Engineered Retargeting of Viral RNA Replication Complexes to an Alternative Intracellular Membrane

Abstract

A universal feature of positive-strand RNA viruses is the involvement of host intracellular membranes in RNA replication complex formation and function. This conclusion is based primarily on four observations. First, immunofluorescence and immunoelectron microscopy have localized viral replicase proteins and nascent viral RNA synthesis to intracellular membranes (16,18,24,28,29,33,42,47,51,53,56). Second, in vitro viral RNA-dependent RNA polymerase (RdRp) activity cofractionates with cellular membranes (6,8,12,51,58). Third, detergents suppress, and in some instances, phospholipids enhance the in vitro activities of viral replicase proteins (2,8,57,58). Fourth, lipid synthesis inhibitors (20,30,36) and mutations in lipid synthesis genes (26) inhibit viral RNA replication. Recent results also show that at least some positive-strand RNA viruses use membrane rearrangements to create virusspecific, membrane-bounded compartments in which RNA replication occurs (51).Despite these observations, many fundamental questions remain about the interaction of viral replication factors with host intracellular membranes and the specific roles of membranes in viral RNA replication complex formation and function.Moreover, while most viruses assemble their replication complexes on a specific membrane or membranes, different viruses use different membranes. For various positive-strand RNA viruses, membranes derived from the endoplasmic reticulum (ER) (26,29,35,42,(46)(47)(48)51), Golgi apparatus (47), lysosomes (18,24,28,47), endosomes (18, 24), vacuoles (52), mitochondria (10,15,32,33), perixosomes (9, 10), and chloroplasts (14) have all been implicated in viral RNA replication complex formation and function. The significance of this diversity of intracellular membranes used by different viruses is unknown.The wide variety of intracellular membrane compartments used by different positive-strand RNA viruses, and their specific targeting, suggests that individual viruses may have unique host factor requirements supplied by specific intracellular membranes. Alternatively, many or all host intracellular membranes could provide the functions necessary for viral RNA replication complex formation and function, and the specific intracellular localization of individual viruses may be related to other steps in the viral life cycle, such as viral protein translation or encapsidation. Results that help distinguish between these competing hypotheses potentially have significant therapeutic implications, as antiviral drugs designed to block viral RNA replication complex formation or function on a specific intracellular membrane compartment will be ineffective if alternative membranes can be used efficiently.To study intracellular viral RNA replication complex targeting, we use Flock House virus (FHV), an alphanodavirus that has been used as a model to investigate viral capsid formation and RNA packaging (49,50,59), viral RNA replication and

Topology of Double-Membraned Vesicles and the Opportunity for Non-Lytic Release of Cytoplasm

“…15 However, when Flock House Virus RNA replication complexes were retargeted to the endoplasmic reticulum (ER), the efficiency of viral RNA replication actually increased. 16 These kinds of data suggest that different RNA viruses may co-opt different subcellular compartments for some reason other than the exigencies of replicating RNA, such as to alter cellular signal transduction in ways that are useful during infections of natural hosts.…”

The Cellular Decapping Activators LSm1, Pat1, and Dhh1 Control the Ratio of Subgenomic to Genomic Flock House Virus RNAs

“…Since LSm1-7, Pat1, and Dhh1 have been shown to localize in P-bodies in close proximity to mitochondria (54), we questioned whether these factors would also affect FHV replication when associated with other membrane compartments. It has been previously shown that FHV RNA replication complexes can be retargeted to the endoplasmic reticulum (ER) through the replacement of the N-proximal sequence by ER-targeting sequences from the HCV NS5B protein (46). These alternative replication complexes were also shown to be fully functional, although FHV replication kinetics were enhanced (46).…”

“…It has been previously shown that FHV RNA replication complexes can be retargeted to the endoplasmic reticulum (ER) through the replacement of the N-proximal sequence by ER-targeting sequences from the HCV NS5B protein (46). These alternative replication complexes were also shown to be fully functional, although FHV replication kinetics were enhanced (46). In these experiments, we focused on protein Dhh1, since it has an effect on the RNA3/RNA1 ratios similar to those of the other two components of the complex without any additional effect on FHV RNA steady-state levels; thus, it shows a clearer phenotype (Fig.…”

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

“…The FHV RdRp and RCs colocalize to spherules within the outer mitochondrial membrane (32,36,37). The RdRp of a second betanodavirus, AHNV, localizes to mitochondria in infected fish cells and in transfected African green monkey kidney COS-7 cells when expressed from a plasmid in the absence of viral RNA replication, but the significance of this localization for the life cycle of the virus is unknown (38).…”