Nodamura Virus RNA Replication in Saccharomyces cerevisiae : Heterologous Gene Expression Allows Replication-Dependent Colony Formation

Abstract

Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 10 4 -fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS ؉ colonies.

A Unique Nodavirus with Novel Features: Mosinovirus Expresses Two Subgenomic RNAs, a Capsid Gene of Unknown Origin, and a Suppressor of the Antiviral RNA Interference Pathway

“…Although alphanodaviruses naturally infect only insects, their RNAs can replicate in vertebrate (9,10), plant (11), and yeast (12) cells. Betanodaviruses cause significant problems in marine fish aquacultures worldwide (13).…”

Two Membrane-Associated Regions within the Nodamura Virus RNA-Dependent RNA Polymerase Are Critical for both Mitochondrial Localization and RNA Replication

“…RNA samples (0.5 g for detection of positive strands or 2 g for detection of negative strands) were separated on denaturing formaldehyde-agarose gels, stained with ethidium bromide, and transferred to charged nylon membranes as previously described (69). Northern blot hybridization was performed as described previously (70, 71), using 32 P-labeled riboprobes specific for the positive or negative strand of NoV RNA3 that also detect RNA1 (47,69,72). The blots were visualized with a Personal Molecular Imager (Bio-Rad) and quantitated using Quantity One 1-D analysis software (Bio-Rad).…”

“…Additionally, the FHV RdRp associates with mitochondrial membranes in infected Drosophila and yeast cells (32,36). To determine whether the NoV RdRp localizes to mitochondria in cells, we used a well-defined reverse genetics system in which NoV RNA replication can be initiated in mammalian cells from cloned cDNA copies of the NoV genomic RNAs (14,47,69,72). For example, the entire replicative cycle can be initiated on expression of the NoV RNA1 and RNA2 cDNAs from T7 promoters in plasmid-transfected baby hamster kidney BSR-T7/5 cells (46) that constitutively express cytoplasmic T7 RNA polymerase (14,47).…”

Host Factors in Viral Life Cycles

“…Whereas the first higher eukaryotic virus able to replicate in yeast was the plant brome mosaic virus (BMV) [35], the list rapidly expanded to other plant viruses but also to viruses that infect insects, mammals and humans [4,57,59,[65][66][67]88]. The BMV-yeast system harbors additional advantages as steps of the viral life cycle can be dissected and studied separately making it possible to identify host factors affecting different steps.…”