed against the α4 and β1 integrin subunits. Furthermore, we demonstrate that expression of the α4 subunit in the α4-deficient BALB/c 3T3 cells increases viral infectivity. Addition of α4 function-blocking antibodies, prior to or after virus adsorption, blocks this increased infectivity without affecting virus binding to cells. Taken together, these data indicate that expression of α4 integrin enhances permissivity to Py, probably by acting as one of the postattachment receptors.

A murine experimental model system aimed at developing potential vaccines to papovavirus infection in immunosuppressed individuals was explored. A VP1-pseudocapsid based on the major capsid protein of the murine polyomavirus A2 strain and a mutant, M17-pseudocapsid as well as four temperature sensitive (ts)-mutants were used as immunogens. T-cells deficient CD4-/-8-/- mice were immunized four times with each immunogen and then together with non-immunized control mice challenged with polyomavirus. In contrast to all control mice, only half of the immunized mice exhibited presence of polyoma DNA when assayed by PCR. The results indicate that pseudocapsids and ts-mutant immunization may potentially protect mice with an impaired T-cell function from polyomavirus infection.

Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated in vivo to a c-MYC promoter region recognized as biologically relevant for the transcriptional regulation of the gene. Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation.

Natural mutants of the DE loop of the Polyomavirus (Py) major coat protein VP1 have been previously shown to display an altered host specificity (L. Ricci, R. Maione, C. Passananti, A. Felsani, and P. Amati, 1992, J. Virol. 66, 7153–7158). To better understand the role of this outfacing loop of the VP1 protein in Py infectivity, we constructed and characterized a Py mutant (Py M17) harboring a deletion of 7 AA within the tip of the DE loop. The mutant virions obtained after DNA transfection were unable to replicate and initiate early transcription in fibroblast cells. Complementation experiments performed to rescue the deficient M17 replication by means of wt functions revealed the cis-dominance of the mutation. In situ cell fractionation experiments demonstrated that the Py mutant, like the Py wt, enters the cells, reaches the nucleus and that both the viral DNA and VP1 protein are found tightly bound to the nuclear matrix. These data suggest that the VP1 protein, associated to the viral DNA, conditions early viral gene expression and that the DE loop of the protein must be involved in this process.

As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way.To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65tpr-met) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65tpr-met highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand–receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65tpr-met kinase. These data indicate that a met kinase signal released from differentiation-dependent control provides a negative stimulus for the onset of myogenic differentiation.

To identify expression plasmids with high efficiency of transcription and with a broad tissue and cell range, we have constructed a recombinant vector combining the late SV40 promoter and the polyomavirus regulatory region derived from a mutant (PyNB11/1) which displays a very wide host range. We show that these recombinant enhancer-promoters are efficient drivers for heterologous gene transcription and expression in vitro in all mouse and human cells tested. The most active combination we identified contained the mutant enhancer (PyNB11/1) in the late orientation. This construct was able to promote a high efficiency of expression without significant fluctuation between cells of different tissutal origin or different differentiative stage. A possible interpretation of these results is discussed.

Polyomavirus mutants capable of replicating in undifferentiated murine C2 myoblasts were selected and characterized. These mutants grow normally in 3T6 mouse fibroblast cells, and they do not complement the wild-type virus in coinfection experiments of C2 myoblasts. Of 12 isolates, 10 possess duplications of the regulatory region including the enhancer A domain. On the bases of the regulatory region structure and the presence and length of the enhancer duplication, the mutant viruses could be grouped into three classes. One mutant class (e.g., PyMB3) possesses an enhancer duplication of 91 bp identical to that of a previously characterized polyomavirus mutant, PyNB11/1. We have demonstrated that this enhancer duplication gives rise at its junction to a novel recognition motif for the transcriptional factor NF-1 (M. Caruso, C. Iacobini, C. Passananti, A. Felsani, and P. Amati, EMBO J. 9:947-955, 1990). The regulatory region PyMB3 virus recombined in a wild-type genome context maintains the mutant phenotype. The other two types of mutants, one with a 30-bp enhancer duplication (e.g., PyMB40) and one with a wild-type enhancer structure (e.g., PyMB27), possess two similar but distinct 6-bp deletions in the same region of the VP1 coding gene. In both cases, the ability to replicate in undifferentiated C2 myoblasts is strictly correlated to the mutation in the VP1 coding region.

