Identification of critical residues in nervous necrosis virus B2 for dsRNA-binding and RNAi-inhibiting activity through by bioinformatic analysis and mutagenesis

Ming Chang Ou, Young Mao Chen, Mei Fen Jeng, Chiau Jun Chu, Huey Lang Yang, Tzong Yueh Chen

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

It is known that the non-structural B2 protein of nervous necrosis virus (NNV) plays an important role in viral replication and can inhibit the RNA interference system of the host cell. Moreover, the mechanism of NNV B2 protein to inhibit RNAi is by sequestration and protection of double strand (ds) RNA. In the flock house virus (FHV), a model alphanodavirus, the structural and mutational analysis of B2 identified that the positively charged Arg54 of the α2 helix mediated the dsRNA-binding activity. According to the betanodavirus B2 protein alignment and modeling results, the amino acid sequences and the predicted structure of betanodavirus B2 are different from alphanodaviruses. It was suggested that the four Arg residues of α3 helix between amino residues 52-60 of B2 may be involved in dsRNA-binding activity. Thus, this study replaced these four Arg residues with Gln at position 52 (R52Q), 53 (R53Q), 59 (R59Q), and 60 (R60Q) by site-directed mutagenesis method. The dsRNA-binding assays of these B2 mutants demonstrated that mB2(R53Q) and mB2(R60Q) mutants are dsRNA-binding defective. Moreover, we have found mB2(R53Q) and mB2(R60Q) could not antagonize RNAi by using HeLa cell as an RNAi inhibition model. These results suggested that Arg53 and Arg60 of betanodavirus B2 protein may be similar to Arg54 of alphanodavirus FHV B2 protein and are critical for dsRNA binding and RNAi-inhibiting. This study may serve as an example where bioinformatic analysis of related viral genomes may lead to meaningful structural and functional clues for certain viral proteins.

Original languageEnglish
Pages (from-to)634-640
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume361
Issue number3
DOIs
Publication statusPublished - 2007 Sep 28

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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