The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2018 |
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| Erschienen: |
2018 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:10 |
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| Enthalten in: |
Viruses - 10(2018), 9 vom: 09. Sept. |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Quirin, Tania [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 12.11.2018 Date Revised 09.12.2020 published: Electronic Citation Status MEDLINE |
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| doi: |
10.3390/v10090483 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM288428226 |
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| 245 | 1 | 4 | |a The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication |
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| 500 | |a Date Completed 12.11.2018 | ||
| 500 | |a Date Revised 09.12.2020 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a RNA capping enzyme | |
| 650 | 4 | |a RNA replication | |
| 650 | 4 | |a alphavirus | |
| 650 | 4 | |a nodavirus | |
| 650 | 4 | |a replication complex | |
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| 700 | 1 | |a Chen, Yu |e verfasserin |4 aut | |
| 700 | 1 | |a Pietilä, Maija K |e verfasserin |4 aut | |
| 700 | 1 | |a Guo, Deyin |e verfasserin |4 aut | |
| 700 | 1 | |a Ahola, Tero |e verfasserin |4 aut | |
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