We have shown that nuclear and cytosolic proteins from embryonal carcinoma F9 cells are able to bind to the early-coding strand of polyoma enhancer A domain. As demonstrated by mobility shift specific competition experiments, DNase I footprinting, depurination and depyrimidation interference, and proteolytic clipping performed with single stranded oligonucleotides, some of these proteins bind specifically to the early-coding PEA1 (AP1) motif. In addition, 'Southwestern' analysis has made possible the identification of a 46 KD nuclear protein that binds to this sequence. These cellular proteins did not bind to the complementary single strand as demonstrated by mobility shift analysis, nor did they bind to RNA synthesized in vitro by using the complementary strand as template. They were also shown to be different from their corresponding double strand binding factors. This new dimension in the functional flexibility and complexity of the polyoma enhancer suggests new properties of the classic regulating sequences that could provide additional modulation of regulating activities.

Poly(ADP-ribosyl)ation, catalysed by a family of poly(ADP-ribose) polymerases (PARPs), plays an important role in a large variety of physiological processes, including cell proliferation, but its role in cell cycle progression is not yet completely defined. As reported here, the examination of early times following serum stimulation of quiescent fibroblasts suggests that poly(ADP-ribosyl)ation is necessary for the transition from the G0 phase to the G1 phase. We show that PARP activity is involved in this step through the regulation of immediate-early response genes, such as c-Fos and c-Myc. This is supported by the finding that exogenous Myc expression substantially restores cell cycle reactivation in the absence of polymer synthesis. Furthermore, using RNA interference, we show that PARP-1 is the PARP family member playing the most prominent role in the upregulation of c-Fos and c-Myc during G0–G1 transition. We report that even in lectin-stimulated peripheral blood mononucleated cells, the inhibition of PARP activity interferes with the upregulation of immediate-early genes and delays the induction of proliferation, suggesting a general role for PARP-1 in linking growth factor signaling with cell cycle entry.

Polyomavirus mutants selected for modified host range exhibit DNA sequence alterations in the regulatory region, which consist mainly of duplications and/or deletions. Single base pair mutations have also been observed, which create or abolish DNA sequence motifs recognized by DNA-binding regulatory factors. The present work deals with the molecular characterization of a Polyoma mutant (PyNB11/1), selected for its high efficiency of growth in neuroblastoma cells. The enhancer region of PyNB11/1 displays a 91 bp tandem duplication harbouring a novel DNA sequence motif created at the boundary of the duplicated fragment. This motif is absent in the wild-type enhancer and is specifically recognized by a nuclear factor that belongs to the NF-1 family of transcription factors. We also report the characterization of an as yet unidentified DNA sequence motif in the D domain of the viral enhancer, that represents the binding site for a nuclear factor that is ubiquitous and comparably abundant in several murine cell types.

The first contact of a virus with the host cell surface and further entry are important steps for a successful outcome of the infection process and for the virus-associated pathogenicity. We have previously shown that the entry of the murine Polyomavirus (Py) into fibroblasts is a multi-step process involving, at least, the attachment to primary sialic acids (SA)-containing cell receptors followed by post-binding interaction with secondary receptors, such as the alpha4beta1 integrin, likely through the VP1-LDV motif. Here we report on the functional role of the VP1-LDV motif in Py infectivity and in vivo virus tissue tropism. For this purpose, we have characterized a recombinant virus mutant, PyLNV, harboring a single aa substitution in this motif (D138N). Although not critical for virus viability, the D138N substitution abrogates the post-attachment Py-alpha4beta1 interaction, rendering the PyLNV mutant virus twofold less infectious than the Py wild-type (Wt) in alpha4beta1-positive fibroblasts. To study the putative role of the VP1-LDV motif in vivo, newborn C57BL/6 mice were inoculated with PyWt or PyLNV and, after six days, organs were analyzed for the presence of viral DNA. Intriguingly, PyLNV showed an altered spectrum of in vivo replication compared with PyWt, particularly in the skin and in the kidney. The implication of Py-alpha4beta1 integrin interaction in conditioning tissue-specificity of virus replication is discussed.

