| Background: All viruses with a positive-strand RNA genome (e.g., hepatitis C virus, dengue virus, West Nile virus, yellow fever virus, SARS coronavirus, enterovirus, rhinovirus, hepatitis A virus, foot-and-mouth-disease virus, rubella, calicivirus, alphavirus, and many other viruses infecting mammals, insects or plants) rearrange intracellular membranes into structures that serve as a scaffold for genomic RNA replication [reviewed in refs 1 and 2]. The organelle origin and structural organization of these membranous replication sites appears to vary for different viruses, but little is known about the mechanisms by which these viruses rearrange cellular membranes. Enteroviruses, a group of important human pathogens that includes poliovirus, coxsackievirus and echovirus, induce a massive accumulation of large vesicular membrane structures in the cytosol. Several lines of evidence suggest that these vesicular structures (which are further referred to as ?replication vesicles?) are derived from the early secretory pathway, but little is known about their exact origin, which cellular and viral proteins are involved in their production, their structure, and how the viral replication complexes are organized at these membranes. Aim: This research proposal is aimed at gaining more insight into how enteroviruses subvert secretory pathway membranes for replication of their genomic RNA. Specifically, the following questions will be addressed: (i) What is the origin of the replication vesicles and what causes their accumulation? (ii) Which cellular proteins / machineries are involved in their production? (iii) What are the cellular targets of the viral proteins implicated in vesicle production? (iv) What is the 3-dimensional structure of these vesicular membranes and what is the architecture of the viral RNA and viral proteins within the viral RNA replication complexes that are associated with these membrane structures? Approach: To elucidate the mechanism used by enteroviruses to rearrange cellular membranes, an integrated approach consisting of four parts is proposed, involving both molecular, biochemical, genetic, cell biological, and microscopical procedures. All experiments will be performed with coxsackievirus B3 (CVB3) in human cell lines that are routinely used to study the molecular and cell biological aspects of enterovirus infection (i.e., HeLa, HepG2, HEK293). These cell lines allow efficient virus replication and can be transfected with high efficiency. (i) To gain more insight into the origin of the enterovirus replication vesicles and accumulation, the replication vesicles induced by coxsackievirus will be characterized by immunofluorescence for the presence / absence of specific markers for secretory pathway (sub)compartments, coat complexes (e.g., COP-II and COP-I) and their regulators (e.g., GTPases like Sar1 and Arf1 as well as their GEFs and GAPs), and targeting/fusion machinery components like Rabs and SNAREs. Moreover, Fluorescence Recover After Photobleaching (FRAP) microscopy will be applied to investigate whether vesicle production/accumulation is due in alterations in the normal dynamic behaviour of Sar1/COP-II or Sarf1/COP-I machinery components. (ii) To investigate which cellular proteins / machineries are involved in vesicle production, an automated and fluorescence-based high-throughput RNA interference (RNAi) screening platform will be used in which replication of a GFP-expressing coxsackievirus will be tested (in collaboration with Dr. R. Pepperkok, Cell Biology and Biophysics Unit, EMBL, Heidelberg, Germany). (iii) Viral proteins 2BC, 3A and 3CD have been implicated in vesicle production but their role and the underlying mechanism are elusive. Microscopical (bimolecular fluorescence complementation) and genetic/biochemical techniques (co-immunoprecipitation and, optionally, a tandem affinity purification procedure) will be used to identify cellular targets of these viral proteins. (iv) To gain more insight into the structure of the replication vesicles (e.g., are the replication vesicles separate structures or are they interconnected?) and the spatial organization of the replication vesicles and the associated viral RNA replication complexes (what is the nature, topology, stoichiometry of the viral RNA and viral proteins that are associated with the replication vesicles?), the 3D organization of the replication vesicles and the association of the viral RNA and the viral proteins with these membranes will be studied by an immuno-electron tomography approach (in collaboration with Dr. J. Klumperman, Dept Cell Biology, Institute of Biomembranes, University Medical Center Utrecht, The Netherlands). Relevance: Unraveling the mechanism by which enteroviruses rearrange intracellular membranes is of utmost importance for a fundamental understanding of how viruses with a plus-strand RNA genome replicate themselves. Elucidation of this mechanism may furthermore reveal new, fundamental key features of intracellular membrane transport that occur in uninfected cells and exploited by viruses. |
