Takayuki Komatsu

In vivo induction of apoptosis by influenza virus.

Intranasal exposure of mice to influenza virus led to cell death in bronchial/bronchiolar epithelial cells, alveolar cells and lymphoid cells. These cells displayed fragmentation of nuclei and chromatin condensation. Nick end-labelling of DNA in situ confirmed that such apoptotic cells had fragmented DNA. These results suggest that the influenza virus induces apoptosis in vivo.

Paramyxovirus Accessory Proteins as Interferon Antagonists

A new role of the Paramyxovirus accessory proteins has been uncovered. The P gene of the subfamily Paramyxovirinae encodes accessory proteins including the V and/or C protein by means of pseudotemplated nucleotide addition (RNA editing) or by overlapping open reading frame. The Respirovirus (Sendai virus and human parainfluenza virus (hPIV)3) and Rubulavirus (simian virus (SV)5, SV41, mumps virus and hPIV2) circumvent the interferon (IFN) response by inhibiting IFN signaling. The responsible genes were mapped to the C gene for SeV and the V gene for rubulaviruses. On the other hand, wild type measles viruses isolated from clinical specimens suppress production of IFN, although responsible viral factors remain to be identified. Both human and bovine respiratory syncytial viruses (RSVs) counteract the antiviral effect of IFN with inhibiting neither IFN signaling nor IFN production. Bovine RSV NS1 and NS2 proteins cooperatively antagonize the antiviral effect of IFN. Studies on the molecular mechanism by which viruses circumvent the host IFN response will not only illustrate co‐evolution of virus strategies of immune evasion but also provide basic information useful for engineering novel antiviral drugs as well as recombinant live vaccine.

The STAT2 Activation Process Is a Crucial Target of Sendai Virus C Protein for the Blockade of Alpha Interferon Signaling

ABSTRACT Sendai virus (SeV) C protein functions as an interferon (IFN) antagonist and renders cells unresponsive to both alpha/beta IFN (IFN-α/β) and IFN-γ. We have recently found the physical association of the C protein with signal transducer and activator of transcription 1 (STAT1) in infected cells. However, involvement of the C-STAT1 interaction in the blockade of IFN signaling has remained unclear. We generated here a series of C mutant proteins that retained or lost the STAT1-binding capacity and examined their effects on IFN-α signaling. All of the C mutant proteins with no STAT1-binding capacity lost the ability to inhibit the IFN-α response. In contrast, the C mutant proteins retaining the STAT1-binding capacity suppressed IFN-α-stimulated tyrosine phosphorylation of both STAT2 and STAT1 to various degrees. Remarkably, their anti-IFN-α capacities correlated well with the inhibitory effect on phosphorylation of STAT2 rather than STAT1. In infected cells, the levels of tyrosine-phosphorylated (pY) STAT2 were below the detection level irrespective of duration of IFN-α stimulation, whereas the levels of pY-STAT1 strikingly increased after long-term IFN-α stimulation. These results suggest that the STAT2 activation process is a crucial target for the blockade of IFN-α signaling. An in vitro binding assay with extracts from (STAT1-deficient) U3A and (STAT1-expressing) U3A-ST1 cells suggested the requirement of STAT1 for the C-STAT2 interaction. Furthermore, expression of STAT1 enhanced the inhibitory effect of the C protein on STAT2 activation in U3A cells. The C protein thus appears to participate in the inhibitory process for STAT2 activation through the STAT1 interaction.

Sendai virus C protein physically associates with Stat1

Background The P/C gene of the Sendai virus (SeV), a member of the family Paramyxoviridae, encodes C protein, which plays a crucial role in counteracting the antiviral effect of interferon (IFN). The C protein blocks IFN signalling to prevent the activation of IFN stimulated genes. However, its underlying molecular mechanism remains to be defined.

Sendai virus C protein impairs both phosphorylation and dephosphorylation processes of Stat1

Sendai virus expresses C protein that blocks interferon (IFN) signaling. We previously reported suppression of IFN‐stimulated tyrosine phosphorylation of signal transducers and activators of transcription (Stats) in infected cells. However this conclusion has remained controversial. To settle it, we re‐examined the effect of C protein expression on phosphorylation of Stat1 in detail. IFN‐stimulated tyrosine phosphorylation of Stat1 was doubtlessly suppressed early in infection, but the suppression was incomplete, suggesting the importance of the unknown blocking mechanism that inactivates the tyrosine‐phosphorylated (pY)‐Stat1 generated as the signaling leak. Interestingly, the dephosphorylation process of pY‐Stat1 was also impaired. These effects on both phosphorylation and dephosphorylation processes were attributable to the function of the C protein.

