5′ cis Elements Direct Nodavirus RNA1 Recruitment to Mitochondrial Sites of Replication Complex Formation

Abstract

Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membraneassociated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in Drosophila cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using Drosophila cells and yeast cells, which also support FHV replication, we also defined the cis-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and Drosophila cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1 cis element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.All positive-strand RNA viruses replicate their genomes in virus-induced replication complexes associated with rearranged intracellular membranes (50). Different viruses replicate in association with different intracellular membranes, with endoplasmic reticulum-derived membranes used most frequently (50). Replication complexes may serve many purposes, including localizing all required viral and cellular factors in close proximity and high concentration to allow effective initiation and efficient progression of replication, protecting replication intermediates such as double-stranded RNA from antiviral responses, and providing a scaffold to organize sequential replication steps (50). Although these complexes are crucial for positive-strand RNA virus replication, their formation is not well understood.For replication complexes to form and function, viral replication proteins, viral genomic RNA, and any required host factors must localize to the relevant intracellular membranes where replication complexes assemble. Some viral replication proteins such as hepatitis C virus (HCV) NS4B, brome mosaic virus (BMV) protein 1a, and tombusvirus p33 direct their own localization to the appropriate membrane (14,49,53). Other viral replication proteins are directed t...

Nodavirus-Induced Membrane Rearrangement in Replication Complex Assembly Requires Replicase Protein A, RNA Templates, and Polymerase Activity

“…However, a previously published real-time RT-PCR analysis (60) showed that, under equivalent conditions, pFA D692E directs the selective recruitment to mitochondrial membranes of ϳ8,000 FHV RNA1 fs templates per Drosophila cell (Van Wynsberghe, personal communication). These ϳ8,000 FHV RNA1 fs templates per Drosophila cell, which have been visualized to be highly localized to mitochondrial membranes (59), are comparable in number to our previous calculation of ϳ20,000 spherules and ϳ16,000 negative-strand RNA1 molecules per FHV-infected cell (28). If even 10% of the RNA1 template so recruited formed spherules, this would have been readily observed by our TEM analysis.…”

“…Note that in panel B generally low but somewhat varying levels of the expected pFA-or pFA D692E -generated, nonreplicatable protein A mRNA transcripts are visible in lanes 3 to 8 at a position slightly above the replicated positive-strand RNA1 band in lane 5. As noted in an earlier study (59), the similarly sized, weak band near RNA1 in panel C, lane 7, appears to represent detection of an incompletely denatured hybrid between negative-strand RNA3 and the positive-strand protein A mRNA. This band does not represent negative-strand RNA1 since, despite its high position in the gel, the band is only detected with a strand-specific probe against the common sequences of negative-strand RNA3 and RNA1, but not with a strand-specific probe against the unique 3Ј-proximal portion of negative-strand RNA1, outside of the sequences shared with RNA3 (59).…”

“…Mitochondria (Mito) and cytoplasm (Cyto) are indicated. recruitment of RNA1 derivatives into a membrane-associated state (59). However, TEM examination of cells transfected with pFA plus pF1-3ЈU showed no detectable spherules, even after examination of sections of 1,382 mitochondria in 107 cells over three independent experiments (Fig.…”

“…Prior fluorescence microscopy and immunogold EM showed that protein A and FHV RNA synthesis localize to the interior of mitochondrial spherules in FHV-infected cells (28) and that, prior to RNA synthesis, protein A recruits FHV RNA templates to mitochondrial membranes (59). To test whether FHV protein A expression alone is sufficient for spherule formation, we transfected Drosophila S2 cells with previously described protein A expression plasmid pFA (60).…”

“…Protein A localizes to mitochondrial outer membranes through an N-terminal mitochondrial targeting and transmembrane sequence (34) and, in a step separable from RNA synthesis, recruits FHV RNA templates to mitochondria (59,60). Pro-tein A also self-interacts in vivo through multiple domains in ways important for RNA replication (16).…”