A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development.

Although alterations in the p53 tumor suppressor gene are detected frequently in human breast cancers, mammary tumors are observed infrequently in p53(null) mice. This has led to the suggestion that absence of p53 alone is not sufficient for induction of mammary tumors. However, early death of p53(null) mice from thymic lymphomas may obscure tumor phenotypes that would develop later. Therefore, p53(null) mammary epithelium was transplanted into cleared mammary fat pads of wild type p53 BALB/c hosts to allow long-term analysis of mammary tumor phenotypes. Five treatments were compared for their effects on tumor incidence in hosts bearing transplants of p53(null) and p53wt mammary epithelium. The treatment groups were: (1) untreated; (2) continuous hormone stimulation with pituitary isografts; (3) multiple pregnancies; (4) DMBA alone; and (5) DMBA+pituitary isografts. The tumor incidences in p53(null) vs p53wt mammary transplants for each treatment group were 62% vs 0%, 100% vs 0%, 68% vs 0%, 60% vs 4% and 91% vs 14%, respectively. The mammary tumors that developed in the p53(null) mammary epithelium were all adenocarcinomas and were frequently aneuploid. These data demonstrate that the absence of p53 is sufficient to cause development of mammary tumors and that hormonal stimulation enhances the tumorigenicity of p53(null) mammary epithelium to a greater extent than DMBA exposure alone. This model provides an in situ approach to examine the molecular basis for the role of p53 in the regulation of mammary tumorigenesis.

Coronavirus polyprotein processing.

MHV gene 1 contains two ORFs in different reading frames. Translation proceeds through ORF 1a into ORF 1b via a translational frame-shift. ORF 1a potentially encodes three protease activities, two papain-like activities and one poliovirus 3C-like activity. Of the three predicted activities, only the more amino terminal papain-like domain has been demonstrated to have protease activity. ORF 1a polypeptides have been detected in infected cells by the use of antibodies. The order of polypeptides encoded from the 5' end of the ORF is p28, p65, p290. p290 is processed into p240 and p50. Processing of ORF1a polypeptides differs during cell free translation of genome RNA and in infected cells, suggesting that different proteases may be active under different conditions. Two RNA negative mutants of MHV-A59 express greatly reduced amounts of p28 and p65 at the non-permissive temperature. These mutants may have defects in one or more viral protease activities. ORF 1b, highly conserved between MHV and IBV, potentially contains polymerase, helicase and zinc finger domains. None of these activities have yet been demonstrated. ORF 1b polypeptides have yet been detected in infected cells.

Characterization of the leader papain-like protease of MHV-A59.

Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteases encoded within the first open reading frame of the replicase gene. The more 5' of these domains, the leader papain-like protease, is responsible for the cleavage of the amino terminal protein, p28. We have defined the core of this protease to between amino acids 1075 and 1344 from the beginning of ORF 1a. Deletion analysis coupled with in vitro expression, was used to study p28 cleavage by this leader protease. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels in some of them. Reduced p28 production resulting from a 0.4 kb deletion positioned between p28 and the protease domain suggests an involvement of this region in catalytic processing. Some mutants display cleavage patterns indicative of a second cleavage site. Interestingly, this newly identified cleavage site maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59 infected cells. Mutagenesis of the catalytic H1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like protease encoded in ORF 1a.

Identification and analysis of MHV-A59 P28 cleavage site.

During translation of Murine hepatitis virus (MHV-A59) ORF1a, p28, the N-terminal polypeptide is cleaved from the growing polypeptide chain. Amino terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly247 and Val248. Site directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg246 (P2) and Gly247 (P1) nearly eliminated cleavage of p28. Single amino acid substitutions of other residues between P7 and P2' were generally permissive for cleavage although a few changes did greatly reduce proteolysis. The amino acids around the p28 cleavage site represent a new sequence recognized by a virus encoded papain-like proteinase.

Intracellular localization of polypeptides encoded in mouse hepatitis virus open reading frame 1A.

We have investigated the intracellular localization of several of the proteolytic cleavage products derived from the 5' portion of mouse hepatitis virus (MHV) gene 1. Antisera UP1 recognizes the N-terminal ORF1a cleavage product p28. Immunofluorescent staining of cells with this antisera resulted in a diffuse punctate pattern of cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm. Immunofluorescent staining of infected cells with antisera which recognize polypeptides p240 and p290 stained discrete vesicular perinuclear structures suggesting that these proteins localized to the Golgi. This was confirmed by double immunofluorescent staining of BHK cells expressing the MHV receptor (BHK-R) with a Golgi specific antibody in addition to our anti-MHV ORF1a antibodies. Antisera UP102 recognizes p28 and the immediately downstream p65 gene product. Double immunofluorescent staining of MHV infected BHK-R cells with UP102 labeled discrete vesicular structures overlapping the Golgi complex. In addition there was punctate staining more widely distributed in the cytoplasm. The simplest explanation for this pattern is that p65 is also localized to the Golgi region of the cell, whereas p28 is more widespread. Plasmids containing the first 4.7 and 6.75 kb of ORF 1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Images obtained by immunofluorescent staining of transfectants with our anti-ORF1a antisera are similar to those obtained during infection with A59. These studies indicate that the signals which direct p290 to the Golgi are likely contained between the C-terminus of p28 and ORF1a residue 1494.

