Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector.

We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34 degrees C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca++-resistant cells did not proliferate at the nonpermissive temperature (40 degrees C), indicating that they depended on T antigen for their proliferation. The temperature-sensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40 degrees C. The serum/Ca++-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca++-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that deltaE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.

Identification of a new element for RNA replication within the internal ribosome entry site of poliovirus RNA.

Several mutants of the Mahoney strain of poliovirus type 1 have been generated by introducing mutations into the stem-loop II (SLII) structure within the internal ribosomal entry site (IRES). Four of these mutants (SLII-1, -4, -5 and -6 mutants) have been characterized previously and are host-range mutants that replicate well in human HeLa cells but not in mouse cells. Two deletion mutants, SLII-2 and SLII-3, were non-viable, even in HeLa cells. It is now reported that SLII-2 was defective in genome RNA synthesis and viral protein synthesis, while SLII-3 was defective only in viral protein synthesis. These results indicate that the SLII region contains a cis-element for RNA replication as well as for IRES-dependent translation and that these two functions lie at the same sites within the SLII region. The host cellular factors that interacted with wild-type SLII and mutant SLII-2 and SLII-3 RNAs were different, suggesting that different host-factor binding regulates expression of mutant phenotypes.

Intracellular redistribution of truncated La protein produced by poliovirus 3Cpro-mediated cleavage.

The La autoantigen (also known as SS-B), a cellular RNA binding protein, may shuttle between the nucleus and cytoplasm, but it is mainly located in the nucleus. La protein is redistributed to the cytoplasm after poliovirus infection. An in vitro translation study demonstrated that La protein stimulated the internal initiation of poliovirus translation. In the present study, a part of the La protein was shown to be cleaved in poliovirus-infected HeLa cells, and this cleavage appeared to be mediated by poliovirus-specific protease 3C (3Cpro). Truncated La protein (dl-La) was produced in vitro from recombinant La protein by cleavage with purified 3Cpro at only one Gln358-Gly359 peptide bond in the 408-amino-acid (aa) sequence of La protein. The dl-La expressed in L cells was detected in the cytoplasm. However, green fluorescence protein linked to the C-terminal 50-aa sequence of La protein was localized in the nucleus, suggesting that this C-terminal region contributes to the steady-state nuclear localization of the intact La protein in uninfected cells. The dl-La retained the enhancing activity of translation initiation driven by poliovirus RNA in rabbit reticulocyte lysates. These results suggest that La protein is cleaved by 3Cpro in the course of poliovirus infection and that the dl-La is redistributed to the cytoplasm. dl-La, as well as La protein, may play a role in stimulating the internal initiation of poliovirus translation in the cytoplasm.

A new internal ribosomal entry site 5' boundary is required for poliovirus translation initiation in a mouse system.

Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1-8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.

Efficient delivery of circulating poliovirus to the central nervous system independently of poliovirus receptor.

The transgenic (Tg) mice carrying the human gene for poliovirus receptor (PVR) are susceptible to poliovirus intravenously (i.v.) inoculated as well as intracerebrally or intraspinally inoculated. Thus, i.v.-inoculated poliovirus may invade the central nervous system (CNS) through the blood-brain barrier (BBB). To know the contribution of PVR to tissue distribution and BBB permeability of i.v.-inoculated polioviruses, these dissemination processes were investigated and compared between the Tg mice and non-Tg mice. Distribution profile of i.v.-inoculated poliovirus in various tissues of the Tg mice is similar to that in non-Tg mice. The data suggest that tissue distribution of the virus occurs independently of the transgene for PVR. The amount of poliovirus delivered to the CNS suggested the existence of a specific delivery system of the virus to the CNS. Virus accumulation in the CNS of the Tg mice was measured up to 7.5 hr after the i.v. inoculation. The viruses, regardless of whether the virulent or attenuated strain, seem to accumulate at a constant rate of approximately 0.2 microliter/min/g tissue. Similar phenomena were observed when the viruses were inoculated into non-Tg mice. The rates of the virus accumulation in the CNS are more than 100 times higher than that of albumin, which is considered not to permeate through the BBB via a specific transport system, and only three times lower than that of monoclonal antibody against transferrin receptor (OX-26), which is a potential candidate as a drug delivery vehicle specific to the CNS. These data suggest that polioviruses permeate through the BBB at a fairly high rate, independently of PVR and virus strains.

