Global activation of oncogenic pathways underlies therapy resistance in diffuse midline glioma.

Diffuse midline gliomas (DMGs) are aggressive pediatric brain tumors with dismal prognosis due to therapy-resistant tumor growth and invasion. We performed the first integrated histologic/genomic/proteomic analysis of 21 foci from three pontine DMG cases with supratentorial dissemination. Histone H3.3-K27M was the driver mutation, usually at high variant allele fraction due to recurrent chromosome 1q copy number gain, in combination with germline variants in ATM, FANCM and MYCN genes. Both previously reported and novel recurrent copy number variations and somatic pathogenic mutations in chromatin remodeling, DNA damage response and PI3K/MAPK growth pathways were variably detected, either in multiple or isolated foci. Proteomic analysis showed global upregulation of histone H3, lack of H3-K27 trimethylation, and further impairment of polycomb repressive complex 2 by ASXL1 downregulation. Activation of oncogenic pathways resulted from combined upregulation of N-MYC, SOX2, p65/p50 NF-κB and STAT3 transcription factors, EGFR, FGFR2, PDGFRα/ß receptor tyrosine kinases, and downregulation of PHLPP1/2, PTEN and p16/INK4A tumor suppressors. Upregulation of SMAD4, PAI-1, CD44, and c-SRC in multiple foci most likely contributed to invasiveness. This integrated comprehensive analysis revealed a complex spatiotemporal evolution in diffuse intrisic pontine glioma, recommending pontine and cerebellar biopsies for accurate populational genetic characterization, and delineated common signaling pathways and potential therapeutic targets. It also revealed an unsuspected activation of a multitude of oncogenic pathways, including cancer cell reprogramming, explaining the resistance of DMG to current therapies.

NHERF1/EBP50 controls lactation by establishing basal membrane polarity complexes with prolactin receptor.

The development of the lactating mammary gland is a complex multifactorial process occurring in mammals during pregnancy. We show here that this process requires NHERF1/EBP50 (Na/H exchanger regulatory factor 1/ERM-binding phosphoprotein 50) expression and that successful lactation depends on NHERF1 allele copy number, with rates of 50 and 20% in NHERF1(+/-) and (-/-) mice, respectively. The prolactin receptor (PRLR)-STAT5 signaling provides the central axis triggering the differentiation of secretory mammary alveolar cells. In successfully lactating glands, NHERF1 is massively upregulated and forms complexes with PRLR, but also with ß-catenin, E-cadherin and ezrin at the alveolar basal membrane, establishing basal polarity. In NHERF1-deficient glands, the basal polarity is disrupted, the PRLR levels and basal membrane localization are abolished, and the downstream STAT5 activation collapses with consequent reduction of milk protein synthesis. NHERF1/EBP50, a protein deregulated in breast cancer, thus emerges as an important physiological mediator of milk secretion, by engagement of PRLR in multimeric complexes at the alveolar basal membrane with subsequent network activation leading to cell differentiation.

PTEN, NHERF1 and PHLPP form a tumor suppressor network that is disabled in glioblastoma.

The phosphatidylinositol-3-OH kinase (PI3K)-Akt pathway is activated in cancer by genetic or epigenetic events and efforts are under way to develop targeted therapies. phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is the major brake of the pathway and a common target for inactivation in glioblastoma, one of the most aggressive and therapy-resistant cancers. To achieve potent inhibition of the PI3K-Akt pathway in glioblastoma, we need to understand its mechanism of activation by investigating the interplay between its regulators. We show here that PTEN modulates the PI3K-Akt pathway in glioblastoma within a tumor suppressor network that includes Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) and pleckstrin-homology domain leucine-rich repeat protein phosphatases 1 (PHLPP1). The NHERF1 adaptor, previously characterized by our group as a PTEN ligand and regulator, shows also PTEN-independent Akt-modulating effects that led us to identify the PHLPP1/PHLPP2 Akt phosphatases as NHERF1 ligands. NHERF1 interacts via its PDZ domains with PHLPP1/PHLPP2 and scaffolds heterotrimeric complexes with PTEN. Functionally, PHLPP1 requires NHERF1 for membrane localization and growth-suppressive effects. PHLPP1 loss boosts Akt phosphorylation only in PTEN-negative cells and cooperates with PTEN loss for tumor growth. In a panel of low-grade and high-grade glioma patient samples, we show for the first time a significant disruption of all three members of the PTEN-NHERF1-PHLPP1 tumor suppressor network in high-grade tumors, correlating with Akt activation and patient's abysmal survival. We thus propose a PTEN-NHERF1-PHLPP PI3K-Akt pathway inhibitory network that relies on molecular interactions and can undergo parallel synergistic hits in glioblastoma.

