Masato Tashiro

Soluble Nef antigen of HIV-1 is cytotoxic for human CD4+ T cells

We have previously shown that Nef‐gene 10 fusion protein induces marked growth arrest of human primary CD4+ T cells. Here, in vitro cytostatic and cytotoxic activities of human immunodeficiency virus type 1 (HIV‐1) Nef against CD4+ T cells were extensively investigated. Growth of human CD4+ cells was inhibited significantly just by the addition of purified full‐length Nef to cultures. When Nef was cross‐linked by anti‐Nef antibodies, it became very cytocidal for CD4+ T cells. A high percentage of sera from HIV‐1‐infected individuals contained soluble Nef. Thus, soluble Nef in vivo may play an important role in immunodysfunction of CD4+ T lymphocytes in HIV‐1 infection.

Pulmonary surfactant is a potential endogenous inhibitor of proteolytic activation of Sendai virus and influenza A virus

The pathogenicities of influenza viruses and paramyxoviruses have been proposed to be primarily determined by a host cell protease(s) that activates viral infectivity by proteolytic cleavage of the envelope glycoproteins. We recently isolated a trypsin‐type endoprotease, named tryptase Clara, from rat bronchial and bronchiolar epithelial Clara cells, which is secreted into the airway lumen and activates Sendai virus and influenza A virus proteolytically. We report here that surfactant in the bronchial fluid inhibited tryptase Clara specifically, having a K i value of 0.13 μM, and inhibited the proteolytic activations by tryptase Clara in vitro and in organ cultures of rat lung. Intranasal infection of rats with Sendai virus was shown to stimulate secretion of tryptase Clara without changing the amount of surfactant in the bronchial lumen, resulting in a preferable condition for proteolytic viral activation and multiplication.

Budding site of sendai virus in polarized epithelial cells is one of the determinants for tropism and pathogenicity in mice

Wild-type Sendai virus fusion (F) glycoprotein requires trypsin or a trypsin-like protease for cleavage-activation in vitro and in vivo, respectively. The virus is pneumotropic in mice and buds at the apical domain of bronchial epithelial cells. On the other hand, the F protein of the protease-activation host range mutant, F1-R, is cleaved by ubiquitous proteases present in different cell lines and in various organs of mice. F1-R causes a systemic infection in mice and the mutant buds bipolarly at the apical and basolateral domains of infected epithelial cells. The enhanced cleavability of the F protein of F1-R has been shown to be a primary determinant for pantropism. Additionally, it has been postulated that bipolar budding of F1-R is required for the systemic spread of the virus and it has been attributed to mutations in the matrix (M) protein of F1-R (Tashiro et al., Virology 184, 227-234, 1991). In this study protease-activation mutants (KD series) were isolated from wild-type virus. They were revealed to bud at the apical domain, and the F protein was cleaved by ubiquitous proteases in mouse organs. The KD mutants were exclusively pneumotropic in mice following intranasal infection, whereas they caused a generalized infection when inoculated directly into the circulatory system. Comparative nucleotide sequence analysis of the F gene of the KD mutants revealed that the deduced amino acid substitutions responsible for enhanced cleavability of the F protein occurred removed from the cleavage site. Mutations were not at all found in the M gene of the KD mutants analyzed, in support of the role of the M protein of F1-R and of a revertant T-9 derived from the latter in bipolar budding. These results suggest that bipolar budding is necessary for the systemic spread of F1-R from the lungs and that apical budding by wild-type virus and the KD mutants leads to respiratory infections. Differential budding at the primary target of infection, in addition to the cleavage-activation of the F protein in mouse organs, is therefore also a determinant for tropism and pathogenicity of Sendai virus in mice.

Human immunodeficiency virus type 1 Nef protein on the cell surface is cytocidal for human CD4+ T cells

We have previously shown that the carboxyl‐terminal region of human immunodeficiency virus type 1 (HIV‐1) Nef antigen present on the outer surface of virus‐infected cells has affinity for uninfected T cells. Here, the in vitro cytotoxic potential of HIV‐1 Nef on the T cell surface against CD4+ T cells was investigated in detail. Human T cells expressing Nef on the cell surface by transfection with non‐infectious mutant HIV‐1 proviruses were demonstrated to kill CD4+ T cells effeciently. Furthermore, it was shown that the carboxyl‐terminal portion of Nef was cytotoxic for CD4+ T cells and that monoclonal antibody against the carboxyl‐terminal region of Nef inhibited Nef induced‐cytolysis. Thus, we concluded that Nef protein on CD4+ T cells may play an important role in the specific loss of CD4+ T lymphocytes during HIV‐1 infection.