Host range phenotype induced by mutations in the internal ribosomal entry site of poliovirus RNA.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.

A new cis-acting element for RNA replication within the 5' noncoding region of poliovirus type 1 RNA.

Mouse cells expressing the human poliovirus receptor (PVR-mouse cells) as well as human HeLa cells are susceptible to poliovirus type 1 Mahoney strains and produce a large amount of progeny virus at 37 degrees C. However, the virus yield is markedly reduced at 40 degrees C in PVR-mouse cells but not in HeLa cells. The reduction in virus yield at 40 degrees C appears to be due to a defective initiation process in positive-strand RNA synthesis (K. Shiroki, H. Kato, S. Koike, T. Odaka, and A. Nomoto, J. Virol. 67:3989-3996, 1993). To gain insight into the molecular mechanisms involved in this detective process, naturally occurring heat-resistant (Hr)-mutants which show normal growth ability in PVR-mouse cells even at 40 degrees C were isolated from a virus stock of the Mahoney strain and their mutation sites that affect the phenotype were identified. The key mutation was a change from adenine (A) to guanine (G) at nucleotide position (nt) 133 within the 5' noncoding region of the RNA. This mutation also gave an Hr phenotype to the viral plus-strand RNA synthesis in PVR-mouse cells. Mutant Mahoney strains with a single point mutation at nt 133 (A to G, C, or T or deletion) were investigated for their ability to grow in PVR-mouse cells at 40 degrees C. Only the mutant carrying G at nt 133 showed an Hr growth phenotype in PVR-mouse cells. These results suggest that a host cellular factor(s) interacts with an RNA segment around nt 133 of the plus-strand RNA or the corresponding region of the minus-strand RNA, contributing to efficiency of plus-strand RNA synthesis.

Transcription stimulation of the adenovirus type 12 E1a gene in vitro by the 266-amino-acid E1A protein.

We previously showed that the 13S but not the 12S mRNA product of the E1a gene of the highly oncogenic type 12 adenovirus (Ad12) stimulates the expression of its own gene. In this study, the mechanism for the autoregulation of the Ad12 E1a gene was investigated in vitro. The 266-amino-acid E1A protein of Ad12 was synthesized in yeast cells and purified as a 57-kDa polypeptide. The purified Ad12 E1A protein stimulated transcription from the proximal promoter of its own gene but had almost no effect on that from the distal promoter. A 35-bp upstream region including a TATA box for the proximal promoter seemed to be sufficient for transcription stimulation by the E1A protein. The Ad12 E1A protein formed a complex with a TATA box-binding protein (TBP), as does the E1A protein of nononcogenic Ad serotypes. Moreover, the E1A protein significantly reduced the binding of TBP to a TATA sequence, while it did not affect the DNA-binding activity of nuclear factor I, a stimulatory protein of the distal transcription of the Ad12 E1a gene. These results suggest that the 13S mRNA product of the Ad12 E1a gene regulates the transcription of its own gene by modulating the activity of TBP.

Negative regulation of the gene for H-2Kb class I antigen by adenovirus 12-E1A is mediated by a CAA repeated element.

Both positive and negative regulatory elements responsive to the product of the adenovirus type 12 E1A gene are located in the promoter region of the gene for mouse H-2Kbm1 major histocompatibility complex (MHC) class I antigen (1). We have analyzed the negative regulatory element that is affected by E1A and identified a target CAA repeated motif, CAA(A)CAAA, within -1725 to -1705 and -1591 to -1568 in a 316-bp sequence located in the far upstream region of H-2Kb promoter (-1837 to -1522; +1 refers to the cap site). The extent of cell surface expression of the MHC class 1 antigen was significantly decreased in the case of transfectants obtained by introducing an expression plasmid that included MHC class 1 cDNA with the CAA repeated element, as compared with that of a plasmid with mutated CAA repeats. We have also characterized the nuclear proteins that bind to this motif. The analysis of the effects of mutations during competition assays of in vivo and gel retardation competition assays demonstrated that the CAA repeated element is essential not only for E1A-dependent repression of transcription but also for the cell surface expression of the product of the mouse H-2Kb class I gene, presumably through nuclear proteins that specifically bind to it.