Autophagy-inducing agents augment the antitumor effect of telerase-selve oncolytic adenovirus OBP-405 on glioblastoma cells.

Oncolytic adenoviruses are a promising tool in cancer therapy. In this study, we characterized the role of autophagy in oncolytic adenovirus-induced therapeutic effects. OBP-405, an oncolytic adenovirus regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad, OBP-301) with a tropism modification (RGD) exhibited a strong antitumor effect on glioblastoma cells. When autophagy was inhibited pharmacologically, the cytotoxicity of OBP-405 was attenuated. In addition, autophagy-deficient Atg5(-/-) mouse embryonic fibroblasts (MEFs) were less sensitive than wild-type MEFs to OBP-405. These findings indicate that OBP-405-induced autophagy is a cell killing effect. Moreover, autophagy-inducing therapies (temozolomide and rapamycin) synergistically sensitized tumor cells to OBP-405 by stimulating the autophagic pathway without altering OBP-405 replication. Mice harboring intracranial tumors treated with OBP-405 and temozolomide survived significantly longer than those treated with temozolomide alone, and mice treated with OBP-405 and the rapamycin analog RAD001 survived significantly longer than those treated with RAD001 alone. The observation that autophagy inducers increase OBP-405 antitumor activity suggests a novel strategy for treating patients with glioblastoma.

Cortical stabilization of beta-catenin contributes to NHERF1/EBP50 tumor suppressor function.

Anchorage-independent growth is a hallmark of tumor growth and results from enhanced proliferation and altered cell-cell and cell-matrix interactions. By using gene-deficient mouse embryonic fibroblasts (MEFs), we showed for the first time that NHERF1/EBP50 (Na/H exchanger regulator factor 1/ezrin-radixin-moesin binding phosphoprotein 50), an adapter protein with membrane localization under physiological conditions, inhibits cell motility and is required to suppress anchorage-independent growth. Both NHERF1 PDZ domains are necessary for the tumor suppressor effect. NHERF1 associates directly through the PDZ2 domain with beta-catenin and is required for beta-catenin localization at the cell-cell junctions in MEFs. Mechanistically, the absence of NHERF1 selectively decreased the interaction of beta-catenin with E-cadherin, but not with N-cadherin. The ensuing disorganization of E-cadherin-mediated adherens junctions as well as the observed moderate increase in beta-catenin transcriptional activity contributed most likely to the anchorage-independent growth of NHERF1-deficient MEFs. In vivo, NHERF1 is specifically localized at the apical brush-border membrane in intestinal epithelial cells and is required to maintain a fraction of the cortical beta-catenin at this level. Thus, NHERF1 emerges as a cofactor essential for the integrity of epithelial tissues by maintaining the proper localization and complex assembly of beta-catenin.

Crk family adaptor proteins trans-activate c-Abl kinase.

BACKGROUND: c-Abl kinase is activated in response to a variety of biological stimuli. Crk family adaptor proteins can interact physically with c-Abl and be involved in the activation of c-Abl kinase. RESULTS: We report that the Crk family of adaptor proteins act as trans-acting activators of c-Abl kinase. The interaction of the amino-terminal Src-homology (SH) 3 domain of c-Crk and the proline-rich motifs of c-Abl is an essential step for the phosphorylation of c-Crk by c-Abl, as well as the activation of c-Abl by c-Crk. The activation of c-Abl by c-Crk is negatively regulated by phosphorylation of the tyrosine 221 of c-Crk. Our data suggest that, in the absence of phosphorylation of the tyrosine Y221, the SH2 domain of c-Crk becomes free to bind to target molecules while the carboxyl-terminal SH3 domain of c-Crk binds to the proline-rich region of c-Abl, inducing the activation of c-Abl by c-Crk. CONCLUSION: This study suggests that the Crk family functions as trans-acting activators of c-Abl kinase. The phosphorylation of c-Crk may regulate c-Abl kinase.

The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction.

