Noboru Uchide

Effect of antioxidants on apoptosis induced by influenza virus infection: inhibition of viral gene replication and transcription with pyrrolidine dithiocarbamate

Influenza virus (IV) infection induced apoptotic DNA fragmentation and the moderate overproduction of reactive oxygen species (ROS) in primary cultured chorion cells prepared from human fetal membranes, and IV particles were released from the infected cells. The antioxidant pyrrolidine dithiocarbamate (PDTC) inhibited the induced DNA fragmentation, ROS overproduction and IV particle release. Although Trolox inhibited ROS overproduction, it did not inhibit DNA fragmentation or IV production. The inhibitory effect of PDTC on DNA fragmentation was manifested when added up to 3 h after infection or by exposing the infected cells to it for only 1 h after infection. PDTC inhibited IV hemagglutinin (HA) viral (vRNA) and complementary (cRNA and mRNA) RNAs synthesis until 6 h after infection and delayed and decreased HA protein synthesis. However, HA RNA synthesis resumed after 12 h even in the presence of PDTC. These results suggested that PDTC inhibited apoptosis by inhibiting viral macromolecule synthesis rather than through its antioxidant effect, because Trolox did not inhibit apoptosis or IV production, although ROS overproduction was inhibited. The synthesis of specific viral macromolecules at the early stage of infection may play a critical role in the mechanism of apoptosis induction and moderate ROS overproduction may not be involved in the mechanism.

Inhibition of influenza-virus-induced apoptosis in chorion cells of human fetal membranes by nordihydroguaiaretic Acid.

Objectives: It has been postulated that the pathogenesis of influenza virus infection involves not only the virus-proliferation-mediated apoptotic cell death in infected cells, but also a direct reactive oxygen species (ROS)-induced cellular injury in the infected organs. We examined effects of an antioxidant, nordihydroguaiaretic acid (NDGA), on apoptosis induction and viral proliferation. Subsequently, the results were compared with those of pyrrolidine dithiocarbamate (PDTC), another antioxidant. Methods: The levels of ROS production were measured with 2′,7′-dichlorofluorescein diacetate; apoptosis induction and viral proliferation were analyzed by DNA fragmentation and plaque-forming assays, respectively. Results: The treatment of infected cells with NDGA inhibited ROS overproduction, apoptotic DNA fragmentation and virus proliferation. The maximum inhibition against DNA fragmentation (76%) was observed with 500 µM NDGA. The antiviral activity of NDGA against influenza virus was more potent than that of PDTC. Conclusions: The present study, therefore, suggests for the first time that NDGA, a known antioxidant reagent, inhibits the induction of apoptosis in human fetal membrane chorion cells infected with influenza virus through the more potent antiviral activity than that of PDTC.

Differentiation of monocytes to macrophages induced by influenza virus-infected apoptotic cells.

The effect of the culture supernatant of influenza virus (IV)-infected apoptotic and non-apoptotic cells on the differentiation of monocytes to macrophages was investigated. IV infection induced apoptotic DNA fragmentation in cultured chorion cells but not in amnion cells prepared from human foetal membrane tissue. To examine the differentiation of monocytes to macrophages, an adhesion assay was employed using the human monocytic leukaemia THP-1 cell line. THP-1 cells became adherent to a substrate by incubation with the culture supernatant of IV-infected chorion cells, but not with that of amnion cells. The spreading THP-1 cells were morphologically characteristic of macrophages and they phagocytosed latex particles. RT-PCR analysis revealed that the expression of class A scavenger receptor mRNA was induced in THP-1 cells by incubation with the culture supernatant of IV-infected chorion cells. These results suggested that monocytic THP-1 cells were morphologically and functionally differentiated to macrophages by IV-infected apoptotic cells due to a soluble factor released from the apoptotic cells.

Semi-quantitative RT-PCR-based assay, improved by Southern hybridization technique, for polarity-specific influenza virus RNAs in cultured cells

Complementary (c) DNAs against viral (v) RNA of negative polarity and complementary and/or messenger (c/m) RNA of positive polarity for influenza virus hemagglutinin (HA) were synthesized from total cellular RNA extracted from influenza virus- and mock-infected cells using polarity-specific primers, respectively. HA vRNA and c/mRNA were amplified readily by polymerase chain reaction (PCR) from influenza virus-infected cells during a virus productive period; however, non-specific PCR product was prone to amplification from mock-infected cells and cells at once after virus infection. Southern blots of the PCR products were hybridized with biotinylated DNA probe, which enabled the generation of specific signals to HA vRNA and c/mRNA. Mock-infected cells produced no signals. Furthermore, titration analyses revealed linear relationships between amount of target RNAs and generated signals. Accordingly, Southern hybridization made possible the quantitation of specific PCR products for HA vRNA and c/mRNA in cell culture and proved the lack of HA RNAs in mock-infected cells in the absence of virus. The RT-PCR based assay combined with Southern hybridization methodology was useful with respect for investigating the processes of replication and transcription of viral genes in cell culture before and during the virus productive period.

Effects of Mitogen-Activated Protein Kinase Inhibitors on Tumor Necrosis Factor-α Gene Expression and Apoptosis Induction in Cultured Human Fetal Membrane Chorion Cells Infected with Influenza Virus

Objectives: We investigated the involvement of p38 mitogen-activated protein (MAP) kinase in tumor necrosis factor (TNF)-α gene expression, apoptosis induction and virus replication in cultured human fetal membrane chorion cells infected with influenza virus. Methods: Influenza virus-infected chorion cells were incubated in the absence or presence of inhibitors of p38 MAP kinase, SB203580 and SB202190. TNF-α mRNA and hemagglutinin viral RNA (HA vRNA) were amplified with reverse transcriptase-polymerase chain reaction techniques. TNF-α protein concentrations were determined by enzyme-liked immunosorbent assay. The extent of apoptosis induction was estimated by DNA agarose gel electrophoresis. Pyrrolidine dithiocarbamate (PDTC) and ribavirin, which have been shown to inhibit apoptosis induction via the inhibition of viral gene replication, were used as positive control reagents. Results: PDTC and ribavirin inhibited the accumulation of TNF-α mRNA and HA vRNA in the virus-infected chorion cells, resulting in the suppression of TNF-α protein secretion. Both SB203580 and SB202190 suppressed TNF-α protein secretion, but not the accumulation of TNF-α mRNA as well as HA vRNA and the induction of apoptosis. Conclusions: These results suggest that p38 MAP kinase pathway is critical in TNF-α gene expression at a post-transcriptional level but not in the apoptosis induction and influenza virus replication in cultured chorion cells.