Sendai Virus C Protein Plays a Role in Restricting PKR Activation by Limiting the Generation of Intracellular Double-Stranded RNA

ABSTRACT Sendai virus (SeV) C protein is a multifunctional protein that plays important roles in regulating viral genome replication and transcription, antagonizing the host interferon system, suppressing virus-induced apoptosis, and facilitating virus assembly and budding. We here report a novel role of SeV C protein, the limitation of double-stranded RNA (dsRNA) generation for maintaining the rate of protein synthesis in infected cells. It was found that the intracellular protein synthesis rate was maintained even after wild-type (wt) SeV infection, but markedly suppressed following C-knockout SeV infection. This indicates the requirement of C protein for maintaining protein synthesis after infection. In contrast to wt SeV infection, C-knockout SeV infection caused phosphorylation of both the translation initiation factor eIF2α and dsRNA-dependent protein kinase (PKR). Phosphorylation of eIF2α occurred mainly due to the action of PKR, since knockdown of PKR by small interfering RNA limited eIF2α phosphorylation. C protein, however, could inhibit neither poly(I):poly(C)-activated nor Newcastle disease virus-induced phosphorylation of PKR and eIF2α, suggesting that C protein does not target common pathways leading to PKR activation. Immunofluorescent staining experiments with a monoclonal antibody specifically recognizing dsRNA revealed generation of a large amount of dsRNA in cells infected with C-knockout SeV but not wt SeV. The dsRNA generation as well as phosphorylation of PKR and eIF2α induced by C-knockout SeV was markedly suppressed in cells constitutively expressing C protein. Taken together, these results demonstrate that the SeV C protein limits generation of dsRNA, thereby keeping PKR inactive to maintain intracellular protein synthesis.

Viremia induced by influenza virus

A mouse model of influenza A/PR/8 virus infection was adopted to investigate the blood and various tissues of intranasally infected mice for the presence of viral RNA by using the nested polymerase chain reaction. The nucleoprotein gene was detected in the red blood cell fraction from 1 to 5 days post-inoculation, while it was found in the lung and brain up to 14 days and in the liver, spleen, kidney, heart, and skeletal muscle up to 7 days. The virus-specific messenger RNA was transiently found in these organs. When mice received the uv-inactivated virus, viremia did not occur. The prior transfer of the hyperimmune serum prevented pneumonia but not bronchitis, and viremia was totally abolished. These results suggest: (1) viremia occurs during the acute phase of infection, (2) the virus is present in various organs and there the virus gene is transiently expressed, and (3) the virus enters the blood stream possibly through capillaries of the infected alveolar wall. Viremia may influence the pathogenesis of influenza.

Parainfluenza virus type 1 infects olfactory neurons and establishes long-term persistence in the nerve tissue.

A mouse model of Sendai virus infection was adopted to examine the in vivo neurovirulence of parainfluenza viruses. A nested polymerase chain reaction detected the Sendai virus nucleoprotein gene in the olfactory bulbs of intranasally infected mice for at least 168 days post-infection (p.i.) and virus-specific messenger RNAs for 28 days p.i. Viral proteins were histochemically detected in some olfactory neurons for 7 days p.i. They were also found in glomeruli of the olfactory bulbs but not in the mitral cells and the tufted cells. No virus was detected in the whole brain not including the olfactory bulbs. When mice were inoculated with UV-inactivated virus, the viral RNA was present in the olfactory bulbs for a short period of 14 days, with no demonstrable viraemia. These results demonstrate that the parainfluenza virus directly accesses the central nervous system via olfactory neurons and establishes long-term persistence in the nerve tissue.

Astrocyte elevated gene‐1 (AEG‐1) is induced by lipopolysaccharide as toll‐like receptor 4 (TLR4) ligand and regulates TLR4 signalling

Astrocyte elevated gene‐1 (AEG‐1) is induced by human immunodeficiency virus 1 (HIV‐1) infection and involved in tumour progression, migration and invasion as a nuclear factor‐κB (NF‐κB) ‐dependent gene. The involvement of AEG‐1 on lipopolysaccharide (LPS) ‐induced proinflammatory cytokine production was examined. AEG‐1 was induced via NF‐κB activation in LPS‐stimulated U937 human promonocytic cells. AEG‐1 induced by LPS subsequently regulated NF‐κB activation. The prevention of AEG‐1 expression inhibited LPS‐induced tumour necrosis factor‐α and prostaglandin E2 production. The AEG‐1 activation was not induced by toll‐like receptor ligands other than LPS. Therefore, AEG‐1 was suggested to be a LPS‐responsive gene and involved in LPS‐induced inflammatory response.

A Tryptophan-Rich Motif in the Human Parainfluenza Virus Type 2 V Protein Is Critical for the Blockade of Toll-Like Receptor 7 (TLR7)- and TLR9-Dependent Signaling

ABSTRACT Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X3-Trp-X9-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.