CMS/CD2AP is a cytoplasmic protein critical for the integrity of the kidney glomerular filtration and the T cell function. CMS contains domains and motifs characteristic for protein-protein interactions, and it is involved in the regulation of the actin cytoskeleton. We report here that the individual SH3 domains of CMS bind to phosphotyrosine proteins of approximately 80, 90, and 180 kDa in cell lysates stimulated with epidermal growth factor. The second SH3 domain of CMS bound specifically to a tyrosine-phosphorylated protein of 120 kDa, which we identified as the proto-oncoprotein c-Cbl. The c-Cbl-binding site for CMS mapped to the carboxyl terminus of c-Cbl and is different from the proline-rich region known to bind SH3-containing proteins. CMS binding to c-Cbl was markedly attenuated in a tyrosine phosphorylation-defective c-Cbl mutant indicating that this interaction is dependent on the tyrosine phosphorylation of CMS. It also implies that CMS interacts with c-Cbl in an inducible fashion upon stimulation of a variety of cell-surface receptors. Immunofluorescence analysis revealed that both proteins colocalize at lamellipodia and leading edges of cells, and we propose that the interaction of CMS with c-Cbl offers a mechanism by which c-Cbl associates and regulates the actin cytoskeleton.

The kinase-deficient Src acts as a suppressor of the Abl kinase for Cbl phosphorylation.

The kinase activity of Abl is known to be regulated by a putative trans-acting inhibitor molecule interacting with the Src homology (SH) 3 domain of Abl. Here we report that the kinase-deficient Src (SrcKD) directly inhibits the tyrosine phosphorylation of Cbl and other cellular proteins by Abl. We found that both the SH2 and SH3 domains of SrcKD are necessary for the suppressor activity toward the Abl kinase phosphorylating Cbl. To suppress the Cbl phosphorylation by Abl, the interaction between the SH3 domain of SrcKD and Cbl is required. This interaction between SrcKD and Cbl is regulated by a closed structure of Cbl. The binding of Abl to the extreme carboxyl-terminal region of Cbl unmasks the binding site of SrcKD to Cbl. This results in a ternary complex that inhibits the Abl-mediated phosphorylation of Cbl by steric hindrance. These results illustrate a mechanism by which the enzymatically inactive Src can exert a biological function in vivo.

Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics.

Dysregulated signal transduction from receptor tyrosine kinases to phosphatidylinositol 3-kinase (PI3K), AKT (protein kinase B), and its effector FKBP-rapamycin-associated protein (FRAP) occurs via autocrine stimulation or inactivation of the tumor suppressor PTEN in many cancers. Here we demonstrate that in human prostate cancer cells, basal-, growth factor-, and mitogen-induced expression of hypoxia-inducible factor 1 (HIF-1) alpha, the regulated subunit of the transcription factor HIF-1, is blocked by LY294002 and rapamycin, inhibitors of PI3K and FRAP, respectively. HIF-1-dependent gene transcription is blocked by dominant-negative AKT or PI3K and by wild-type PTEN, whereas transcription is stimulated by constitutively active AKT or dominant-negative PTEN. LY294002 and rapamycin also inhibit growth factor- and mitogen-induced secretion of vascular endothelial growth factor, the product of a known HIF-1 target gene, thus linking the PI3K/PTEN/AKT/FRAP pathway, HIF-1, and tumor angiogenesis. These data indicate that pharmacological agents that target PI3K, AKT, or FRAP in tumor cells inhibit HIF-1alpha expression and that such inhibition may contribute to therapeutic efficacy.

Stabilization and productive positioning roles of the C2 domain of PTEN tumor suppressor.

PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancer. It acts as a phosphoinositide phosphatase and consists of an amino-terminal phosphatase domain tightly linked to a COOH-terminal C2 domain involved in lipid membrane-binding. We investigated the functions of the C2 domain and their relevance for tumor growth. To discriminate between PTEN C2 domain ability to recruit or to position the active site to the membrane, we artificially membrane-targeted PTEN by a myristoylation signal. This modification increased wild-type PTEN growth inhibition but did not rescue a C2 mutant defective in lipid-binding, suggesting a model in which PTEN C2 domain positions the active site productively with respect to the membrane-bound phosphoinositide substrate. When tumor-derived mutations in the loops that connect the C2 beta-strands were analyzed, we found that these generally destabilized the protein but had variable effects on the phosphatase activity and tumor growth. The magnitude of these effects was dependent on the presence of the COOH-terminal PEST sequences and on the cell type where the mutant proteins were expressed, suggesting the existence of fluctuating structural defects of the mutant protein. One of the C2 loop mutants induced a total loss of PTEN tumor-suppressor function, most likely by affecting both the membrane binding and the protein stability. These data support a double role for PTEN C2 domain in protein stability and in productive orientation of the catalytic site.

Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association.

The PTEN tumor suppressor is mutated in diverse human cancers and in hereditary cancer predisposition syndromes. PTEN is a phosphatase that can act on both polypeptide and phosphoinositide substrates in vitro. The PTEN structure reveals a phosphatase domain that is similar to protein phosphatases but has an enlarged active site important for the accommodation of the phosphoinositide substrate. The structure also reveals that PTEN has a C2 domain. The PTEN C2 domain binds phospholipid membranes in vitro, and mutation of basic residues that could mediate this reduces PTEN's membrane affinity and its ability to suppress the growth of glioblastoma tumor cells. The phosphatase and C2 domains associate across an extensive interface, suggesting that the C2 domain may serve to productively position the catalytic domain on the membrane.

The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region.

PTEN is a recently identified tumor suppressor inactivated in a variety of cancers such as glioblastoma and endometrial and prostate carcinoma. It contains an amino-terminal phosphatase domain and acts as a phosphatidylinositol 3,4,5-trisphosphate phosphatase antagonizing the activity of the phosphatidylinositol 3-OH kinase. PTEN also contains a carboxyl-terminal domain, and we addressed the role of this region that, analogous to the amino-terminal phosphatase domain, is the target of many mutations identified in tumors. Expression of carboxyl-terminal mutants in PTEN-deficient glioblastoma cells permitted the anchorage-independent growth of the cells that otherwise was suppressed by wild-type PTEN. The stability of these mutants in cells was reduced because of rapid degradation. Although the carboxyl-terminal region contains regulatory PEST sequences and a PDZ-binding motif, these specific elements were dispensable for the tumor-suppressor function. The study of carboxyl-terminal point mutations affecting the stability of PTEN revealed that these were located in strongly predicted beta-strands. Surprisingly, the phosphatase activity of these mutants was affected in correlation with the degree of disruption of these structural elements. We conclude that the carboxyl-terminal region is essential for regulating PTEN stability and enzymatic activity and that mutations in this region are responsible for the reversion of the tumor-suppressor phenotype. We also propose that the molecular conformational changes induced by these mutations constitute the mechanism for PTEN inactivation.

CMS: an adapter molecule involved in cytoskeletal rearrangements.

Cas ligand with multiple Src homology (SH) 3 domains (CMS) is an ubiquitously expressed signal transduction molecule that interacts with the focal adhesion protein p130(Cas). CMS contains three SH3 in its NH2 terminus and proline-rich sequences in its center region. The latter sequences mediate the binding to the SH3 domains of p130(Cas), Src-family kinases, p85 subunit of phosphatidylinositol 3-kinase, and Grb2. The COOH-terminal region contains putative actin binding sites and a coiled-coil domain that mediates homodimerization of CMS. CMS is a cytoplasmic protein that colocalizes with F-actin and p130(Cas) to membrane ruffles and leading edges of cells. Ectopic expression of CMS in COS-7 cells resulted in alteration in arrangement of the actin cytoskeleton. We observed a diffuse distribution of actin in small dots and less actin fiber formation. Altogether, these features suggest that CMS functions as a scaffolding molecule with a specialized role in regulation of the actin cytoskeleton.

Biological effects of c-Mer receptor tyrosine kinase in hematopoietic cells depend on the Grb2 binding site in the receptor and activation of NF-kappaB.