Temperature-sensitive mouse cell factors for strand-specific initiation of poliovirus RNA synthesis.

Two cell lines, TgSVA and TgSVB, were established from the kidneys of transgenic mice carrying the human gene encoding poliovirus receptor. The cells were highly susceptible to poliovirus infection, and a large amount of infectious particles was produced in the infected cells at 37 degrees C. However, the virus yield was greatly reduced at 40 degrees C. This phenomenon was common to all mouse cells tested. To identify the temperature-sensitive step(s) of the virus infection cycle, different steps of the infection cycle were examined for temperature sensitivity. The results strongly suggested that the growth restriction observed at 40 degrees C was due to reduced efficiency of the initiation process of virus-specific RNA synthesis. Furthermore, this restriction appeared to occur only on the synthesis of positive-strand RNA. Virus-specific RNA synthesis in crude replication complexes was not affected by the nonpermissive temperature of 40 degrees C. In vitro uridylylation of VPg seemed to be temperature sensitive only after prolonged incubation at 40 degrees C. These results indicate that a specific host factor(s) is involved in the efficient initiation process of positive-strand RNA synthesis of poliovirus and that the host factor(s) is temperature sensitive in TgSVA and TgSVB cells.

Adenovirus E1A proteins stimulate inositol phospholipid metabolism in PC12 cells.

To study the influence of nuclear oncogenes on inositol phospholipid metabolism, we examined the various parameters of inositol phospholipid metabolism in PC12 cells expressing adenovirus type 12 or adenovirus type 5 E1A. Although the inositol 1,4,5-trisphosphate content was increased only slightly, the diacylglycerol content was 2.4-fold higher in E1A-expressing PC12 cells. Furthermore, we found that the activity of phospholipase C, one of the key enzymes in inositol phospholipid metabolism, was increased at least five- to eightfold. Diacylglycerol kinase activity in the membrane fraction was 10 to 15% of that in parental PC12 cells. Overall protein kinase C activities in E1A-expressing PC12 cells were decreased, but the activity of membrane-bound protein kinase C was significantly increased. These observations clearly indicate that inositol phospholipid metabolism is stimulated in cells producing E1A and suggest that nuclear oncogene E1A has the ability to stimulate inositol phospholipid metabolism.

Activation of the mouse proliferating cell nuclear antigen gene promoter by adenovirus type 12 E1A proteins.

A plasmid carrying the 5'-flanking region (-1584 to +47 with respect to the transcription initiation site) of the mouse proliferating cell nuclear antigen (PCNA) gene was fused with the chloramphenicol acetyltransferase (CAT) gene, and then cotransfected into mouse N18TG2 cells with expression plasmids for the adenovirus type 12 E1 genes. Expression of E1A gene products elevated the CAT expression by 5- to 9-fold, but expression of the E1B gene product did not. RNase protection analysis revealed that the activation of the PCNA gene promoter by E1A was at the transcription step. Both the 13S E1A and the 12S E1A activated the PCNA gene promoter, indicating that the activation domain of E1A resides in a common region(s) of 13S and 12S E1A products. The major target region of E1A was mapped within the 68 base-pair region (-21 to +47) of the PCNA gene, which includes consensus sequences for transcription factors PEA3 and E2F, although the upstream region (-83 to -21) including ATF(CREB)-binding consensus had an additional effect in the transactivation.

Activation of the mouse DNA polymerase beta gene promoter by adenovirus type 12 E1A proteins.

A plasmid carrying the 5'-flanking region (-1852 to +33 with respect to the transcription initiation site) of the mouse DNA polymerase beta gene fused with the chloramphenicol acetyltransferase (CAT) gene was cotransfected into mouse N18TG2 cells with adenovirus type 12 E1 genes-expressing plasmids. Expression of E1A gene products resulted in the elevation of the CAT expression by 3 to 7 folds, but that of E1B gene product was much less effective. RNase protection analysis revealed that the activation by E1A was at the transcription process. Both the 13S E1A and the 12S E1A activated the DNA polymerase beta gene promoter, indicating that the activation domain of E1A is in a common region(s) of 13S and 12S E1A products. The major target sequence of E1A was mapped within the 10 base pair-region (-30 to -20) of the DNA polymerase beta gene promoter, which overlapped with the palindromic sequence known as the ATF(CREB)/E4F-binding consensus. The results suggest that the palindromic sequence is essential for E1A-induced transcriptional activation of the mouse DNA polymerase beta gene.

