Analysis of interaction of Sendai virus V protein and melanoma differentiation-associated gene 5

Abstract

Sendai virus (SeV), a pneumotropic virus of rodents, has an accessory protein, V, and the V protein has been shown to interact with MDA5, inhibiting IRF3 activation and interferon-β production. In the present study, interaction of the V protein with various IRF3-activating proteins including MDA5 was investigated in a co-immunoprecipitation assay. We also investigated interaction of mutant V proteins from SeVs of low pathogenicity with MDA5. The V protein interacted with at least retinoic acid inducible gene I, inhibitor of κB kinase epsilon and IRF3 other than MDA5. However, only MDA5 interacted with the V protein dependently on the C-terminal V unique (Vu) region, inhibiting IRF3 reporter activation. The Vu region has been shown to be important for viral pathogenicity. We thus focused on interaction of the V protein with MDA5. Point mutations in the Vu region destabilized the V protein or abolished the interaction with MDA5 when the V protein was stable. The V-R 320 G protein was highly stable and interacted with MDA5, but did not inhibit activation of IRF3 induced by MDA5. Viral pathogenicity of SeV is related to the inhibitory effect of the V protein on MDA5, but is not always related to the binding of V protein with MDA5. Key words MDA5, Sendai virus-V protein.SeV, which belongs to the genus Respirovirus in the family Paramyxoviridae, is a respiratory tract pathogen of rodents. It is an enveloped virus with a single-stranded, negative-sense RNA genome of approximately 15.4 kilobases. The SeV genome comprises six genes encoding structural proteins, including N (nucleocapsid), P (phospho-), M (matrix), F (fusion), HN (hemagglutininneuraminidase), and L (large) proteins (1).The P gene, unlike the other genes, encodes not a single protein but multiple proteins. The colinear transcript encodes the P protein as well as C' , C, Y 1 and Y 2 proteins; the latter four proteins are translated in a shifted frame by alternative translational starts and a common stop codon. The P gene also directs synthesis of an additional mRNA for the V protein by inserting a pseudotemplated G residue at the specific editing site (2, 3). Consequently, P and V proteins share the same 317 residues at the amino terminus (P/V common region), while the two proteins have unique carboxyl termini. The V protein contains a 67-residue unique carboxyl terminus (Vu region), which is 760

Inhibition of Interferon Regulatory Factor 3 Activation by Paramyxovirus V Protein

“…We previously showed interactions of multiple molecules, including RIG-I, MDA5, and IRF3, with the V protein by coimmunoprecipitation assays (36). Here we reproduced those results showing that the SeV V protein physically interacted with RIG-I and IRF3 as well as MDA5 by IP-Western blotting (Fig.…”

“…Protein expression plasmids pCAG-FL-RIG-I and pCAG-FL-MDA5 were described previously (36). An expression plasmid for a C-terminally truncated FLAG-tagged RIG-I (residues 1 to 129) was constructed to generate pCAG-FL-RIG-I-CA (the constitutive active [CA] form) according to a previous report (51).…”

“…The cDNA was processed for three rounds of in vitro mutagenesis to generate IRF3-5D (S396D, S398D, S402D, T404D, and S405D) by using an AMAP multisitedirected mutagenesis kit (Amalgaam, Tokyo, Japan) following the manufacturer's instructions. A reporter plasmid, p-55C1B-EGFP, that has 8 tandem IRF3-binding motifs upstream of the enhanced green fluorescent protein (EGFP) gene was described previously (36).…”

“…The IRF3 reporter assay was performed as described previously (36). Briefly, subconfluent 293T cells in 6-well plates were transfected with p-55C1B-EGFP, an IRF3 signal-inducing plasmid such as pCAG-FL-MDA5, pCAG-FL-RIG-I-CA, pCAG-FL-TRIF-CA, or pCAG-HA-IRF3-5D, and an expression plasmid for the V protein.…”

“…We have recently analyzed the interaction of melanoma differentiation-associated gene 5 (MDA5) with V proteins derived from SeV mutants with different pathogenicities and have shown that SeV pathogenicity appears to be related to interaction of the V protein with MDA5 (36). It has also been reported that MDA5 is involved in activation of innate immunity in mice (9).…”