Enhancer-activated plasmid transcription complexes contain constrained supercoiling.

It has been proposed that transcriptionally active chromatin contains totally unconstrained supercoiling. The results of recent studies have raised the possibility that this topological state is the property of highly transcribed genes. Since the transcription rate of RNA polymerase II genes can be dramatically increased by the presence of an enhancer, we have determined if the transcription complex of an enhancer-activated plasmid contains totally unconstrained supercoils. Following transfection into COS7 cells, the topology of the transcription complex DNA was determined directly by agarose gel electrophoresis. We find that an enhancer-activated plasmid transcription complex is supercoiled, and these supercoils remain following topoisomerase I treatment. Thus the transcribing complexes contain constrained supercoils, and the level of supercoiling suggests a nucleosome-like organization. However, we cannot rule out the possibility that unconstrained supercoils exist in addition to these constrained supercoils in the transcription complex in the cell.

Characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain A59.

The 21.7-kb replicase locus of mouse hepatitis virus strain A59 (MHV-A59) encodes several putative functional domains, including three proteinase domains. Encoded closest to the 5' terminus of this locus is the first papain-like proteinase (PLP-1) (S. C. Baker et al., J. Virol. 67:6056-6063, 1993; H.-J. Lee et al., Virology 180:567-582, 1991). This cysteine proteinase is responsible for the in vitro cleavage of p28, a polypeptide that is also present in MHV-A59-infected cells. Cleavage at a second site was recently reported for this proteinase (P. J. Bonilla et al., Virology 209:489-497, 1995). This new cleavage site maps to the same region as the predicted site of the C terminus of p65, a viral polypeptide detected in infected cells. In this study, microsequencing analysis of the radiolabeled downstream cleavage product and deletion mutagenesis analysis were used to identify the scissile bond of the second cleavage site to between Ala832 and Gly833. The effects of mutations between the P5 and P2' positions on the processing at the second cleavage site were analyzed. Most substitutions at the P4, P3, P2, and P2' positions were permissive for cleavage. With the exceptions of a conservative P1 mutation, Ala832Gly, and a conservative P5 mutation, Arg828Lys, substitutions at the P5, P1, and P1' positions severely diminished second-site proteolysis. Mutants in which the p28 cleavage site (Gly247 / Val248) was replaced by the Ala832 / Gly833 cleavage site and vice versa were found to retain processing activity. Contrary to previous reports, we determined that the PLP-1 has the ability to process in trans at either the p28 site or both cleavage sites, depending on the choice of substrate. The results from this study suggest a greater role by the PLP-1 in the processing of the replicase locus in vivo.

Identification of the murine coronavirus p28 cleavage site.

Mouse hepatitis virus strain A59 encodes a papain-like cysteine proteinase (PLP-1) that, during translation of ORF1a, cleaves p28 from the amino terminus of the growing polypeptide chain. In order to determine the amino acid sequences surrounding the p28 cleavage site, the first 4.6 kb of murine hepatitis virus strain A59 ORF1a was expressed in a cell-free transcription-translation system. Amino-terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly-247 and Val-248. Site-directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg-246 (P2) and Gly-247 (P1) nearly eliminated cleavage of p28. Single-amino-acid substitutions of other residues between P7 and P2' were generally permissive for cleavage, although a few changes did greatly reduce proteolysis. The relationship between the p28 cleavage site and other viral and cellular papain proteinase cleavage sites is discussed.

Mouse hepatitis virus strain A59 RNA polymerase gene ORF 1a: heterogeneity among MHV strains.

Gene 1, the putative RNA replicase gene of coronaviruses, is expressed via two large overlapping open reading frames (ORF 1a and ORF 1b). We have determined the nucleotide sequence of ORF 1a, encoded within the first 13.7 kb of gene 1, for the coronavirus mouse hepatitis virus strain A59 (MHV-A59). Putative papain-like protease domains, a picornavirus 3C-like protease domain, two hydrophobic domains, and a domain "X" of unknown function, previously identified in other coronaviruses (1-3), are also present in ORF 1a of MHV-A59. Comparison between the ORF 1a sequence of MHV-A59 and the published sequence of the JHM strain of MHV (2) showed a high degree of similarity with the exception of several short regions. We sequenced one region of MHV-JHM that contained an 18 amino acid insertion relative to A59 and four other regions in which the sequences of the two strains differed. The MHV-2 and MHV-3 strains were also sequenced in some of these regions. Our analysis confirmed the presence of only one heterogeneous region in ORF 1a of MHV-A59 and MHV-JHM which is also present in MHV-2. Our findings indicate the need to modify the published sequence of MHV-JHM.

Characterization of the leader papain-like proteinase of MHV-A59: identification of a new in vitro cleavage site.

Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteinases encoded within the first open reading frame (ORF 1a) of the replicase gene. The more 5' of these domains, the leader papain-like proteinase, is responsible for the cleavage of the amino terminal protein, p28. The core of this proteinase domain was defined to between amino acids 1084 and 1316 from the beginning of ORF 1a. Through the use of deletion analysis coupled with in vitro expression, we studied the role of the coding region between p28 and the leader papain-like proteinase on the cleavage of p28 itself. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels. Reduced p28 production resulting from a 0.4-kb deletion positioned between p28 and the proteinase domain suggests an involvement of this region in catalytic processing. Some mutants displayed cleavage patterns indicative of a second cleavage site. Interestingly, this new cleavage site identified in vitro maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59-infected cells. Mutagenesis of the catalytic His1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like proteinase encoded in ORF 1a.

Efficient autoproteolytic processing of the MHV-A59 3C-like proteinase from the flanking hydrophobic domains requires membranes.

The replicase gene of the coronavirus MHV-A59 encodes a serine-like proteinase similar to the 3C proteinases of picornaviruses. This proteinase domain is flanked on both sides by hydrophobic, potentially membrane-spanning, regions. Cell-free expression of a plasmid encoding only the 3C-like proteinase (3CLpro) resulted in the synthesis of a 29-kDa protein that was specifically recognized by an antibody directed against the carboxy-terminal region of the proteinase. A protein of identical mobility was detected in MHV-A59-infected cell lysates. In vitro expression of a plasmid encoding the 3CLpro and portions of the two flanking hydrophobic regions resulted in inefficient processing of the 29-kDa protein. However, the efficiency of this processing event was enhanced by the addition of canine pancreatic microsomes to the translation reaction, or removal of one of the flanking hydrophobic domains. Proteolysis was inhibited in the presence of N-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indicating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhance the trans processing of a precursor containing the inactive proteinase domain and both hydrophobic regions by a recombinant 3CLpro expressed from Escherichia coli. Membrane association assays demonstrated that the 29-kDa 3CLpro was present in the soluble fraction of the reticulocyte lysates, while polypeptides containing the hydrophobic domains associated with the membrane pelletes. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the proteolytic product containing the amino-terminal hydrophobic domain.

Localization of mouse hepatitis virus open reading frame 1A derived proteins.

We have investigated the intracellular localization of proteolytic cleavage products encoded in the 5' portion of mouse hepatitis virus (MHV) gene 1. Immunofluorescent labeling of cells with an antiserum which recognizes p28, the ORF1a N-terminal cleavage product, resulted in widespread somewhat granular cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm of MHV-infected, but not control uninfected cells. Immunofluorescent staining of infected cells with antisera which recognize the downstream polypeptides, p65, p240 and p290 labeled discrete vesicular perinuclear structures. Double immunofluorescent labeling of BHK cells expressing the MHV receptor (BHK(MHVR1)) and infected with MHV-A59 with a Golgi-specific anti-mannosidase II monoclonal antibody and with antiserum recognizing each of these anti-MHV ORF1a polypeptides, showed that the p240 and p290 polypeptides were localized in discrete vesicular structures that overlapped the Golgi complex. Labeling with antibodies specific for p65 colocalized with the Golgi region, and showed staining of the perinuclear cytoplasm as well. Plasmids containing sequences contained in the first 6.75 kb of ORF1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Immunofluorescent labeling of transfectants with the anti-ORF1a antisera showed patterns of antigen distribution similar to those observed in cells infected with MHV-A59. A deletion analysis with constructs containing only portions of the ORF1a sequence indicated that 303 amino acids containing the first papain-like protease domain (PLP-1) was sufficient to associate this protein with the Golgi.

Radiation-induced tumorigenesis in preneoplastic mouse mammary glands in vivo: significance of p53 status and apoptosis.

In mouse mammary tumorigenesis, p53 mutations facilitate tumorigenesis in concert with other oncogenic alterations. Ionizing radiation enhances tumorigenesis in preneoplastic mammary outgrowth lines and induces p53-dependent apoptosis. We asked if normal p53 function modulates radiation-induced tumorigenesis in preneoplastic mammary lesions by affecting the apoptotic pathway of cell deletion. Three different hyperplastic outgrowth lines were compared. Outgrowth line D1 overexpressed wild-type p53 and responded to irradiation with enhanced tumorigenicity but no induction of apoptosis. Outgrowth line TM12 exhibited normal wild-type p53 expression and responded to irradiation with no alteration in tumorigenicity but with a marked increase in apoptosis. Outgrowth line TM2L also exhibited normal wild-type p53 expression and responded to irradiation with a marked enhancement in both tumorigenicity and apoptosis. These results indicate that the two radiation-induced responses, apoptosis and tumorigenesis, are dissociable events in the mammary gland. Furthermore, radiation-induced tumorigenicity was not abrogated by either enhanced wild-type p53 expression or a robust apoptotic response. The radiation dose of 5 Gy most likely induces multiple genetic alterations in surviving cells, including genomic instability, and this may account for the tumorigenicity. Future experiments will examine lower doses of irradiation that still induce a significant apoptotic response but significantly less genomic instability.