The c-Mer receptor tyrosine kinase (RTK) is most closely related to chicken c-Eyk and belongs to the Axl RTK subfamily. Although not detected in normal lymphocytes, c-Mer is expressed in B- and T-cell leukemia cell lines, suggesting an association with lymphoid malignancies. To gain an understanding of the role of this receptor in lymphoid cells, we expressed in murine interleukin-3 (IL-3)-dependent Ba/F3 pro-B-lymphocyte cells a constitutively active receptor, CDMer, formed from the CD8 extracellular domain and the c-Mer intracellular domain. Cells transfected with a plasmid encoding the CDMer receptor became IL-3 independent. When tyrosine (Y)-to-phenylalanine (F) mutations were introduced into c-Mer, only the Y867 change significantly reduced the IL-3-independent cell proliferation. The Y867 residue in the CDMer receptor mediated the binding of Grb2, which recruited the p85 phosphatidylinositol 3-kinase (PI 3-kinase). Despite the difference in promotion of proliferation, both the CDMer and mutant F867 receptors activated Erk in transfected cells. On the other hand, we found that both transcriptional activation of NF-kappaB and activation of PI 3-kinase were significantly suppressed with the F867 mutant receptor, suggesting that the activation of antiapoptotic pathways is the major mechanism for the observed phenotypic difference. Consistent with this notion, apoptosis induced by IL-3 withdrawal was strongly prevented by CDMer but not by the F867 mutant receptor.

Direct binding of p130(Cas) to the guanine nucleotide exchange factor C3G.

p130(Cas) (Cas; crk-associated substrate) belongs to a new family of docking molecules. It contains one Src homology (SH) 3 domain in its amino-terminal region followed by a region containing binding motifs for SH2 and SH3 domains. To gain further insight into Cas signaling we used the SH3 domain of Cas in a two-hybrid screen to search a human placenta library for binding partners. The screen confirmed a previous finding of its binding to the focal adhesion kinase (FAK) but also identified C3G, a guanine nucleotide exchange factor. We found direct interaction between Cas and C3G in vitro and in vivo. A series of analysis with C3G deletion mutants revealed a proline-rich Cas-binding site (Ala0-Pro1-Pro2-Lys3-Pro4-Pro5-Leu6-Pro7) located NH2-terminal to the previously characterized Crk binding motifs in C3G. Mutagenesis studies showed that Pro1, Lys3, and Pro4 within the ligand-binding site are critical for high affinity interaction. These results, combined with sequence alignments of proline-rich binding elements from proteins known for Cas binding, define the consensus sequence XXPXKPX which is recognized by the CasSH3 domain. Cas shows structural characteristics of a docking molecule and may serve to bring C3G to specific compartments within the cell.

Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis.

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and in rare cases cause vaccine-associated paralytic poliomyelitis (VAPP). Reversion of vaccine strains toward a pathogenic phenotype is probably one of the main causes of VAPP, a disease most frequently associated with type 3 and type 2 strains and more rarely with the type 1 (Sabin 1) strain. To identify the determinants and mechanisms of safety versus pathogenicity of the Sabin 1 strain, we characterized the genetic and phenotypic changes in six Sabin 1-derived viruses isolated from immunocompetent patients with VAPP. The genomes of these strains carried either few or numerous mutations from the original Sabin 1 genome. As assessed in transgenic mice carrying the human poliovirus receptor (PVR-Tg mice), all but one strain had lost the attenuated phenotype. Four strains presented only a moderate neurovirulent phenotype, probably due at least in part to reversions to the wild-type genotype, which were detected in the 5' noncoding region of the genome. The reversions found in most strains at nucleotide position 480, are known to be associated with an increase in neurovirulence. The construction and characterization of Sabin 1 mutants implicated a reversion at position 189, found in one strain, in the phenotypic change. The presence of 71 mutations in one neurovirulent strain suggests that a vaccine-derived strain can survive for a long time in humans. Surprisingly, none of the strains analyzed were as neurovirulent to PVR-Tg mice as was the wild-type parent of Sabin 1 (Mahoney) or a previously identified neurovirulent Sabin 1 mutant selected at a high temperature in cultured cells. Thus, in the human gut, the Sabin 1 strain does not necessarily evolve toward the genetic characteristics and high neuropathogenicity of its wild-type parent.

Random selection: a model for poliovirus infection of the central nervous system.

