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Viruses 2018, 10(9), 483; https://doi.org/10.3390/v10090483

The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication

1
Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki, Viikinkaari 9 (P.O. Box 56), 00014 Helsinki, Finland
2
State Key Laboratory of Virology, Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan 430072, China
3
School of Medicine, Sun Yat-sen University, Guangzhou 510080, China
*
Author to whom correspondence should be addressed.
Received: 23 May 2018 / Revised: 5 September 2018 / Accepted: 5 September 2018 / Published: 9 September 2018
(This article belongs to the Section Insect Viruses)
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Abstract

The nodavirus flock house virus (FHV) and the alphavirus Semliki Forest virus (SFV) show evolutionarily intriguing similarities in their replication complexes and RNA capping enzymes. In this study, we first established an efficient FHV trans-replication system in mammalian cells, which disjoins protein expression from viral RNA synthesis. Following transfection, FHV replicase protein A was associated with mitochondria, whose outer surface displayed pouch-like invaginations with a ‘neck’ structure opening towards the cytoplasm. In mitochondrial pellets from transfected cells, high-level synthesis of both genomic and subgenomic RNA was detected in vitro and the newly synthesized RNA was of positive polarity. Secondly, we initiated the study of the putative RNA capping enzyme domain in protein A by mutating the conserved amino acids H93, R100, D141, and W215. RNA replication was abolished for all mutants inside cells and in vitro except for W215A, which showed reduced replication. Transfection of capped RNA template did not rescue the replication activity of the mutants. Comparing the efficiency of SFV and FHV trans-replication systems, the FHV system appeared to produce more RNA. Using fluorescent marker proteins, we demonstrated that both systems could replicate in the same cell. This work may facilitate the comparative analysis of FHV and SFV replication. View Full-Text
Keywords: RNA replication; nodavirus; alphavirus; RNA capping enzyme; replication complex RNA replication; nodavirus; alphavirus; RNA capping enzyme; replication complex
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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Quirin, T.; Chen, Y.; Pietilä, M.K.; Guo, D.; Ahola, T. The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication. Viruses 2018, 10, 483.

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