Kiyohisa Mizumoto

Molecular Determinants for Subcellular Localization of Hepatitis C Virus Core Protein

ABSTRACT Hepatitis C virus (HCV) core protein is a putative nucleocapsid protein with a number of regulatory functions. In tissue culture cells, HCV core protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HCV core combined with green fluorescent proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the core protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic α-helix structure. With regard to the nuclear targeting of the core protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-α. Differences in the cellular trafficking of HCV core protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the core protein.

Proteomic analysis of human brain identifies α-enolase as a novel autoantigen in Hashimoto’s encephalopathy

Hashimotos encephalopathy (HE) is a rare autoimmune disease associated with Hashimotos thyroiditis (HT). To identify the HE‐related autoantigens, we developed a human brain proteome map using two‐dimensional electrophoresis and applied it to the immuno‐screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI‐TOF‐MASS analysis, immuno‐positive spots of 48 kDa (pI 7.3–7.8) detected from HE patient sera were identified as a novel autoimmuno‐antigen, α‐enolase, harboring several modifications. Specific high reactivities against human α‐enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut‐off level. Although a few HT patients showed faint reactions to α‐enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti‐α‐enolase antibody is useful for defining HE‐related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.

Sendai virus C proteins are categorically nonessential gene products but silencing their expression severely impairs viral replication and pathogenesis

The P/C mRNA of Sendai virus (SeV), a prototypic member of the family Paramyxoviridae in the Mononegavirales superfamily comprising a large number of nonsegmented negative strand RNA viruses, encodes a nested set of accessory proteins, C′, C, Y1 and Y2, referred to collectively as C proteins, initiating, respectively, at ACG/81 and AUGs/114, 183, 201 in the +1 frame relative to the ORF of phospho (P) protein, the smaller subunit of RNA polymerase. Among them, C is the major species expressed in infected cells at a molar ratio which is several‐fold higher than the other three. However, their function has remained an enigma. It has not even been established whether or not the C proteins are essential for viral replication. Many other viruses in Mononegavirales encode C‐like proteins, but their roles also remain to be defined.

Minimum molecular architectures for transcription and replication of the influenza virus.

The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. The PB1 subunit plays a key role in both the assembly of three P protein subunits and the catalytic function of RNA polymerization. We have established a simultaneous expression system of three P proteins in various combinations using recombinant baculoviruses, and isolated the PA–PB1–PB2 ternary (3P) complex and two kinds of the binary (2P) complex, PA–PB1 and PB1–PB2. The affinity-purified 3P complex showed all of the catalytic properties characteristic of the transcriptase, including capped RNA-binding, capped RNA cleavage, model viral RNA binding, model viral RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. The PB1–PB2 binary complex showed essentially the same catalytic properties as does the 3P complex, whereas the PA–PB1 complex catalyzed de novo initiation of RNA synthesis in the absence of primers. Taken together we propose that the catalytic specificity of PB1 subunit is modulated to the transcriptase by binding PB2 or the replicase by interaction with PA.

Monocytic differentiation modulates apoptotic response to cytotoxic anti-Fas antibody and tumor necrosis factor alpha in human monoblast U937 cells.

Interferon‐γ (IFN‐γ), vitamin D3 (VD), and retinoic acid (RA) induce differentiation of human monoblastic leukemia U937 cells to macrophage‐like cells with potential superoxide anion‐generating activity upon further stimulation. Here we report that U93 7 cells thus differentiated show various responses to apoptotic induction with a cytotoxic anti‐Fas antibody and tumor necrosis factor (TNF). VD‐or RA‐treated U937 cells acquired resistance against Fas‐ or TNF receptor (TNFR)‐mediated apoptosis, whereas apoptotic cell death was accelerated in IFN‐γ‐treated cells. By flow cytometric analyses, no decrease in expression of surface Fas antigen or p55 TNFR was observed in differentiated U937 cells. Cell surface expression of CD11b was seen only when differentiation was induced with VD or RA but not with IFN‐γ. The growth of VD‐ or RA‐treated cells was retarded but IFN‐γ‐treated cells were prolific. These findings suggest that the differentiation state differs with the inducer and that the cellular response to apoptotic induction is closely related to the state including the cell cycle. J. Leukoc. Biol. 60: 778–783; 1996.

Involvement of a Cellular Glycolytic Enzyme, Phosphoglycerate Kinase, in Sendai Virus Transcription

In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors). Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin. In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparentM r of 46,000 (p46). From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46. Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation. On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated. The enzymatic activity per se of PGK did not seem to be required for its activity. West-Western blot analysis showed that PGK could directly interact with tubulin. These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex.

