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.

Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

“…Protein A is the only viral protein required for nodavirus RNA replication complex formation and a major component of the crown complex ( 19 , 22 , 24 , 25 ). Moreover, protein A is a highly self-multimerizing, OMM-associated protein that supports RNA synthesis and RNA-capping enzymatic activities during the viral life cycle ( 19 , 22 – 24 ) ( Fig. 1 ).…”

“…Protein A also has two membrane-association domains: A wellestablished N-terminal transmembrane domain and, in keeping with significant membrane interaction after deletion of this transmembrane segment, a predicted membrane-interaction region within the Iceberg domain ( Fig. 1) (20)(21)(22)(23). Finally, various distinct multimerization regions within the capping, Iceberg, and RdRp domains are required for protein A self-interaction and function in RNA replication (24).…”

Cryo-electron microscopy of nodavirus RNA replication organelles illuminates positive-strand RNA virus genome replication

“…FHV’s two component, ∼4.5 kb genome expresses four known proteins ( Figure 1 ). RNA1 encodes protein A, a 998 aa, multi-functional protein with membrane-association, multimerization, RNA-dependent RNA polymerase and 5′ RNA capping activities [ 12 , 13 , 14 ]. RNA1-derived subgenomic RNA3 encodes proteins B1 and B2.…”

Design and Use of Chikungunya Virus Replication Templates Utilizing Mammalian and Mosquito RNA Polymerase I-Mediated Transcription

“…Decoupling the viral replicase protein(s) expression from viral RNA synthesis represents a powerful approach to study viral RNA replication. This has been proven useful to study the replication of influenza virus (24,25), the flavivirus Kunjin virus (26), and the nodavirus Flock House virus (27). In addition, the ability of viral replicase to amplify the RNA template provided in trans has also allowed construction of Saccharomyces cerevisiae yeast-based replication systems for bromo-, noda-, and tombusviruses (28)(29)(30).…”