Positive-strand RNA viruses such as poliovirus replicate their genomes on intracellular membranes of their eukaryotic hosts. The RNA-dependent RNA polymerase of poliovirus, 3D, can be linked to intracellular membranes via interaction with a membrane-associated viral protein, 3AB. The surfaces by which polymerase 3D binds to 3AB, and through which the polymerase oligomerizes to form large planar lattices, have been defined by x-ray crystallography, site-directed mutagenesis and electron microscopy. Membranous vesicles isolated from infected cells contain structures consistent with the presence of two-dimensional polymerase arrays on their surfaces. However, the hypothesis that RNA replication in poliovirus-infected cells occurs on catalytic lattices of polymerase remains to be tested explicitly.
In Specific Aim 1, a collection of defined temperature-sensitive viruses will be used to monitor the recruitment of 3AB and 3D polymerase to membranes during infection, to identify the intermolecular contacts required for this recruitment, to visualize the resulting complexes by freeze-etch electron microscopy, and to test the genetic consequences of polymerase oligomerization.
In Specific Aim 2, assays to identify the path of bound template RNA along a polymerase oligomer and tests to probe the mechanism by which long RNA templates can be copied by immobilized polymerase oligomers are described.
In Specific Aim 3, the surprising finding that the protein primer, 3B, binds to a polymerase surface distinct from the known binding sites of nucleic acid primers observed with other polymerases, is explored. Experiments are presented to determine how a polymerase can catalyze phosphodiester bond formation for substrates bound at two different sites, and whether this requires the formation of polymerase-containing oligomers. For one other genome, that of DNA bacteriophage phi29, the use of oligomeric lattices during membrane-associated replication has been proposed. It has also been reported recently that human telomerase and hepatitis C virus RNA-dependent RNA polymerase undergo oligomerization that is likely to be required for their function. Thus, these studies with poliovirus, for which an abundance of structural, cell biological and genetic tools exists, may provide a paradigm for membrane-associated nucleic acid synthesis in many systems. Finally, we provide a genetic rationale for choosing oligomeric proteins as antiviral targets. ? ?
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