High-level expression of exogenous genes by replication-competent retrovirus vectors with an internal ribosomal entry site

We report the construction of two types of Rous sarcoma virus (RSV)-based replication-competent avian retrovirus vectors, IR1 and IR2 to express an exogenous gene at a very high level. In these vectors, the internal ribosomal entry site (IRES) derived from encephalomyocarditis virus (EMCV) was inserted between the env gene and an exogenous gene. The IR1 vector retains the splicing acceptor site that is present in the downstream of the env gene while the IR2 vector lacks it. Using a v-fos mutant (v-fos-CD3) as an example of exogenous genes, we show here that both IR1 and IR2 vectors expressed the gene product, CD3, at expression levels 5- and 8-fold higher than that of their parental vector without IRES, respectively. These vectors were moderately stable and kept a high-level expression of CD3 for at least three passages through the cells. Analysis of viral transcripts indicate that exogenous genes carried by both IR vectors were translated exclusively from the IRES that is present in all the species of the viral transcripts. High-level expression of exogenous genes was also observed in the case of the Hoxa-13 gene in the IR1 vector or the fra-2 gene in the IR2 vector, indicating that the extremely high-level expression characteristic of these vectors is applicable to several exogenous genes.

p53-independent apoptosis is induced by the p19ARF tumor suppressor.

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.

Interaction of S-adenosylhomocysteine hydrolase of Xenopus laevis with mRNA(guanine-7-)methyltransferase: implication on its nuclear compartmentalisation and on cap methylation of hnRNA

S-adenosylhomocysteine hydrolase (SAHH) is the only enzyme known to cleave S-adenosylhomocysteine (SAH), a product and an inhibitor of all S-adenosylmethionine-dependent transmethylation reactions. Xenopus SAHH is a nuclear enzyme in transcriptionally active cells and inhibition of xSAHH prevents cap methylation of hnRNA [Mol. Biol. Cell 10 (1999) 4283]. Here, we demonstrate that inhibition of xSAHH in Xenopus XTC cells results in a cytoplasmic accumulation of the shuttling hnRNPs, while xSAHH itself remains in the nucleus. The functional link between xSAHH and mRNA cap methylation is further supported by a physical association between xSAHH and mRNA(guanine-7-)methyltransferase (CMT). We show by co-immunoprecipitation of tagged proteins that both enzymes interact in vivo. Direct interaction in vitro is shown by pull-down experiments that further demonstrate that the N-terminal 55 amino acids of xSAHH are sufficient for binding to CMT. Since CMT is known to bind to the hyperphoshorylated C-terminal domain (CTD) of its large subunit of RNA polymerase II, we have studied the co-localisation of RNA polymerase II and xSAHH in oocyte nuclei. Immunolocalisation on spreads of lampbrush chromosomes shows xSAHH on the loops of the transcriptionally active lampbrush chromosomes, in Cajal bodies and in B-snurposomes, the nuclear compartments that are most likely engaged in storage and recycling of RNA polymerase II and its cofactors. We therefore suggest that a subfraction of the nuclear xSAHH remains associated with the RNA polymerase holoenzyme complexes, also while these are not actively engaged in transcription.

Rescue of Sendai virus from viral ribonucleoprotein-transfected cells by infection with recombinant vaccinia viruses carrying Sendai virus L and P/C genes.

The Sendai virus ribonucleoprotein (RNP) showed only very low plaque-forming titers upon transfection and the virus yields after one-step growth were quite limited. We tried to enhance the Sendai virus yield by supplying the viral L and P/C gene products through vaccinia vectors. A combination of the recombinant vaccinia viruses carrying the L gene (Vac-HL) and the P/C gene (Vac-HPC), both of which were driven by the promoter of the vaccinia virus 7.5K protein gene, enhanced the yield only a little whereas another combination of Vac-HLd7.5, the L gene insert of which was driven by the promoter of the vaccinia virus thymidine kinase gene in place of the 7.5K promoter, and Vac-HPC greatly enhanced the Sendai virus yield. This seemed to correlate with the fact that the Vac-HL interfered with Sendai virus growth markedly while the Vac-HLd7.5 did not. These results strongly suggest that the L and P/C gene products act in cooperation as the RNA polymerase, and overproduction of the L protein is inhibitory for Sendai virus growth. This system seems to be of value as a tool for analyzing the functions of L and P/C genes of Sendai virus.