Abstract
| Original language | English |
|---|---|
| Article number | 483 |
| Journal | Viruses |
| Volume | 10 |
| Issue number | 9 |
| Number of pages | 18 |
| ISSN | 1999-4915 |
| DOIs | |
| Publication status | Published - 9 Sep 2018 |
| MoE publication type | A1 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
}
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 journal › Article › Scientific › peer-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 -