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

Abstract

Viruses with positive-strand RNA genomes amplify their genomes in replication complexes associated with cellular membranes. Little is known about the mechanism of replication complex formation in cells infected with Nodamura virus. This virus is unique in its ability to lethally infect both mammals and insects. In mice and in larvae of the greater wax moth (Galleria mellonella), Nodamura virus-infected muscle cells exhibit mitochondrial aggregation and membrane rearrangement, leading to disorganization of the muscle fibrils on the tissue level and ultimately in hind limb/segment paralysis. However, the molecular basis for this pathogenesis and the role of mitochondria in Nodamura virus infection remains unclear. Here, we tested the hypothesis that Nodamura virus establishes RNA replication complexes that associate with mitochondria in mammalian cells. Our results showed that Nodamura virus replication complexes are targeted to mitochondria, as evidenced in biochemical, molecular, and confocal microscopy studies. More specifically, we show that the Nodamura virus RNA-dependent RNA polymerase interacts with the outer mitochondrial membranes as an integral membrane protein and ultimately becomes associated with functional replication complexes. These studies will help us to understand the mechanism of replication complex formation and the pathogenesis of Nodamura virus for mammals. IMPORTANCEThis study will further our understanding of Nodamura virus (NoV) genome replication and its pathogenesis for mice. NoV is unique among the Nodaviridae in its ability to infect mammals. Here we show that NoV establishes RNA replication complexes (RCs) in association with mitochondria in mammalian cells. These RCs contain newly synthesized viral RNA and feature a physical interaction between mitochondrial membranes and the viral RNA-dependent RNA polymerase (RdRp), which is mediated by two membrane-associated regions. While the nature of the interaction needs to be explored further, it appears to occur by a mode distinct from that described for the insect nodavirus Flock House virus (FHV). The interaction of the NoV RdRp with mitochondrial membranes is essential for clustering of mitochondria into networks that resemble those described for infected mouse muscle and that are associated with fatal hind limb paralysis. This work therefore provides the first link between NoV RNA replication complex formation and the pathogenesis of this virus for mice.

Expression and partial characterisation of rabbit haemorrhagic disease virus non-structural proteins

“…Expression of the polymerase of Nodamura virus, also a nodavirus, induced mitochondria to cluster into distinct cytoplasmic networks. It was shown that nodavirus RNA synthesis localises to these polymerase-induced mitochondrial networks, establishing the polymerase as a key protein for the assembly of membrane-anchored replication complexes (Gant et al, 2014). Our unexpected finding of a redistribution of Golgi membranes in cells expressing the RHDV polymerase, or the entire virus polyprotein, strongly indicates that RHDV employs the polymerase to utilise modified membranes of the secretory pathway for its replication.…”

Arachidonic Acid Induces the Migration of MDA-MB-231 Cells by Activating Raft-associated Leukotriene B4 Receptors

“…7C, in which green (CTXB) and red (BLT1) signals are assigned to the x and y axes, respectively. Each pixel is represented as a dot, and pixels with well co-localized signals appear as a scattered diagonal line [41, 42]. The average Manders’ overlap coefficients for MCF-7 cells (0.41 ± 0.013) and MDA MB 231 cells (0.90 ± 0.045) suggest strong colocalization between CTXB and BLT1 along the cell membrane of MDA MB 231.…”

RNA-Dependent RNA Polymerases of Both Virulent and Benign Rabbit Caliciviruses Induce Striking Rearrangement of Golgi Membranes

“…Although polymerase-dependent membrane rearrangements were described for members of Nodaviridae family, such as Flock house virus and Nodamura virus , it should be noted that the RdRps of these viruses are twice as large as their RHDV and RCV counterparts and that they carry distinct functional domains in addition to the core polymerase structure present in all RdRps [26, 27]. Rabbit calicivirus RdRps, on the other hand do not have any obvious domains or motifs that could be easily linked to the observed Golgi membrane rearrangements.…”