The encapsidated genome of Microplitis demolitor bracovirus integrates into the host Pseudoplusia includens

Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism.     

Characterization and kinetic analysis of protein tyrosine phosphatase-H2 from Microplitis demolitor bracovirus

The polydnavirus Microplitis demolitor bracovirus (MdBV) encodes 13 genes that share homology with classical protein tyrosine phosphatases (PTPs). Prior sequence analysis suggested that five members of the MdBV PTP gene family (ptp-H2, -H3, -H5, -N1 and -N2) encode PTPs, seven family members encode pseudophosphatases, and one family member is a pseudogene. Prior experimental studies further implicated PTP-H2 in disabling the function of host hemocytes following infection by MdBV. Here we report expression of PTP-H2 and selected mutants in Escherichia coli cells as non-fusion or thioredoxin-fusion proteins. Following purification by nickel affinity chromatography, the full-length and mutant proteins ran as single bands of predicted size on SDS-PAGE gels under reducing conditions. The non-fusion form of PTP-H2 exhibited classical Michaelis–Menten kinetics using the phosphopeptide END(pY)INASL and difluoro-4-methylumbiliferyl phosphate (DiFMUP) as substrates. As expected, the non-fusion mutant PTP-H2^C236S had no enzymatic activity, while the thioredoxin-fusion form of PTP-H2 had low levels of activity. PTP-H2 exhibited optimal activity at pH 4.0 and 26 °C in sodium acetate buffer, and its activity was diminished by increasing buffer ionic strength. Activity was also greatly reduced by the presence of copper, heparin, and the classical PTP inhibitor vanadate. Using an anti-PTP-H2 antibody, immunoblotting and immunocytochemical studies only detected PTP-H2 in hemocytes from MdBV-infected Pseudoplusia includens. Overall, our results indicate that PTP-H2 is a functional tyrosine phosphatase that is specifically expressed in MdBV-infected hemocytes.     

Widespread Genome Reorganization of an Obligate Virus Mutualist

The family Polydnaviridae is of interest because it provides the best example of viruses that have evolved a mutualistic association with their animal hosts. Polydnaviruses in the genus Bracovirus are strictly associated with parasitoid wasps in the family Braconidae, and evolved ∼100 million years ago from a nudivirus. Each wasp species relies on its associated bracovirus to parasitize hosts, while each bracovirus relies on its wasp for vertical transmission. Prior studies establish that bracovirus genomes consist of proviral segments and nudivirus-like replication genes, but how these components are organized in the genomes of wasps is unknown. Here, we sequenced the genome of the wasp Microplitis demolitor to characterize the proviral genome of M. demolitor bracovirus (MdBV). Unlike nudiviruses, bracoviruses produce virions that package multiple circular, double-stranded DNAs. DNA segments packaged into MdBV virions resided in eight dispersed loci in the M. demolitor genome. Each proviral segment was bounded by homologous motifs that guide processing to form mature viral DNAs. Rapid evolution of proviral segments obscured homology between other bracovirus-carrying wasps and MdBV. However, some domains flanking MdBV proviral loci were shared with other species. All MdBV genes previously identified to encode proteins required for replication were identified. Some of these genes resided in a multigene cluster but others, including subunits of the RNA polymerase that transcribes structural genes and integrases that process proviral segments, were widely dispersed in the M. demolitor genome. Overall, our results indicate that genome dispersal is a key feature in the evolution of bracoviruses into mutualists.     

Establishment of a cell line from Chrysodeixis chalcites permissive for Chrysodeixis chalcites and Trichoplusia ni nucleopolyhedrovirus

A new cell line was established from the embryos of the insect Chrysodeixis chalcites (Lepidoptera, Noctuidae, Plusiinae). The cell line contains several morphologically different cell types and was distinguished from three other lepidopteran cell lines propagated in the laboratory by DNA amplification fingerprinting. The cultured cells, which we officially named WU-CcE-1 cells, were permissive for infection by C. chalcites nucleopolyhedrovirus (ChchNPV) and large numbers of occlusion bodies were produced that retained their infectivity for C. chalcites larvae. The CcE-1 cells were also permissive for Trichoplusia ni single nucleopolyhedrovirus (TnSNPV). ChchNPV could be passaged in these cells for at least four passages indicating that budded virus production was supported. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa armigera (Hear) NPV both induced apoptosis in these cells. The results obtained indicate that the CcE-1 cell line will be a useful tool in the study of both ChchNPV and TnSNPV.     

Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens

The braconid wasp Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of several noctuid moths. A key function of MdBV in parasitism is suppression of the host's cellular immune response. Prior studies in the host Pseudoplusia includens indicated that MdBV blocks encapsulation by preventing two types of hemocytes, plasmatocytes and granulocytes, from adhering to foreign targets. The other main immune response mediated by insect hemocytes is phagocytosis. The goal of this study was to determine which hemocyte types were phagocytic in P. includens and to assess whether MdBV infection affects this defense response. Using the bacterium Escherichia coli and inert polystyrene beads as targets, our results indicated that the professional phagocyte in P. includens is granulocytes. The phagocytic responses of granulocytes were very similar to those of High Five cells that prior studies have suggested are a granulocyte-like cell line. MdBV infection dose-dependently disrupted phagocytosis in both cell types by inhibiting adhesion of targets to the cell surface. The MdBV glc1.8 gene encodes a cell surface glycoprotein that had previously been implicated in disruption of adhesion and encapsulation responses by immune cells. Knockdown of glc1.8 expression by RNA interference (RNAi) during the current study rescued the ability of MdBV-infected High Five cells to phagocytize targets. Collectively, these results indicate that glc1.8 is a key virulence determinant in disruption of both adhesion and phagocytosis by insect immune cells.     

Systematic analysis of a wasp parasitism arsenal

Parasitoid wasps are among the most diverse insects on earth with many species causing major mortality in host populations. Parasitoids introduce a variety of factors into hosts to promote parasitism, including symbiotic viruses, venom, teratocytes and wasp larvae. Polydnavirus‐carrying wasps use viruses to globally suppress host immunity and prevent rejection of developing parasites. Although prior results provide detailed insights into the genes viruses deliver to hosts, little is known about other products. RNAseq and proteomics were used to characterize the proteins secreted by venom glands, teratocytes and larvae from Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). These data revealed that venom glands and teratocytes secrete large amounts of a small number of products relative to ovaries and larvae. Venom and teratocyte products exhibited almost no overlap with one another or MdBV genes, which suggested that M. demolitor effector molecules are functionally partitioned according to their source. This finding was well illustrated in the case of MdBV and teratocytes. Many viral proteins have immunosuppressive functions that include disruption of antimicrobial peptide production, yet this study showed that teratocytes express high levels of the antimicrobial peptide hymenoptaecin, which likely compensates for MdBV‐mediated immunosuppression. A second key finding was the prevalence of duplications among genes encoding venom and teratocyte molecules. Several of these gene families share similarities with proteins from other species, while also showing specificity of expression in venom glands or teratocytes. Overall, these results provide the first comprehensive analysis of the proteins a polydnavirus‐carrying wasp introduces into its host.     

Intrinsic interspecific competition between the polyembryonic parasitoid Copidosoma floridanum and solitary endoparasitoid Microplitis demolitor in Pseudoplusia includens

Multiparasitism studies were conducted in the laboratory between the egg-larval polyembryonic parasitoid Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae) and the larval endoparasitoid Microplitis demolitor Wilkinson (Hymenoptera: Braconidae) in the mutual host Pseudoplusia includens (Walker) (Lepidoptera: Noctuidae). Most multiparasitized hosts produced either a C. floridanum brood or died without any parasitoid emergence. Most multiparasitized hosts underwent supernumerary molting; however, multiparasitized hosts that produced a C. floridanum brood exhibited growth characteristics associated with C. floridanum parasitism while multiparasitized hosts that died exhibited growth characteristics more typical of M. demolitor parasitism. Multiparasitized hosts that produced a C. floridanum brood increased in weight, and usually exhibited the behavioral and morphological characteristics associated with initiation of metamorphosis. In contrast, multiparasitized hosts that ultimately died gained little weight and exhibited no characteristics associated with metamorphosis. M. demolitor progeny died in multiparasitized hosts as eggs or first instar larvae, but no direct evidence was found to indicate that physical attack by C. floridanum precocious larvae was responsible for their elimination.

