Ryuji Fukuda

Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells.

The process of cell death caused by influenza virus infection in cultured MDCK and HeLa cells was analysed. This infection gave rise to nuclear fragmentation and chromatin condensation accompanied by chromosomal DNA fragmentation into oligonucleosomes. Chromosomal DNA fragmentation progressed concomitantly with cell lysis of MDCK cells and HeLa cells, producing high and low yields of virus particles, respectively, indicating that the extent of cell lysis was not proportional to the virus production. The endonuclease inhibitor zinc blocked DNA fragmentation in MDCK cells. Cycloheximide inhibited DNA fragmentation as well as cell lysis. Inhibition occurred when the drug was added to the medium within 2 h after infection but not efficiently at 4 h or later. Infection induced the Fas Ag gene, which encodes a possible apoptosis-mediating molecule, in the early infectious stage followed by the expression of Fas Ag on the cell surface. These results suggested that influenza virus infection causes apoptotic death of cultured cells, and their fate might be determined at an early stage of the infection by induction of an apoptotic gene.

dinP, a new gene in Escherichia coli, whose product shows similarities to UmuC and its homologues.

A new gene, designated dinP, was found during E. coli genomic sequencing around the 5.5 min region. Its coding region is preceded by a sequence similar to the consensus binding sequence for LexA, the so-called SOS box sequence. The amino acid sequence of DinP (351 amino acid residues) has a strong similarity to the C. elegans hypothetical protein F22B7.6 and weaker similarities to the UmuC homologues in E. coli and Salmonella typhimurium and also to REV1 of Saccharomyces cerevisiae. Another SOS operon (dinJ1 and dinJ2 genes) found in this region is also described.

Effects of Mutation in Hepatitis C Virus Nonstructural Protein 5A on Interferon Resistance Mediated by Inhibition of PKR Kinase Activity in Mammalian Cells

The IFN‐induced double‐stranded RNA (dsRNA)‐activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A‐expressing cell lines derived from human osteosarcoma (Saos‐2). The cells expressing NS5A derived from an IFN‐resistant clone (NS5A‐1b) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A‐2a) from an IFN‐sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A‐1b (NS5A‐ΔISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A‐2a nor the C‐terminal truncated mutant of NS5A‐1b (NS5A‐ΔC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C‐terminal region of NS5A‐1b is important for the suppression of PKR activity.

Analysis of influenza A virus temperature-sensitive mutants with mutations in RNA segment 8.

Temperature-sensitive (ts) mutants of influenza virus strain A/Udorn/72 (H3N2 subtype) with lesions in RNA segment 8 exhibited intrasegmental complementation, and were divided in two complementation groups (H1 and H2) on MDCK cells. The nucleotide sequence of segment 8 was determined for three of these mutants. The H1 strains, ICR1629 and SPC45, have a single amino acid substitution in the coding region of the non-structural protein NS1, whereas the H2 strain, ICR516, has a substitution in the NS2-coding region. With both NS1 ts mutants, the synthesis of two late proteins, the matrix protein (M1) and haemagglutinin (HA), was greatly reduced and NS1 synthesis also decreased at 40 degrees C (non-permissive temperature) compared to that at 34 degrees C (permissive temperature). The synthesis of each virus-specific RNA was analysed using a quantitative hybridization method. However, at 40 degrees C, the levels of individual mRNAs including those for the late proteins, were almost the same as those at 34 degrees C, and attained the wild-type levels later in the infection (5 h post-infection) when the synthesis of the late proteins and the NS1 protein was severely reduced. The observations suggest that the NS1 protein, which is a nuclear protein, is involved in some post-transcriptional processes in the synthesis of the late proteins and the NS1 protein.

Tyrosine phosphorylation-induced changes in absorption and UV resonance Raman spectra of Src-peptides

To elucidate possible conformational changes of proteins induced by tyrosine phosphorylation, the phosphorylation-induced changes in the absorption and UV resonance Raman (UVRR) spectra of the two kinds of 13-residue peptides in pp60c-src protein containing either Tyr416 (autophosphorylation site) or Tyr527 (C-terminal phosphorylation site) were investigated and compared with the changes for free L-tyrosine. Phosphorylation caused a blue shift and a marked decrease in the 275 nm absorption band (Lb transition) for both peptides and L-tyrosine. In the UVRR spectra, although wavenumber downshifts of several modes were observed upon phosphorylation, the most pronounced-change was the distinct intensity reduction of the Y1 band around 850 cm-1 and appearance of the Y13 band around 760 cm-1. These phosphorylation-induced changes were totally restored upon dephosphorylation with alkaline phosphatase. A Raman band characteristic of the Tyr527-peptide was also detected. A band at 1461 cm-1, which appeared regardless of the phosphorylation state, was assigned to amide IIp from comparison with the spectra of other peptides containing proline residues. The effects of H/D exchange on the UVRR spectra were also examined. This is the first application of UVRR spectroscopy to the detection of possible structural changes of the tyrosine residue on phosphorylation.

Involvement of the influenza A virus PB2 protein in the regulation of viral gene expression

To determine the function(s) of the PB2 protein of influenza A virus, six temperature-sensitive (ts) mutants of A/Udorn/72 (H3N2) virus, each carrying a ts mutation in the PB2 gene, were analysed for virus RNA and protein synthesis. One of the mutants, ICRC27, exhibited unique phenotypes and was characterized in detail. At the non-permissive temperature, 40 degrees C, the accumulation of mRNA for each genome segment was reduced severely, leading to delayed and reduced synthesis of viral proteins, complementary and viral RNAs (cRNAs and vRNAs). At the permissive temperature, 34 degrees C, the mutant virus produced several-fold greater concentrations of both mRNAs and cRNAs of PB2, PB1 and PA segments than wild-type virus. The synthesis of the three polymerase proteins and the induction of RNA polymerase activity were also greatly increased. By contrast, the expression of the haemagglutinin (HA) gene was severely suppressed. The over-production of the polymerase mRNAs was not observed during primary transcription, i.e. in the presence of cycloheximide. The ts+ revertants of ICRC27 did not exhibit the ts defects and also lost most of the non-ts phenotypes at 34 degrees C. These observations indicate that the PB2 protein participates not only in the synthesis of viral RNAs, but also in the regulation of viral gene expression, i.e. in the down-regulation of the three polymerase genes and the up-regulation of the HA gene during secondary transcription.

Interactions of phosphatidylinositol 3-kinase Src homology 3 domain with its ligand peptide studied by absorption, circular dichroism, and UV resonance Raman spectroscopies

Absorption, circular dichroism (CD), and UV resonance Raman (UVRR) spectroscopies were applied to selectively examine the environmental and structural changes of Trp and Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain induced by ligand association. Comparison of the spectra of PI3K SH3 in the presence or absence of its ligand peptide RLP1 (RKLPPRPSK) indicated that RLP1 binding changed the environment of Trp55 of the SH3 to be more hydrophilic and its H bonding weaker and that of Tyr residues to be more hydrophobic. The D21N mutant (Asp21 --> Asn) of the SH3 yielded a UV CD distinct from that of the wild type, and its spectral changes induced by RLP1 binding were smaller and different from those of the wild type in absorption, CD, and UVRR spectra, suggesting that the mutation of conserved Asp21 affected the conformation of the ligand binding cleft and thus might lead to the decrease in the ligand affinity. These data provide direct evidence for the occurrence of environmental and structural changes of PI3K SH3 by the association of a ligand and the D21N mutation.