Clustered localization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell surface

We studied human immunodeficiency virus type 1 (HIV‐1) Nef protein biochemically and histologically. HIV‐1 Nef, derived from baculosystem and from cells infected with HIV‐1, formed homomeric monomers, dimers, trimers, and further polymers. These oligomers were non‐covalently associated. In cells infected with HIV‐1, Nef molecules were clustered at the cell surface as well as cytoplasm. Our previous results have indicated that the Nef on the surface of cells infected with HIV‐1 is cytotoxic against uninfected CD4+ T cells. Thus, it is very likely that the HIV‐1‐mediated cytotoxic reaction is due, at least in part, to the clustered localization of oligomeric Nef on the cell surface.

Sendai virus infection changes the subcellular localization of tryptase Clara in rat bronchiolar epithelial cells

Tryptase Clara activates the infectivity of Sendai and influenza viruses proteolytically. In this study, we investigated changes in the subcellular localization of tryptase Clara in rat bronchioles with progression of Sendai virus infection. Tryptase Clara and Sendai virus F2 antigen were localized by light and electron immunohistochemical studies. In the uninfected rat lung, tryptase Clara was specifically localized in the secretory granules of respiratory bronchiolar epithelial nonciliated cells, but not in bronchiolar ciliated, or alveolar cells. In the initial stage of Sendai virus infection with slight pathological changes, however, anti-tryptase Clara was highly reactive in luminal peripheral membranes of both nonciliated and ciliated epithelial cells of the bronchioles together with some Sendai virus envelope glycoprotein, F2 antigen. In the progressed stage, tryptase Clara was hard to detect, with heavy accumulation of F2 antigen in the epithelial cells. These immunohistochemical results support our previous findings that in the bronchial lavage fluid tryptase Clara is significantly increased both in amount and activity after viral infection. These results suggest that Sendai virus stimulates the secretion of tryptase Clara from nonciliated bronchiolar epithelial cells to the airway lumen. Accumulation of tryptase Clara on the luminal surface of the bronchiolar epithelial cells and/or in the airway lumen may produce favourable conditions for proteolytic viral activation and multiplication.

Inhibitory effect of pulmonary surfactant on Sendai virus infection in rat lungs

SummaryIntranasal infection of rats with active (infectious) Sendai virus enhances secretion of tryptase Clara, a Sendai virus-activating protease, into the bronchial lumen by Clara cells of the bronchial epitheliums, and inversely suppresses secretion of pulmonary surfactant, an inhibitor of the protease, into the lumen [Kido H et al. (1993) FEBS Lett 322: 115–119]. A trypsin-resistant mutant, TR-2, showed similar effects, although its replication was restricted to a single cycle in the lungs. In contrast, neither nonactive (noninfectious) wild-type virus possessing receptor-binding activity and lacking envelope fusion activity nor UV-inactivated virus retaining receptor binding and envelope fusion activities altered the mode of secretions. These results indicate that viral replication is required for producing a condition in the bronchial lumen for proteolytic activation of progeny virus, thereby infection is extended to a fatal pneumonia. On the other hand, intranasal administration of infected rats with pulmonary surfactant suppressed activation of progeny virus and pathological changes in the lungs, suggesting a therapeutic use of pulmonary surfactant for influenza pneumonia.

Significance of basolateral domain of polarized MDCK cells for Sendai virus-induced cell fusion

SummaryFusion (fusion from within) of polarized MDCK monolayer cells grown on porous membranes was examined after infection with Sendai viruses. Wild-type virus, that buds at the apical membrane domain, did not induce cell fusion even when the F glycoprotein expressed at the apical domain was activated with trypsin. On the other hand, a protease activation mutant, F 1-R, with F protein in the activated form and that buds bipolarly at the apical and basolateral domains, caused syncytia formation in the absence of exogenous protease. Anti-Sendai virus antibodies added to the basolateral side, but not at the apical side, inhibited cell fusion induced by F 1-R. In addition, T-9, a mutant with bipolar budding phenotype of F 1-R but with an uncleavable F protein phenotype like wild-type virus, induced cell fusion exclusively when trypsin was added to the basolateral medium. By electron microscopy, cell-to-cell fusion was shown to occur at the lateral domain of the plasma membrane. These results indicate that in addition to proteolytic activation of the F protein, basolateral expression of Sendai virus envelope glycoproteins is required to induce cell fusion.