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

Research output: Contribution to journalArticleScientificpeer-review

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.
Original languageEnglish
Article number483
JournalViruses
Volume10
Issue number9
Number of pages18
ISSN1999-4915
DOIs
Publication statusPublished - 9 Sep 2018
MoE publication typeA1 Journal article-refereed

Fields of Science

  • 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

Cite this

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title = "The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication",
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.",
keywords = "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",
author = "Tania Quirin and Yu Chen and Pietil{\"a}, {Maija K.} and Deyin Guo and Tero Ahola",
year = "2018",
month = "9",
day = "9",
doi = "10.3390/v10090483",
language = "English",
volume = "10",
journal = "Viruses",
issn = "1999-4915",
publisher = "MDPI",
number = "9",

}

The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication. / Quirin, Tania; Chen, Yu; Pietilä, Maija K.; Guo, Deyin; Ahola, Tero.

In: Viruses, Vol. 10, No. 9, 483, 09.09.2018.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication

AU - Quirin, Tania

AU - Chen, Yu

AU - Pietilä, Maija K.

AU - Guo, Deyin

AU - Ahola, Tero

PY - 2018/9/9

Y1 - 2018/9/9

N2 - 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.

AB - 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.

KW - 1182 Biochemistry, cell and molecular biology

KW - 1183 Plant biology, microbiology, virology

KW - RNA replication

KW - nodavirus

KW - alphavirus

KW - RNA capping enzyme

KW - replication complex

KW - SEMLIKI-FOREST-VIRUS

KW - ALPHAVIRUS NONSTRUCTURAL PROTEINS

KW - IN-VITRO

KW - SACCHAROMYCES-CEREVISIAE

KW - VIRAL NANOPARTICLE

KW - NODAVIRUS RNA

KW - GENOMIC RNA

KW - EXPRESSION

KW - MEMBRANE

KW - MESSENGER

KW - RNA replication

KW - nodavirus

KW - alphavirus

KW - RNA capping enzyme

KW - replication complex

KW - SEMLIKI-FOREST-VIRUS

KW - ALPHAVIRUS NONSTRUCTURAL PROTEINS

KW - IN-VITRO

KW - SACCHAROMYCES-CEREVISIAE

KW - VIRAL NANOPARTICLE

KW - NODAVIRUS RNA

KW - GENOMIC RNA

KW - EXPRESSION

KW - MEMBRANE

KW - MESSENGER

U2 - 10.3390/v10090483

DO - 10.3390/v10090483

M3 - Article

VL - 10

JO - Viruses

JF - Viruses

SN - 1999-4915

IS - 9

M1 - 483

ER -