Cytoplasmic Granule Formation and Translational Inhibition of Nodaviral RNAs in the Absence of the Double-Stranded RNA Binding Protein B2

Abstract

bFlock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3= end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.T he nodaviruses are a family of positive-strand RNA viruses that naturally infect insects and fish. They have a bipartite genome that contains approximately 4,500 bases and encodes three proteins. The most thoroughly studied nodavirus is the insect virus Flock House virus (FHV), which has served as an important model system to investigate the structural and molecular basis of RNA replication, virus assembly, and inhibition of antiviral host responses (1). The larger of the two genomic RNAs, RNA1 (3.1 kb), encodes the RNAdependent RNA polymerase (RdRp; 112 kDa), which is located on the outer membrane of mitochondria in infected cells (2, 3). Viral RNA synthesis induces so-called spherules (4), i.e., membrane invaginations, which are thought to sequester the replication complexes and double-stranded RNA (dsRNA) intermediates to protect them from RNA silencing, a major antiviral pathway activated in insects upon infection with RNA viruses (5). Further protection from RNA silencing is afforded by FHV protein B2 (11.6 kDa), a dsRNA binding protein that is translated from RNA3 (387 nucleotides), a subgenomic RNA derived from the 3= end of RNA1 (6-8). The capsid protein, protein alpha (43 kDa), is translated from RNA2 (1.4 kb), the second genomic RNA segment.Although a seemingly simple virus, FHV uses a sophisticated regulatory system to control its gene expression. This regulation occurs at several levels and is currently incompletely understo...

Differential segregation of nodaviral coat protein and RNA into progeny virions during mixed infection with FHV and NoV

“…Using antibodies against the polymerase combined with mitotracker staining, we reproduced earlier results that RdRp is located on mitochondria in these cells (Fig. 2A) and that the organelles exhibit the expected clustering around the nucleus that has been previously noted in insect cells (Miller et al, 2001; Petrillo et al, 2013). More importantly, electron microscopy of thin sections prepared from BHK21 cells transfected with FHV RNA1, the RdRp message, revealed extensive architectural reorganization of the organelles and the presence of numerous replication spherules in the outer membrane (Fig.…”

“…As previously observed (Petrillo et al, 2013), cells transfected with FHV RNA contained coat protein throughout the cytoplasm and distributed in a somewhat reticular pattern (Fig. 1A).…”

“…The RNA replication incompatibility between the two viruses enabled us to conclude that cells containing both types of coat protein had acquired all four RNAs. Coat protein in FHV-infected BHK21 cells was known from a previous study to be distributed throughout the cytoplasm in a reticular pattern (Petrillo et al, 2013), which is also observed in FHV-infected Drosophila cells (Venter et al, 2009). Here we found that NoV coat protein is distributed in the same manner and that it appears to co-localize with that of FHV in dually infected cells.…”

“…For FISH analysis of Drosophila cells, 2×10 6 S2 cells were infected with F+N particles at an moi of 100 and plated on ConA-coated 35-mm glass bottom dishes (MatTek Corporation P35G-1.5-14-C). Cells were fixed at 12 hpi and FISH was carried out using the QuantiGene® ViewRNA ISH Cell Assay kit (Affymetrix QVC0001) with custom-designed probes directed against positive-sense FHV RNA1 (VF4-14080-01), FHV RNA2 (VF1-13994-01), NoV RNA1 (AF174533) and NoV RNA2 (AF174534) according to the manufacturer's instructions and as described previously (Petrillo et al, 2013). …”

The Mammalian Response to Virus Infection Is Independent of Small RNA Silencing

“…Together, these papers put forth some provocative data hinting at the existence of an RNAi pathway in mammals, but neither paper demonstrated that the small RNAs detected mediated viral cleavage in an antiviral fashion (Li et al, 2013). Furthermore, both papers utilize multifunctional virus antagonists (B2 of Nodavirus and VP35 of Ebola), which have been shown to profoundly impact the mammalian host response to virus independently of small RNAs (Petrillo et al, 2013; Prins et al, 2010). Given the importance of this question, we sought to formally evaluate the mammalian response to virus to determine whether RNAi was a physiological contributor to this process.…”