Three-Dimensional Analysis of a Viral RNA Replication Complex Reveals a Virus-Induced Mini-Organelle

Abstract

Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)–infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside ∼50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of ∼10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of ∼100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence

“…It was described that BVDV NS4B alone can rearrange the host membrane (Weiskircher et al, 2009) as well as HCV NS4B (Egger et al, 2002), suggesting that pestivirus NS4B might act as a trigger for building replication complex. So far, however, pestivirus replication is considered not to remodel cytoplasmic host cell membranes (Schmeiser et al, 2014) as opposed to HCV, which induces organellelike structures designated the membranous web where the formation of the viral replication complex is triggered (Romero-Brey et al, 2012), and to other positive-strand RNA viruses (Knoops et al, 2008;Kopek et al, 2007;Spuul et al, 2007;Welsch et al, 2009). Therefore, we hypothesize that the role of pestivirus NS4B may be partially different from that in other members of the Flaviviridae.…”

Dicing of viral replication intermediates during silencing of latent Drosophila viruses

“…The recent report that FHV replication is spatially restricted to spherules within the mitochondrion offers a picture in which the host RNAi machinery may have limited access to replicating virus (21). FHV encodes a double-stranded RNA binding protein (B2) that protects it from the host RNAi response (1,2,19,20).…”

Hepatitis c virus and host cell lipids: An intimate connection

“…81,108 The origin of these membranes varies depending on the virus and they might be derived from e.g., the ER (Dengue virus), 109 the outer mitochondrial membrane (Flock house virus), 110 or the plasma membrane (alphaviruses). 111,112 It is assumed that the specific protein and lipid composition of these membranes play distinct roles in the replication cycle of a given virus.…”