Poly(ADP-ribose)polymerases are involved in fundamental cellular events as well as they seem to be associated to some viral infection process. In this work, the poly(ADP-ribose)polymerase-1 (PARP-1) role in the polyomavirus life cycle has been investigated. Early viral transcription was reduced by competitive inhibitors of PARPs in Swiss 3T3 cells and almost abolished in PARP-1 knockout fibroblasts and in wild-type fibroblasts when PARP-1 was silenced by RNA interference. In vivo chromatin immunoprecipitation assays showed that poly(ADP-ribosyl)ation (poly(ADP-ribose)) facilitates the release of the capsid protein viral protein 1 (VP1) from the chromatin of infecting virions. In vitro experiments demonstrated that VP1 stimulates the enzymatic activity of PARP-1 and binds non-covalently both protein-free and PARP-1-bound poly(ADP-ribose). Our studies suggest that PARP-1 promotes the complete VP1 displacement from viral DNA favouring the viral early transcription.

In this work, we sought to determine whether the components of the murine polyomavirus capsid establish specific interactions with the minichromosome encapsidated into the mature viral particles by using the cis -diamminedichloroplatinum(II) cross-linking reagent. Our data indicated that VP1, but not minor capsid proteins, interacts with the viral genome in vivo. In addition, semiquantitative PCR assays performed on cross-linked DNA complexes revealed that VP1 binds to all regions of the viral genome but significantly more to the regulatory region. The implications of such an interaction for viral infectivity are discussed.

Medullary thyroid carcinoma (MTC) is a rare human tumor affecting the calcitonin-secreting c-cells of the thyroid. Here we report that two independent strains of transgenic mice expressing a Polyomavirus (Py) truncated middle-T antigen (deltaMT), consisting of the amino-terminal 304 amino acids, and the full length Py small-T antigen, developed multifocal bilateral MTCs with 100\% penetrance. Occasionally one strain also developed mammary and bone tumors. Furthermore, offspring from both transgenic lines displayed pronounced waviness of the whiskers and fur, previously associated with defective epidermal growth factor receptor signaling. Transgene transcription, driven by the homologous early promoter/enhancer, and the corresponding translation products were detected in tumors and in many other organs which did not develop pathologies. The subcellular distribution of deltaMT and its interactions with the adapter proteins of the SHC family have also been analysed. Our study describes a novel murine model of MTC and provides evidence that the N-terminal 304 amino acid fragment of Py middle-T antigen, possibly in co-operation with small-T antigen, acts as a potent oncogene in c-cells of the thyroid.

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.

Mouse fibroblasts were stably transfected with expression plasmids in which sequences of the early region of polyomavirus were inserted both in sense and antisense orientation. The cell lines that synthesize in the antisense orientation, a 1195-bp viral genome fragment covering the Ori, Cap, ATG, and all of the early mRNA splicing sites acquire resistance to viral infection. Smaller fragments covering Ori, Cap, and ATG sites or the splicing sites, as well as fragments cloned in sense orientation, failed to confer cell immunity to polyoma infection. The resistance proved to be directly dependent upon the specific antisense RNA and to be inversely proportional to the multiplicity of infecting polyoma.