Highly efficient focus formation by Rous sarcoma virus on adenovirus type 12 E1A-transformed rat 3Y1 cells.

When rat 3Y1 cells were infected with Rous sarcoma virus (RSV) variant SR-RSV-D(H), many 3Y1 cells acquired a stable provirus but only few of them formed transformed foci. In contrast, 12E1AY cells (3Y1 cells expressing the adenovirus type 12 [Ad12] E1A protein) formed transformed foci upon RSV infection with the same high frequency as did chicken embryo fibroblast cells. This enhancement of focus-forming efficiency was specifically observed in 3Y1 cells expressing Ad12 E1A protein but was not observed in 3Y1 cells expressing simian virus 40 T, c-myc, p53, c-fos, or v-fos protein. This enhancement was not evident in 5E1AY cells (3Y1 cells expressing the Ad5 E1A protein). Judging from the experiment using Ad12-Ad5 hybird E1A DNAs, the N-terminal half of the Ad12 E1A protein was responsible for this enhancement. The promoter activity of the RSV long terminal repeat measured by pLTR-CAT did not correlate to the efficiency of focus formation by RSV in these 3Y1 cells. Moreover, RSV containing the neo gene instead of the src gene produced G418-resistant cells equally efficiently among 3Y1, E1AY, and chicken embryo fibroblast cells. These results suggest that the enhancement of focus formation by RSV is not due to the increased expression of the src gene by the E1A protein. src mRNA and src protein were lower in RSV-transformed E1AY (RSVE1AY) cells than in RSV-transformed 3Y1 (RSV3Y1) cells. The phosphotyrosine-containing proteins were also less abundant in RSVE1AY cells than in RSV3Y1 cells, suggesting that E1AY cells require a lower threshold dose of p60v-src for transformation than do 3Y1 cells. E1AY cells were found to be more sensitive to lysis by detergents. The results suggest that the enhancement is due to changes in membrane structures in E1AY cells.

Elevation of nerve growth factor synthesis by constitutive expression of v-src oncogene in cultured rat fibroblasts.

Rat fibroblast 3Y1 cells transformed by Rous sarcoma virus (RSV) or transfected with the v-src gene showed a highly constitutive v-src gene expression. Simultaneously, marked increases in the cellular level of nerve growth factor (NGF) mRNA and NGF content in the culture medium were observed. The levels of NGF mRNA and NGF secreted into the medium were correlated with the expression level of v-src mRNA gene in both transformants and control 3Y1 cells. These results suggest that v-src gene expression is relevant to regulation of NGF synthesis in rat 3Y1 fibroblasts.

Altered protein glycosylation of rat 3Y1 cells induced by activated c-myc gene.

N-linked sugar chains of rat 3Y1 cells and tumorigenic cells derived by transfection with activated c-myc gene were quantitatively released as oligosaccharides from membrane preparations by hydrazinolysis. Structural analyses revealed that cells of both types contain bi-, tri- and tetra-antennary complex-type oligosaccharides as well as high-mannose-type oligosaccharides. However, the c-myc-transfected cells showed an increase in tri- and tetra-antennary oligosaccharides having the GlcNAc beta 1----4Man alpha 1----and/or the GlcNAc beta 1----6Man alpha 1----linkages with a decrease in biantennary oligosaccharides compared to control 3Y1 cells. The data suggest that c-myc gene has a potential role in the regulation of cellular protein glycosylation and that an elevated expression of c-myc gene in the cells leads to increased branch formation of outer chains in N-linked oligosaccharides concomitant with the acquisition of tumorigenicity.

Nonlethal G0-ts mutant tsJT60 becomes lethal at the nonpermissive temperature after transformation: a hint for new cancer chemotherapeutics.

tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G0 mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G0 and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34 degrees C. However, most of these clones grew slowly at 40 degrees C, producing many floating dead cells, and some clones were killed at 40 degrees C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40 degrees C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G0 phase and are metabolically imbalanced at 40 degrees C during self-stimulation from the G0 to S phase. We propose that a drug which exclusively block, G0-G1 transition would be cytocidal to transformed cells but cytostatic to normal cells.