s8ORF2 protein of infectious salmon anaemia virus is a RNA-silencing suppressor and interacts with Salmon salar Mov10 (SsMov10) of the host RNAi machinery
The infectious salmon anaemia virus (ISAV) is a piscine virus, a member of Orthomyxoviridae family. It encodes at least 10 proteins from eight negative-strand RNA segments. Since ISAV belongs to the same virus family as Influenza A virus, with similarities in protein functions, they may hence be characterised by analogy. Like NS1 protein of Influenza A virus, s8ORF2 of ISAV is implicated in interferon antagonism and RNA-binding functions. In this study, we investigated the role of s8ORF2 in RNAi suppression in a well-established Agrobacterium transient suppression assay in stably silenced transgenic Nicotiana xanthi. In addition, s8ORF2 was identified as a novel interactor with SsMov10, a key molecule responsible for RISC assembly and maturation in the RNAi pathway. This study thus sheds light on a novel route undertaken by viral proteins in promoting viral growth, using the host RNAi machinery.
KeywordsViral suppressor of RNA silencing Infectious Salmon Anaemia Virus Segment 8 Open Reading Frame 2 Salmon salar Mov10 SsMov10
This research was funded by the Department of Biotechnology Indo-Norwegian grant, Govt. of India; the Indian Council for Medical Research fellowship for VT, research grant 183196/S40 from the Research Council of Norway, and internal funds from the School of Science, Monash University Malaysia. The authors are grateful for technical help from Stine Braaen.
SKL, ACB and SKM conceived, designed the experiments. VT and BV performed the EMSA experiments and analysed the results. VT designed and analysed Mutants in EMSA, GFP-reversal assay Phage display and Yeast two-hybrid analysis. VT has written the manuscript. SSP performed Dose-dependent analysis of the s8ORF2 protein, its NM-s8ORF2 mutant and other mutants. SKMi performed the experiment to establish s8ORF2 as a suppressor in GFP-silenced N. xanthi in Reversal of Silencing leaf assay. RBR performed and analysed fish cell studies and participated in writing the manuscript. CMO and ER planned the fish cell study and contributed to writing the manuscript. CMO analysed the EPC data and performed the WB and immunostaining concerning NM-s8ORF2. SKL, RZ, ACB and SKM helped in the analysis of the results and editing the manuscript. All co-authors read and approved the final manuscript.
Compliance with ethical standards
Conflicts of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
- 1.McCauley, J.W., Hongo, S., Kaverin, N.V., Kochs, G., Lamb, R.A., Matrosovich, M.N., Perez, D.R., Palese, P., Presti, R.M., Rimstad, E., Smith, G.J.D. Orthomyxoviridae. eds. by M.Q. King, Michael J. Adams, Eric B. Carstens, and Elliot J. Lefkowitz. Virus Taxonomy Classification and Nomenclature of Viruses Ninth Report of the International Committee on Taxonomy of Viruses Editors Andrew 2012. Elsevier 2012Google Scholar
- 4.K.E. Thorud, H.O. Djupvik, Infectious salmon anemia in Atlantic salmon (Salmo salar L.). Bull. Eur. Assoc. Fish Pathol. 8, 109–111 (1988)Google Scholar
- 8.E. Garcia-Rosado, T. Markussen, Ø. Kileng, E.S. Baekkevold, B. Robertsen, S. Mjaaland, E. Rimstad, Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity. Virus Res. 133(2), 228–238 (2008). https://doi.org/10.1016/j.virusres.2008.01.008 CrossRefPubMedGoogle Scholar
- 9.E. Bierin, K. Falk, E. Hoel, J. Thevarajan, M. Joerink, A. Nylund, C. Endresen, B. Krossøyl, Segment 8 encodes a structural protein of infectious salmon anaemia virus (ISAV); the co-linear transcript from segment 7 probably encodes a non-structural or minor structural protein. Dis. Aquat. Organ. 49(2), 117–122 (2002)CrossRefPubMedGoogle Scholar
- 31.W.X. Li, H. Li, R. Lu, F. Li, M. Dus, P. Atkinson, E.W. Brydon, K.L. Johnson, A. García-Sastre, L.A. Ball, P. Palese, S.W. Ding, Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc. Natl. Acad. Sci. USA. 101(5), 1350–1355 (2004)CrossRefPubMedPubMedCentralGoogle Scholar
- 32.S. Qian, X. Zhong, L. Yu, B. Ding, P. de Haan, K. Boris-Lawrie, HIV-1 Tat RNA silencing suppressor activity is conserved across kingdoms and counteracts translational repression of HIV-1. Proc. Natl. Acad. Sci. USA. 106(2), 605–610 (2009). https://doi.org/10.1073/pnas.0806822106 CrossRefPubMedPubMedCentralGoogle Scholar
- 35.M.C. Ou, Y.M. Chen, M.F. Jeng, C.J. Chu, H.L. Yang, T.Y. Chen, Identification of critical residues in nervous necrosis virus B2 for dsRNA-binding and RNAi-inhibiting activity through by bioinformatic analysis and mutagenesis. Biochem. Biophys. Res. Commun. 361(3), 634–640 (2007)CrossRefPubMedGoogle Scholar
- 36.S. Karjee, A. Minhas, V. Sood, S.S. Ponia, A.C. Banerjea, V.T. Chow, S.K. Mukherjee, S.K. Lal, The 7a accessory protein of severe acute respiratory syndrome coronavirus acts as an RNA silencing suppressor. J. Virol. 84(19), 10395–10401 (2010). https://doi.org/10.1128/JVI.00748-10 CrossRefPubMedPubMedCentralGoogle Scholar
- 39.P. James, J. Haliaday, E.A. Craig, Genomic libraries and a host strain designed for highly efficient two hybrid selection in yeast. Genetics. 144, 1425–1436 (1996)Google Scholar
- 53.J. Zhang, F. Huang, L. Tan, C. Bai, B. Chen, J. Liu, J. Liang, C. Liu, S. Zhang, G. Lu, Y. Chen, H. Zhang, Host protein moloney leukemia virus 10 (MOV10) acts as a restriction factor of influenza a virus by inhibiting the nuclear import of the viral nucleoprotein. J. Virol. 90(8), 3966–3980 (2016)CrossRefPubMedPubMedCentralGoogle Scholar
- 56.Y.H. Han, Y.J. Luo, Q. Wu, J. Jovel, X.H. Wang, R. Aliyari, C. Han, W.X. Li, S.W. Ding, RNA-based immunity terminates viral infection in adult Drosophila in the absence of viral suppression of RNA interference: characterization of viral small interfering RNA populations in wild-type and mutant flies. J. Virol. 85(24), 13153–13163 (2011)CrossRefPubMedPubMedCentralGoogle Scholar