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Résumé Lors de l'étude au laboratoire du multiparasitisme de Pseudoplusia includens Walker (Lépido. Noctuidae) par le parasitoïde ovolarvaire polyembryonnaire C. floridanum Ashmead (Hyméno. Encyrtidae) et l'endoparasitoïde larvaire M. demolitor Wilkinson (Hyméno. Braconidae), soit les hôtes donnent en majorité C. floridanum, soit ils meurent sans que quoi que ce soit émerge. P. includens non parasité présente 5 stades larvaires; les hôtes parasités par C. floridanum subissent 5 ou 6 stades avant le développement complet du parasitoïde, et les hôtes parasités par M. demolitor muent en stade 5 avant émergence du parasitoïde. Par contre, les hôtes multiparasités muent en stade 7; ceux qui produisent une couvée de C. floridanum ont présenté la croissance caractéristique des hôtes parasités par C. floridanum, tandis que les hôtes multiparasités qui sont morts avaient présenté la croissance caractéristique des hôtes parasités par M. demolitor. La plupart des hôtes multiparasités desquels C. floridanum a émergé ont augmenté de poids et présenté les caractères associés au début de la métamorphose. La descendance de M. demolitor est morte dans les hôtes multiparasités au stade uf ou chenille du premier stade, mais il n'y avait aucum indice d'attaque physique par des larves de C. floridanum qui auraient été responsables de leur élimination.

     

Intrinsic competition and its effects on the survival and development of three species of endoparasitoid wasps

In natural systems, pre‐adult stages of some insect herbivores are known to be attacked by several species of parasitoids. Under certain conditions, hosts may be simultaneously parasitized by more than one parasitoid species (= multiparasitism), even though only one parasitoid species can successfully develop in an individual host. Here, we compared development, survival, and intrinsic competitive interactions among three species of solitary larval endoparasitoids, Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae), Microplitis demolitor Wilkinson, and Microplitis croceipes (Cresson) (Hymenoptera: Braconidae), in singly parasitized and multiparasitized hosts. The three species differed in certain traits, such as in host usage strategies and adult body size. Campoletis sonorensis and M. demolitor survived equally well to eclosion in two host species that differed profoundly in size, Pseudoplusia includens (Walker) and the larger Heliothis virescens (Fabricius) (both Lepidoptera: Noctuidae). Egg‐to‐adult development time in C. sonorensis and M. demolitor also differed in the two hosts. Moreover, adult body mass in C. sonorensis (and not M. demolitor) was greater when developing in H. virescens larvae. We then monitored the outcome of competitive interactions in host larvae that were parasitized by one parasitoid species and subsequently multiparasitized by another species at various time intervals (0, 6, 24, and 48 h) after the initial parasitism. These experiments revealed that M. croceipes was generally a superior competitor to the other two species, whereas M. demolitor was the poorest competitor, with C. sonorensis being intermediate in this capacity. However, competition sometimes incurred fitness costs in M. croceipes and C. sonorensis, with longer development time and/or smaller adult mass observed in surviving wasps emerging from multiparasitized hosts. Our results suggest that rapid growth and large size relative to competitors of a similar age may be beneficial in aggressive intrinsic competition.     

Biosynthesis of plasma membrane components by SV40-virus-transformed 3T3 mouse cells temperature sensitive for expression of some transformed cell properties

We have studied the plasma membranes of an SV40-transformed 3T3 cell line temperature sensitive for the transformed growth phenotype (ts H6-15 cells), and have found that they vary little as a function of temperature of cultivation. Analysis by polyacrylamide gel electrophoresis was performed on plasma membranes prepared from ts H6-15 cell cultured at the permissive (32 °C) and non-permissive (39 °C) temperatures and radioactively-labelled in several ways. No significant differences were seen when the electrophoretic patterns of polypeptides of the plasma membranes of ts H6-15 cells, grown through 3–4 generations in medium containing radioactive leucine (32 °C and 39 °C temperatures) were compared. Plasma membranes derived from cells similarly grown in medium with radioactive glucosamine indicated that extensive alterations in the intrinsic glycopeptides occurred in association with alteration in growth phenotype. A shift towards decreased synthesis of large molecular weight (? 100 000–160 000) glycopeptides occurred in cells grown at the temperature of non-transformed growtn (39 °C). A decrease in amount of a 1200 000 molecular weight glycopeptide at 39 °C was the most prominent of these alterations.We have studied the surface exposure of polypeptides and glycopeptides of intact cells grown at 32 and 39 °C, using lactoperoxidase-catalyzed iodination, NaBH₄ reduction of galactose oxidase-treated cells, and metabolic-labelling with glucosamine of trypsin-sensitive molecules. We found no major qualitative differences between whole cell extracts or between plasma membrane preparations of cells cultivated at the permissive and non-permissive temperatures. Of special interest was the observation that the formation and surface exposure of a trypsin-sensitive, 240 000 molecular weight polypeptide appeared not to be ts in ts H6-15 cells. The significance of these observations will be discussed.     

