Characterization of the amino acid residues of sendai virus C protein that are critically involved in its interferon antagonism and RNA synthesis down-regulation.

Sendai virus (SeV) encodes two accessory proteins, V and C, in the alternative reading frames in the P gene that are accessed transcriptionally (V) or translationally (C). The C protein is expressed as a nested set of four C-coterminal proteins, C', C, Y1, and Y2, that use different initiation codons. Using HeLa cell lines constitutively expressing the various C proteins, we previously found that the smallest (the 175-residue Y2) of the four C proteins was fully capable of counteracting the antiviral action of interferons (IFNs) and inhibiting viral RNA synthesis and that the C-terminal half of 106 residues was sufficient for both of these inhibitory functions (A. Kato et al., J. Virol. 75:3802-3810, 2001, and A. Kato et al., J. Virol. 76:7114-7124, 2002). Here, we further generated HeLa cell lines expressing the mutated C (Cm) proteins with charged amino acids substituted for alanine residues at either positions 77 and 80; 114 and 115; 139 and 142; 151, 153, and 154; 156; or 173, 175, and 176. We found that only the mutations at positions 151, 153, and 154 abolished IFN antagonism. All the Cm proteins lost the ability to bind with STAT1 under our assay conditions, regardless of their ability to inhibit IFN signaling. On the other hand, the Cm proteins that altered the tyrosine phosphorylation and dephosphorylation of STAT1 and STAT2 always retained IFN antagonism. Thus, the abnormality of phosphorylation or dephosphorylation appeared to be a cause of the IFN antagonism by SeV C. Regarding viral RNA synthesis inhibition, all mutants but the mutant with replacements at positions 114 and 115 greatly reduced the inhibitory activity, indicating that anti-RNA synthesis by the C protein is governed by amino acids scattered across its C-terminal half. Thus, amino acid sequence requirements differ greatly between IFN antagonism and RNA synthesis inhibition. In addition, we confirmed that another SeV accessory protein, V, does not antagonize IFN.

Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling.

A monoclonal antibody (mAb) against human Toll-like receptor (TLR) 3 was established and its effect on TLR3-mediated responses was tested using human fibroblast cell lines expressing TLR3 on the cell surface. Fibroblasts are known to produce IFN-beta upon viral infection or treatment with double-stranded RNA (dsRNA) through distinct signaling pathways. Here, we show the mAb to TLR3 suppressed poly(I):poly(C)-mediated IFN-beta production by human fibroblasts naturally expressing TLR3 on their surface. By reporter gene assay using HEK293 cells transfected with a human TLR3 expression vector, TLR3 recognized dsRNA to activate NF-kappaB and the IFN-beta promoter. TLR3 signaling was not elicited by either single-stranded RNA (ssRNA) or dsDNA. Thus, specific recognition of dsRNA by extracellular TLR3 is essential for induction of type I IFN: the interassociation between dsRNA and TLR3, regardless of direct or indirect binding, should be disrupted by mAb being attached to TLR3. The mAb against TLR3 reported herein may serve as a regulator for virus-mediated immune response via an alternative pathway involving the dsRNA-TLR3 recognition which might occur on host cells.

The amino-terminal half of Sendai virus C protein is not responsible for either counteracting the antiviral action of interferons or down-regulating viral RNA synthesis.

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.

Dephosphorylation failure of tyrosine-phosphorylated STAT1 in IFN-stimulated Sendai virus C protein-expressing cells.

Sendai virus C protein (SeV C) has been reported to counteract the antiviral activities of interferons (IFNs) by inhibiting the expression of IFN-stimulated gene products. In SeV C-expressing cells, formation of an active ISGF3 complex and translocation of STAT1 into the nucleus were not observed. STAT1 was continuously phosphorylated at tyrosine 701 by IFN signaling; however, its serine phosphorylation was suppressed. In addition, tyrosine-phosphorylated STAT1 grew to form abnormally huge complexes. These findings suggest that the counteraction of IFN in SeV C-expressing cells is caused by disordered phosphorylation and dephosphorylation of STAT1.