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

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Quirin , T , Chen , Y , Pietilä , M K , Guo , D & Ahola , T 2018 , ' The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication ' , Viruses-Basel , vol. 10 , no. 9 , 483 . https://doi.org/10.3390/v10090483

Title: The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication
Author: Quirin, Tania; Chen, Yu; Pietilä, Maija K.; Guo, Deyin; Ahola, Tero
Contributor: University of Helsinki, RNA virus replication and antivirals
University of Helsinki, Department of Microbiology
University of Helsinki, Department of Microbiology
Date: 2018-09-09
Language: eng
Number of pages: 18
Belongs to series: Viruses-Basel
ISSN: 1999-4915
URI: http://hdl.handle.net/10138/265097
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.
Subject: 1182 Biochemistry, cell and molecular biology
1183 Plant biology, microbiology, virology
RNA replication
nodavirus
alphavirus
RNA capping enzyme
replication complex
SEMLIKI-FOREST-VIRUS
ALPHAVIRUS NONSTRUCTURAL PROTEINS
IN-VITRO
SACCHAROMYCES-CEREVISIAE
VIRAL NANOPARTICLE
NODAVIRUS RNA
GENOMIC RNA
EXPRESSION
MEMBRANE
MESSENGER
RNA replication
nodavirus
alphavirus
RNA capping enzyme
replication complex
SEMLIKI-FOREST-VIRUS
ALPHAVIRUS NONSTRUCTURAL PROTEINS
IN-VITRO
SACCHAROMYCES-CEREVISIAE
VIRAL NANOPARTICLE
NODAVIRUS RNA
GENOMIC RNA
EXPRESSION
MEMBRANE
MESSENGER
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