The V protein of canine distemper virus is required for virus replication in human epithelial cells.

Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells

Functionally distinct effects of the C-terminal regions of IKKε and TBK1 on type I IFN production.

Inhibitor of κB kinase ε (IKKε) and TANK binding kinase 1 (TBK1), so-called non-canonical IKKs or IKK-related kinases, are involved in the cellular innate immunity by inducing type I IFNs. Two kinases commonly phosphorylate transcription factors IRF3 and IRF7 in type I IFN production pathway. In contrast to TBK1, underlying mechanisms of IKKε activation and regions required for activation of downstream molecules are poorly understood. In this study, we investigated regions of IKKε required for the activation of type I IFN promoter specially, by focusing on the C-terminal region. To show the functional significance of the IKKε C-terminal region on type I IFN production, we employed various mutant forms of IKKε and compared to corresponding region of TBK1. We identified the specific regions and residues of IKKε involved in the activation of downstream signaling. Interestingly, corresponding region and residues are not required for activation of downstream signaling by TBK1. The results highlight the importance of the C-terminal region in the functional activity of IKKε in innate immune response and also the difference in activation mechanisms between IKKε and the closely related TBK1

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

Acquired and selected nucleotides during passages in NCI-H358 cells.

<p>^a Acquired and selected nucleotides are underlined.</p

IFN-related responses in the parental and V protein-expressing H358 cells.

<p>(A) The parental H358, H358-V-Tyr₂₆₇-11, and H358-V-Cys₂₆₇-6 cells were infected with 007Lm-VDS, and CDV antigens (green) and IRF-3 (red) were detected by immunofluorescence confocal microscopy. The nuclei (blue) were detected by counterstaining with DAPI. (B) The parental H358, H358-V-Tyr₂₆₇-11, and H358-V-Cys₂₆₇-6 cells were untreated (0 h) or treated with 100 U/ml of IFN-β for 2, 4, and 8 h. Total RNAs were purified and the relative amounts of mRNAs for three ISGs (ISG15, OAS1, and ISG56) were measured by RT-qPCR.</p

Virus growth in the parental and V protein-expressing H358 cells.

<p>(A) Immunoblotting. The parental H358, H358-V-Tyr₂₆₇-11, H358-V-Cys₂₆₇-5, and H358-V-Cys₂₆₇-6 cells were lysed in RIPA buffer, and subjected to SDS-PAGE followed by immunoblotting for detection of the V protein. Tubulin was detected as an internal control. (B, C) The parental H358, H358-V-Tyr₂₆₇-11, and H358-V-Cys₂₆₇-6 cells were infected with 007Lm-VDS (B) or MVΔV (C) at a MOI of 0.01. At 5 days p.i., the virus titers (PFUs and FFUs, respectively) were determined. (D) Observation of EGFP-expressing MVΔV-infected cells (syncytia) using a fluorescence microscope.</p