Armed and accurate: engineering cytotoxic T cells for eradication of leukemia

After publication we discovered an error in the identification of the origin of the cell line reported in our article in BMC Biotechnology (2010, 10:50), entitled "Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins". Upon analysis of primary A. odorata cultures, we found that they were contaminated with cells of Trichoplusia ni origin. The origin of the Ao38 cell line was determined as T. ni using three marker genes and the Ao38 cell line was renamed BTI-Tnao38. References to the origin of the cell line as Ascalapha odorata should be replaced with "a cell line of Trichoplusia ni origin". The absence of TNCL virus detection in the BTI-Tnao38 (Ao38) cell line was confirmed using a highly sensitive RT-PCR protocol capable of detecting TNCL virus RNA at approximately 0.018 copies/cell. Because of these observations, we have revised the title of the original article to "Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins" and two additional authors were added to reflect their contributions to the analysis of this cell line.     

Metabolic Labeling of Caenorhabditis elegans Primary Embryonic Cells with Azido-Sugars as a Tool for Glycoprotein Discovery

Glycobiology research with Caenorhabditis elegans (C. elegans) has benefitted from the numerous genetic and cell biology tools available in this system. However, the lack of a cell line and the relative inaccessibility of C. elegans somatic cells in vivo have limited the biochemical approaches available in this model. Here we report that C. elegans primary embryonic cells in culture incorporate azido-sugar analogs of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), and that the labeled glycoproteins can be analyzed by mass spectrometry. By using this metabolic labeling approach, we have identified a set of novel C. elegans glycoprotein candidates, which include several mitochondrially-annotated proteins. This observation was unexpected given that mitochondrial glycoproteins have only rarely been reported, and it suggests that glycosylation of mitochondrially-annotated proteins might occur more frequently than previously thought. Using independent experimental strategies, we validated a subset of our glycoprotein candidates. These include a mitochondrial, atypical glycoprotein (ATP synthase α-subunit), a predicted glycoprotein (aspartyl protease, ASP-4), and a protein family with established glycosylation in other species (actin). Additionally, we observed a glycosylated isoform of ATP synthase α-subunit in bovine heart tissue and a primate cell line (COS-7). Overall, our finding that C. elegans primary embryonic cells are amenable to metabolic labeling demonstrates that biochemical studies in C. elegans are feasible, which opens the door to labeling C. elegans cells with other radioactive or azido-substrates and should enable the identification of additional post-translationally modified targets and analysis of the genes required for their modification using C. elegans mutant libraries.     

A Subclone of HuH-7 with Enhanced Intracellular Hepatitis C Virus Production and Evasion of Virus Related-Cell Cycle Arrest

Hepatitis C virus (HCV) cell culture system with JFH-1 strain and HuH-7 cells enabled us to produce infectious HCV particles in vitro, and such system is useful to explore the anti-HCV compounds and to develop the vaccine against HCV. In the present study, we describe the derivation of a cell line that permits improved production of HCV particles. Specifically, we characterized several subclones that were isolated from the original HuH-7 cell line by limiting dilution. These HuH-7 subclones displayed a notable range of HCV production levels following transfection by full-genome JFH-1 RNA. Among these subclones, HuH-7T1 produced HCV more efficiently than other subclones and Huh-7.5.1 that is known to be highly permissive for HCV replication. Upon transfection with full-genome RNA, HCV production was increased ten-fold in HuH-7T1 compared to Huh-7.5.1. This increase in viral production correlated with increased efficiency of intracellular infectious virus production. Furthermore, HCV replication did not induce cell cycle arrest in HuH-7T1, whereas it did in Huh-7.5.1. Consequently, the use of HuH-7T1 as host cells could provide increased population of HCV-positive cells and elevated viral titer. In conclusion, we isolated a HuH-7 subclone, HuH-7T1, that supports efficient HCV production. High efficiency of intracellular infectious virus production and evasion of cell cycle arrest were important for this phenotype. We expect that the use of this cell line will facilitate analysis of the underlying mechanisms for HCV particle assembly and the cell cycle arrest caused by HCV.     

