Nodamura Virus Nonstructural Protein B2 Can Enhance Viral RNA Accumulation in both Mammalian and Insect Cells

Abstract

During infection of both vertebrate and invertebrate cell lines, the alphanodavirus Nodamura virus (NoV) expresses two nonstructural proteins of different lengths from the B2 open reading frame. The functions of these proteins have yet to be determined, but B2 of the related Flock House virus suppresses RNA interference both in Drosophila cells and in transgenic plants. To examine whether the NoV B2 proteins had similar functions, we compared the replication of wild-type NoV RNA with that of mutants unable to make the B2 proteins. We observed a defect in the accumulation of mutant viral RNA that varied in extent from negligible in some cell lines (e.g., baby hamster kidney cells) to severe in others (e.g., human HeLa and Drosophila DL-1 cells). These results are consistent with the notion that the NoV B2 proteins act to circumvent an innate antiviral response such as RNA interference that differs in efficacy among different host cells.Nodamura virus (NoV) is the type species of the genus Alphanodavirus of the Nodaviridae, a family of small riboviruses with bipartite, positive-strand RNA genomes that also includes Flock House virus (FHV). NoV is unique among alphanodaviruses in its ability to lethally infect both insects and mammals, including the mosquitoes Aedes aegypti, Aedes albopictus, and Toxorhynchites amboinensis (4, 36, 41), suckling mice, and suckling hamsters (17,35,36). NoV infects cultured mosquito cells from Aedes pseudoscutellaris, A. aegypti, and A. albopictus (1, 3, 41) and cultured baby hamster kidney BHK21 cells (3,23,30). When NoV genomic RNAs are introduced by transfection, they can replicate in a wide range of cultured cells (6).The divided nodavirus genome naturally separates the replicative and packaging functions onto two different positivesense RNA molecules, RNA1 and RNA2, respectively. These two genomic RNAs are copackaged into the same virion, and both are required for infectivity (24,30,38). RNA1 encodes protein A, the RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of both genome segments. RNA2 encodes the viral capsid precursor protein, ␣. RNA2 and protein ␣ are dispensable for RNA1 replication. Protein A also catalyzes the synthesis of a single subgenomic RNA3 from an RNA1 template. RNA3 is not packaged into virus particles. For FHV, RNA3 encodes two small proteins, B1 and B2, in overlapping reading frames. Protein B1 is in the same reading frame as protein A and thus represents its C-terminal fragment, whereas protein B2 is in the ϩ1 reading frame relative to protein A (11). For NoV, the first and second AUG codons of RNA3 initiate the translation of two forms of B2 (B2-137 and B2-134) that differ only at the N terminus, whereas B1 initiates at the third AUG codon. As for FHV, the B2 proteins are in the ϩ1 reading frame relative to the A/B1 open reading frame (ORF) (23). All three proteins are detected in cells transfected with NoV RNA1 (NoV1), which replicates autonomously and leads to the synthesis of RNA3 (23).The functions of the nodavirus B1 and B2 prote...

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

“…Additionally, titers of LV vectors encoding short hairpin RNAs could be rescued by cotransfecting NovB2 to the packaging cells. 21,40,49 As NovB2's mode of action is the unspecific binding of long dsRNA and subsequent protection against cytoplasmic Dicer-mediated cleavage, 21 it is likely that the gain in titer is the result of increased amounts of packageable genomic RNA (Supplementary Figure S3). Of note, the titer increasing effect was not only observed for GV and LV vectors but also for a multitude of transgenes supporting the hypothesis of rather unspecific protection of dsRNA Improving bidirectional vector performance T Maetzig et al Improving bidirectional vector performance T Maetzig et al against degradation.…”

Double-Stranded RNA Binding May Be a General Plant RNA Viral Strategy To Suppress RNA Silencing

“…Indeed, it has been demonstrated that size-independent dsRNA-binding proteins could act as silencing suppressors in insect and mammalian cells (5,9,14,28,33,55). The flock house virus B2 suppressor is a dsRNA-binding protein which interacts with the backbone of dsRNA as a dimer (10,35).…”

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

“…For example, no morphological changes were observed in BHK21 cells transfected with purified NoV genomic RNA1 and RNA2 even at 24 hpt, when abundant RNA replication and progeny virus particles were detected (11). Furthermore, during the time course of this and our previous studies (14,47), cells expressing the NoV RdRp did not exhibit any of the morphological hallmarks of apoptosis, including cytoplasmic vacuolization, membrane blebbing, rounding, nuclear fragmentation, or detachment from the growth surface. Instead, in this study the cells appeared healthy for up to 24 hpt, and in a previous study they remained healthy for 12 days postinfection (47).…”

“…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). The replicative cycle launched from this system mirrors the kinetics seen during NoV infection (47).…”

“…RNA3 is 3= coterminal with RNA1 and encodes proteins B1 and B2 from overlapping open reading frames (ORFs). The function of B1 is unknown, while the B2 proteins of FHV and NoV suppress the cellular RNA interference (RNAi) response in insect, plant, and mammalian cells (13)(14)(15)(16).…”