The structural and biological properties of a polyomavirus mutant selected in Friend erythroleukemic cells were investigated. The growth efficiency of this mutant (PyFL78) was compared with that of the parental PyA2 strain by a growth competition assay in Friend erythroleukemic and 3T3 (or 3T6) cell lines. The results reveal that PyFL78 displays a cis-acting growth advantage over the PyA2 parental strain in Friend erythroleukemic cells but not in 3T3 or 3T6 cells. This cell-specific cis advantage is shown to be due to modifications within the polyomavirus noncoding regulatory region.

Polyomavirus reaches the nucleus in a still encapsidated form, and the viral genome is readily found in association with the nuclear matrix. This association is thought to be essential for viral replication. In order to identify the protein(s) involved in the virus-nuclear matrix interaction, we focused on the possible roles exerted by the multifunctional cellular nuclear matrix protein Yin Yang 1 (YY1) and by the viral major capsid protein VP1. In the present work we report on the in vivo association between YY1 and VP1. Using the yeast two-hybrid system we demonstrate that the VP1 and YY1 proteins physically interact through the D-E region of VP1 and the activation domain of YY1.

Chloramphenicol added at various times during intracellular development of various bacteriophages causes a reduction in the rate of synthesis of phage DNA. The reduction is faster the smaller the size of the DNA molecule in the mature phage.

The mRNAs for myogenic functions are coordinately transcribed with polyomavirus (Py) early mRNA during in vitro differentiation of mouse C2 myoblast cells. Sequence analysis shows that the A domain of the Py enhancer includes an E1A-like consensus sequence that is also found in the 5' upstream region of two genes expressed during myoblast differentiation: alpha-actin and myosin light chain. Therefore, the coordinate expression of such genes with Py early mRNA may be activated by a common cellular regulatory factor. In the present work, we report that C2 cells surviving Py infection are unable to differentiate and do not express alpha-actin and myosin light-chain mRNAs. Hybrids between such Py-resistant myoblast cells and the parental cells exhibited dominance of the permissibility to Py growth and of the expression of myogenic mRNAs. In C2 cells transiently transfected with a chimeric plasmid (pSVPy12CAT) harboring the bacterial chloramphenicol acetyltransferase (CAT) gene driven by the Py enhancer-promoter region, the CAT gene was expressed irrespective of their stage of differentiation. Moreover, undifferentiated stably transfected cells expressing the CAT gene restricted viral growth. Py-resistant C2 myoblasts transiently transfected with pSVPy12CAT also expressed the CAT gene driven by the Py enhancer. This contradictory finding is similar to results previously obtained by other investigators with cloned genes specific for myogenic functions, and it may be explained by a structural difference between the pSVPy12CAT and the Py genomic organizations in which the viral enhancer operates.