Baculovirus infection influences host protein expression in two established insect cell lines

We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus-host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and semi-permissive to H. zea single nucleopolyhedrovirus (HzSNPV). Hz-AM1 cells are non-permissive to AcMNPV. We challenged each cell line with baculovirus infection and after 24 h determined protein identities by MALDI TOF/TOF mass spectrometry. For Hv-AM1 cells, 21 proteins were identified, and for Hz-AM1 cells, 19 proteins were newly identified (with 8 others having been previously identified). In the permissive relationship, 18 of the proteins changed in expression by 70% or more in AcMNPV infected Hv-AM1 cells as compared with non-infected controls; 12 were significantly decreased and 6 cellular proteins were significantly increased. We also identified 3 virus-specific proteins. In the semi-permissive infections, eight proteins decreased by 2-fold or more. Non-permissive interactions did not lead to substantial changes in host cell protein expression. We hypothesize that some of these proteins act in determining host cell specificity for baculoviruses.     

Propagation of Arthropod-Borne Rickettsia spp. in Two Mosquito Cell Lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Fate of Unintegrated Viral DNA in Fv-1 Permissive and Resistant Mouse Cells Infected with Murine Leukemia Virus

We have found that levels of unintegrated linear viral DNA were nearly identical in several Fv-1 resistant cell lines, whereas levels of closed circular viral DNA are markedly reduced in these resistant cells, to the same extent as virus production (P. Jolicoeur and E. Rassart, J. Virol. 33:183-195, 1980). To determine the fate of linear viral DNA made in resistant cells we performed pulse-chase experiments, labeling viral DNA with 5-bromodeoxyuridine and following it with a thymidine chase. 5-Bromodeoxyuridine-labeled viral DNA (HH) recovered by banding on cesium chloride gradients was sedimented on neutral sucrose density gradients or separated by the agarose gel-DNA transfer procedure and detected by hybridization with complementary DNA. Levels of linear viral DNA made in Fv-1^b/b (JLS-V9 and SIM.R) and Fv-1^n/n (NIH/3T3 and SIM) cells were found to decrease during the chase period at about the same rate in permissive and nonpermissive conditions, indicating that linear viral DNA is not specifically degraded in Fv-1 resistant cells. Levels of the two species of closed circular viral DNA made in Fv-1 permissive cells increased relative to the levels of linear DNA during the chase period. This confirmed the precursor-product relationship between linear DNA and the two species of circular DNA. In Fv-1 resistant cells, this apparent conversion of linear viral DNA into circular forms was not seen, and no supercoiled viral DNA could be detected. To determine whether the transport of linear viral DNA from the cytoplasm into the nucleus was prevented by the Fv-1 gene product, SIM.R cells were fractionated into cytoplasmic and nuclear fractions, and viral DNA was detected in each fraction by the agarose gel-DNA transfer procedure. Levels of linear viral DNA were nearly identical in both cytoplasmic and nuclear fractions of permissive or resistant cells. Circular viral DNA could be detected in the nuclear fraction of permissive cells, but not in that of resistant cells. A pulse-chase experiment was also performed with SIM.R cells. During the thymidine chase period, linear viral DNA was seen to accumulate in nuclei of both permissive and resistant cells, whereas supercoiled viral DNA accumulated only in nuclei of permissive cells. These results indicate that the Fv-1 gene product does not interfere with the transport of linear viral DNA into the nucleus. Our data also suggest that the Fv-1 restriction does not operate through a degradation process. Therefore, the Fv-1 gene product could either block the circularization of linear viral DNA directly or promote the synthesis of a faulty linear viral DNA whose defect (yet undetected) would prevent its circularization.     

Development of Oral Epithelial Cell Line ROE2 with Differentiation Potential from Transgenic Rats Harboring Temperature-Sensitive Simian Virus40 Large T-Antigen Gene

We have developed an immortalized oral epithelial cell line, ROE2, from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene. The cells grew continuously at either a permissive temperature of 33°C or an intermediate temperature of 37°C. At the nonpermissive temperature of 39°C, on the other hand, growth decreased significantly, and the Sub-G1 phase of the cell cycle increased, indicating that the cells undergo apoptosis at a nonpermissive temperature. Histological and immunocytochemical analyses revealed that ROE2 cells at 37°C had a stratified epithelial-like morphology and expressed cytokeratins Krt4 and Krt13, marker proteins for oral nonkeratinized epithelial cells. Global-scale comprehensive microarray analysis, coupled with bioinformatics tools, demonstrated a significant gene network that was obtained from the upregulated genes. The gene network contained 16 genes, including Cdkn1a, Fos, Krt13, and Prdm1, and was associated mainly with the biological process of skin development in the category of biological functions, organ development. These four genes were validated by quantitative real-time polymerase chain reaction, and the results were nearly consistent with the microarray data. It is therefore anticipated that this cell line will be useful as an in vitro model for studies such as physiological functions, as well as for gene expression in oral epithelial cells.