Mixed infections occur in the natural environment, and also result from the use of mixed live vaccines. Some recipients of the trivalent oral poliovirus vaccine develop vaccine-associated paralytic poliomyelitis (VAPP). Numerous serotypes and recombinant genotypes of vaccine-derived polioviruses may be found in stool samples from such cases. To investigate the relationship between the multiplication of various genotypes at the primary replication site in the gut and the infection outcome in the central nervous system (CNS), the viruses excreted on consecutive days by two patients with VAPP were compared with the viruses isolated from the CNS. The genotypes from stools were numerous and varied with time in both cases, suggesting a multiplication of the viruses in multiple foci in the gut. Where the CNS isolated virus clearly corresponded to one of the many viruses detected in stool, this virus was unexpectedly less neurovirulent than others isolated from stool. To assess the mechanism by which viruses with different degrees of neurovirulence are selected in the CNS, transgenic mice sensitive to poliovirus infection were inoculated extraneurally with mixtures of two phenotypically different viruses at different neuropathogenic doses. The virus(es) inducing neurological disease was then isolated from the CNS. At less than 100% input neuropathogenic dose of both inoculated viruses, individual mice were affected stochastically by the virus variants from the mixture. Extrapolated to humans, this selection pattern might explain the occurrence of CNS infections with less neurotropic viruses derived from an extraneural pool containing also highly neurotropic viruses.

The replacement of water with deuterium oxide significantly improves the thermal stability of the oral poliovirus vaccine.

The poliomyelitis eradication programme relies largely on the massive administration of the oral poliovirus vaccine (OPV). The major difficulty in assuring good vaccine coverage, especially in hot climates, is the thermostability of the vaccine. Several attempts have been made to stabilize the OPV with limited benefits. In this report, we describe a heavy water based stabilization procedure, which has been shown to increase the thermostability of the vaccine, notably at temperatures which are commonly encountered during usual transportation in conditions of cold chain failure. Safety considerations regarding the human use of heavy water containing bioproducts are discussed.

Tripartite genome organization of a natural type 2 vaccine/nonvaccine recombinant poliovirus.

Intertypic vaccine/vaccine recombinant polioviruses are frequently isolated from vaccine-associated paralytic poliomyelitis cases (VAPP). We identified a vaccine/nonvaccine poliovirus recombinant as the causative agent of a lethal VAPP. Partial RNA sequencing revealed a tripartite recombinant structure of the viral genome. This consisted of a central capsid core of vaccine origin flanked by two units of nonvaccine origin. The first nonvaccine genomic unit spanned the whole 5' noncoding region, and the second one almost the entire nonstructural protein-coding region and the 3' noncoding region. Amino acid and nucleotide sequence similarities in the 3' and 5' unidentified regions indicated that the viral donor(s) were poliovirus species, suggesting recombination between a vaccine-derived and a wild poliovirus. The nonvaccine donor(s) could not be identified among the investigated wild polioviruses cocirculating in the same geographical area. This is the first report of a natural recombination event occurring in the 5' genomic extremity of poliovirus. The neurovirulence for transgenic mice and the pathogenicity for humans of the recombinant suggested that the modular genomic organization of this virus might have conferred a selective advantage over its vaccine parent.

Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus.

The temperature-sensitive and attenuated phenotypes of the Sabin type 1 vaccine strain of poliovirus result from numerous point mutations which occurred in the virulent Mahoney virus parent. One of these mutations is located in a 3D polymerase (3Dpol) codon (U-6203-->C, Tyr-73-->His) and is involved in attenuation in common mice (M. Tardy-Panit, B. Blondel, A. Martin, F. Tekaia, F. Horaud, and F. Delpeyroux, J. Virol. 67:4630-4638, 1993). This mutation also appears to contribute to temperature sensitivity, in association with at least 1 other of the 10 mutations of the 3'-terminal part of the genome including the 3Dpol coding and 3' noncoding regions. To map the other mutation(s), we constructed poliovirus mutants by mutagenesis and recombination of Mahoney and Sabin 1 cDNAs. Characterization of these poliovirus mutants showed that a second mutation in a 3Dpol codon (C-7071-->U, Thr-362-->Ile) contributes to temperature sensitivity. A mutation in the 3' noncoding region of the genome (A-7441-->G), alone or linked to another mutation (U-7410-->C), also appeared to be involved in this phenotype. The temperature-sensitive effect associated with the 3'-terminal part of the Sabin 1 genome results from the cumulative and/or synergistic effects of at least three genetic determinants, i.e., the His-73 and Ile-362 codons of 3Dpol and nucleotide G-7441. Sequence analysis of strains isolated from patients with vaccine-associated paralytic poliomyelitis showed that these genetic determinants are selected against in vivo, although the Ile-362 codon appeared to be more stable than either the His-73 codon or G-7441. These genetic determinants may contribute to the safety of Sabin 1 in vaccines.