Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

Abstract

Viruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infects Drosophila cells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ϳ50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infected Drosophila cells. These preparations can be programmed to synthesize both single-and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis. IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infected Drosophila cells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication

“…The system can be used as a basis for in vitro replication experiments, in which, similar to alphaviruses, mainly positive strand RNAs are produced ( Figure 3 ) [ 34 , 49 ]. In contrast to a recent study [ 36 ], we find that the endogenous template present inside the cells is sufficient for replication activity in vitro. As a side note, we have used PEI as a transfection reagent for FHV and it was seen to give good transfection results.…”

“…RNA products were already generated within the first 30 min of the reaction ( Figure 3 b). The reaction conditions used in the IVRA were based on previous studies done using FHV [ 36 ] or alphaviruses [ 42 , 43 ]. Additionally, we wanted to investigate the effect of varying temperatures on FHV replication in vitro.…”

“…The FHV in vitro replication assay (IVRA) described here was adapted from Short et al [ 36 ]. Briefly, 8 µL of CMP was added to a replication buffer (50 mM Tris, pH 8.0; 18 mM MgCl 2 ; 30 mM KCl; 16 mM NaCl; 250 mM sucrose; 10 µg/mL actinomycin D; 0.2 U/µL RiboLock; 1 mM ATP/GTP/UTP; 10 µM CTP; 5 µCi of CTP[α-32P] (specific activity, 3000 Ci/mmol, Perkin Elmer) to a final volume of 50 µL.…”

Lipid Tales of Viral Replication and Transmission

“…Electron microscopic studies of replication organelles reveal complex vesiculo-tubular structures with both positive and negative curvature membranes that resemble little the parental organelles from which they derived [9,15]. Host ESCRT proteins play critical roles in shaping replication organelle membranes during BMV and TBSV infections.…”

Inspirations on Virus Replication and Cell-to-Cell Movement from Studies Examining the Cytopathology Induced by Lettuce infectious yellows virus in Plant Cells

“…FHV is the first virus whose replication complexes were visualized three-dimensionally in detail by ET, numerous spherules of 50–70 nm in diameter are shown as outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked structure of ∼10 nm in diameter ( Kopek et al, 2007 ; Figure 3C ). FHV RNA replication intermediates, nascent RNA and replication protein A were detected within membrane-bound spherules by immunofluorescence microscopy, thus validating the spherules as FHV-induced compartments for vRNA synthesis ( Kopek et al, 2007 ; Short et al, 2016 ). Protein A was found to be necessary, but not sufficient for spherule formation, the presence of a replication-competent RNA template is also required.…”