| Viruses rely on the host cell?s infrastructure and metabolism during essentially all stages of their replication cycle and therefore have to coordinate a variety of molecular interactions in both time and intracellular space. Positive-stranded RNA (+RNA) viruses are the most abundant virus group and include many medically important and fundamentally interesting pathogens. Following translation of the incoming genome, the key event in the +RNA virus life cycle is the assembly of a cytoplasmic replication/transcription complex (RTC) for viral RNA synthesis. Since RNA-templated RNA synthesis is foreign to the host cell, this process is driven by virus-encoded enzymes, including a subunit with RNA-dependent RNA polymerase (RdRp) activity and, in most viruses, several other RTC ?core enzymes?. Consequently, these proteins are also attractive targets for antiviral drug development. Over the past decade it has become clear that +RNA virus RTCs are invariably associated with virus-induced membrane structures, which derive from drastically rearranged intracellular membranes. Although their ultrastructure and function are only beginning to be understood, these structures are presumed to provide a framework for RNA synthesis by facilitating the concentration and cooperation of viral macromolecules on a dedicated membrane surface. RTC membrane association is mediated by specific replicase subunits that either contain transmembrane domains or associate with the cytoplasmic face of the membrane. These poorly characterized proteins can alter membrane properties and structure and may serve as ?point of membrane attachment? for the RTC?s core enzymes. The membrane-associated RTC can thus be viewed as the natural ?working environment? of the core enzymes, but is essentially also the cradle of +RNA virus evolution, which occurs at an unparalleled rate due to the RdRp?s intrinsically high error frequency. The resulting genetic variation is a major factor in RNA-virus evolution and emergence of novel virus infections. The structural and functional characterization of viral RTCs will be critical to understand the biochemistry of virus replication and develop innovative antiviral control strategies. This project proposes to study +RNA virus RTC structure and function using two distantly related nidoviruses, the SARS-coronavirus (SARS-CoV) and the arterivirus equine arteritis virus (EAV). Among +RNA viruses, nidoviruses stand out for their complex genome organization and life cycle. Both corona- and arteriviruses employ a unique mechanism of discontinuous RNA synthesis to transcribe a nested set of subgenomic mRNAs. The nidovirus replicase/transcriptase is expressed as two large polyprotein precursors, which are autoproteolytically cleaved into 13 to 16 functional subunits, including core enzymes and membrane-anchoring subunits, which coordinate and drive replication and transcription. Nidovirus RTCs associate with elaborate ER-derived modified membranes, whose formation is presumably mediated by three transmembrane replicase subunits. Translation, replicase polyprotein processing, and membrane insertion are thought to be highly coordinated processes, probably involving regulation by the viral proteases that direct replicase maturation. The other RTC subunits, like the RdRp, subsequently associate with the modified membranes, likely by interacting with the cytoplasmic domains of the transmembrane proteins. Interestingly, the enzyme complex of both corona- and arteriviruses was recently found to contain a unique second RdRp activity, which was postulated to act as RNA primase and most likely interacts with the RdRp to form a ?tandem? that plays a central role in RTC activity. In addition to providing an optimized micro-environment for RNA synthesis, the nidovirus-induced membrane structures may serve to separate different steps in the virus life cycle and to prevent detection and clearance of infection by antiviral host cell responses. The central theme of this project is that nidoviruses, through the action of their transmembrane RTC subunits, hijack intracellular membranes to convert them into novel structures that accommodate viral RNA synthesis. Using a combination of biochemistry, structural and molecular biology, and advanced electron microscopy, we aim to dissect structure and function of the nidovirus RTC, with a special focus on the role of transmembrane replicase subunits and membranes. The central research topics to be studied in this project are the following: " Membrane topology and membrane-modifying properties of transmembrane replicase subunits " Host factors and membrane trafficking pathways involved in nidovirus RNA synthesis " Pinpointing the active nidovirus RTC and defining the modified membranes with which it is associated " Composition, activity parameters and structural features of the purified nidovirus RTC " The role of the unique nidovirus primase and its interactions with RdRp and other RTC subunits The results obtained in this project will constitute a major step toward the realization of one of our long-term research objectives, the development of an in vitro reconstitution system allowing the dissection of the molecular mechanisms governing nidovirus replication/transcription. The ultrastructural and biochemical dissection of the RTC will be critical to take our understanding of the complex nidovirus life cycle to the next level. |