The noncoding regulatory region of polyoma (Py) virus regulates early and late mRNA transcription as well as DNA replication (Tyndall et al., NAR 9, 6231-6250, 1961). This segment of the viral genome also enhances the transcription of heterologous genes in chimeric plasmids (de Villiers and Schaffner, NAR 9,6251-6264, 1961) in a manner analogous to the SV40 enhancer and the LTR region of retroviruses (for review, see Khoury and Gruss, Cell 33, 313-314, 1963). The Py enhancer is of particular interest because sequence rearrangements in this region of the genome have been found frequently among “natural” viral strains (Ruley and Fried, J. Virol. 47 233-237, 1983). Moreover, the activity of the enhancer can be studied in the mouse, the most genetically well characterized mammal. Recently, much attention has been focused on the regulatory region of Py mutants that have a broader host range than wild type (wt) virus strains (such as those growing on embryonal carcinoma or trophoblast undifferentiated cells; Kelly and Condamine, BBA 657, 105-141, 1982; Tanaka et al., EMBO J. 7, 1521-1527, 1982); and Py mutants that grow at a higher rate on differentiated cells which further differentiate in vitro (such as neuroblastoma and Friend erythroleukemic cells; Maione et al., EMBO J. 4, in press; Delli Bovi et al., J. Virol. 49,566-571, 1984; De Simone et al., MCB 5, 2142-2146, 1985). Most of these mutants are characterized by rearrangements of the regulatory region. These rearrangements consist of either duplications or deletions that affect one or both of the two domains involved in the control of DNA replication and mRNA transcription, variously termed a and /I (Muller et al., J. Virol. 47, 586-599, 1983) or A and B (Herbomel et al., Cell 39, 653-662, 1984). The simplest wt Py strain, PyA2, carries a single copy of each of the two domains. Other naturally occurring Py strains (TOR, P16, and CSP) contain a duplication of the A domain, in addition to a few base pair changes (see figure). Sequence analysis of the regulatory region of Py mutants selected for their ability to grow in nonpermissive (undifferentiated) cells indicates that the rearrangements required for viral gene expression and replication in a new environment are host-specific and involve changes that are extensive in some cases and minor in others. The ability of Py mutants to grow on embryonal carcinoma PCC4 cells is always associated with extensive rearrangements (Vasseur et al., PNAS 77; 1068-1072, 1980; Katinka et al., Cell 20, 393-399, 1980; Melin et al., EMBO J. 4, 17991803,1985). Limited base alterations are sufficient for viral growth on the embryonal carcinoma F9 cell line, although Minireview

To determine the relative growth advantages of polyomavirus regulatory region mutants selected from Friend leukemic and neuroblastoma cells persistently infected with polyomavirus A2 wild type, different murine cell lines, some of which are capable of further differentiation in vitro, were used as hosts in mixed infection and transfection. The tests allowed the determination, through the measurement of cis-advantage in replication, of the most effective polyomavirus regulatory region constitutions for a given cell line and, in some cases, for specific stages of cell differentiation. Furthermore, different domains of the viral regulatory region were shown to have different effects--positive, neutral, or negative--depending on the host analyzed.

By using the zinc-finger region of human cHF.10 cDNA as a probe at low-stringency hybridization conditions, several individual phages from a mouse skeletal muscle cDNA library have been isolated. The amino acid sequences of the "zinc-finger" domains derived from the DNA sequences of three cDNA clones are shown. The expression of the corresponding mRNAs in three cell lines (NIH 3T3, F9 teratocarcinoma, and C2 myoblast cells) at different stages of differentiation and in eight adult mouse tissues has been analyzed. The transcription of these genes is induced during the in vitro differentiation of the cell lines tested. These three genes are widely and evenly expressed in adult mouse tissues, with the remarkable exception of one that is expressed predominantly in the testis.

Two mouse neuroblastoma cell lines were analyzed for their permissivity for polyoma virus growth. One (N18) is fully permissive for polyoma replication, the other (41A3) shows limited permissivity and the viral genome persists, without noticeable cell death. Virus persistence does not seem to alter the cells' ability to differentiate in vitro and leads to selection of viral mutants altered in the untranscribed regulatory region of the genome. The mutant types obtained appear to be related to the degree of host cell differentiation. Nucleotide sequence analysis of the restriction fragment covering the regulatory region shows that duplications are present in all mutants, while deletions in the non-duplicated segment are only present in mutants selected from less differentiated cells. These alterations involve both domains of the regulatory region that are considered to be essential for DNA replication and for enhancer activity. Mixed infections with polyoma wild type show that the selected mutants have cis-advantage in replication in neuroblastoma cells and not in 3T6 cells. Mutants carrying the deletion in the non-duplicated segment of the enhancer show a selective advantage in replication over the undeleted one in mixed infection. This advantage is much stronger in neuroblastoma cells in suspension (less-differentiated stage) than in monolayer cells (more-differentiated stage). An interpretation of the overall structure of the regulatory enhancer region, based on the observed differences between the mutants selected at different stages of differentiation in neuroblastoma and previously described mutants selected in undifferentiated cells, is discussed.