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Chapter 8 : Genome Replication II: the Process
Category: Viruses and Viral Pathogenesis
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In this chapter the authors attempt to dissect the mechanisms that underlie picornavirus RNA replication by first addressing how the viral template RNA may be specifically recognized. They then discuss the coordination of viral translation with negative-strand RNA synthesis. This discussion is followed by a description of the organization of proteins in the RNA replication complex on two-dimensional membrane surfaces, with an emphasis on viral proteins that rearrange cytoplasmic membrane structures and tether RNA replication complexes to the rearranged membranous vesicles as well as viral polymerase proteins and the proteins with which they interact. Then, the chapter describes how the cascade of proteolytic processing contributes to the formation and maturation of picornavirus RNA replication complexes. Finally, steps in the synthesis and utilization of the protein-nucleotidyl primer for initiation of picornavirus RNA synthesis are presented as a lead-in to a discussion of RNA chain elongation and the topology of the RNA in the viral RNA replication complex. Given the unique viral protein-protein and protein-RNA interfaces highlighted in the processes, picornavirus RNA replication remains an attractive target for the development of small-molecule inhibitors that disrupt this crucial part of the viral replication cycle.
Ways to achieve tethering of a newly synthesized protein to its mRNA. (A) RNA display. Short RNAs with randomized regions were translated in vitro. When the sequence is engineered so that the last amino acid is unique, and present in the mixture as a puromycin derivative, the newly synthesized polypeptide can become covalently attached to the RNA. Then, selection for the functions of the peptide also selects for its “recipe,” to which it is covalently bound. The illustration was adapted from reference 69 with permission of the publisher. (B) Model for the requirement for translation in cis for an approximately 7,500-nucleotide picornavirus positive-strand RNA to assemble into an RNA replication complex. Certain newly synthesized proteins display high nonspecific binding affinities both for RNA and for membranes. As a consequence, the mRNA that encodes the new proteins is brought to the target membranes with them. (Modified from reference 57 .)
Polymerase-VPg complexes. (A) UMP-VPg-binding site at the polymerase active site, as seen in the cocrystal with FMDV polymerase and FMDV VPg in the presence of UTP. (B) VPg-binding site on the “back” of the polymerase, as seen in the cocrystal of CVB3 polymerase bound to CVB3-encoded VPg in the absence of UTP. (Panels A and B were modified from illustrations published in reference 47 .) (C) The locations of the two VPg-binding sites on an assemblage of poliovirus polymerase molecules interacting along Interface I; as neither of these complexes has been observed structurally for poliovirus, the locations are indicated with shaded circles. (Modified from reference 75 .) T, thumb contacts of Interface I; P, palm contacts of Interface I.
Replication of double-stranded and single-stranded templates with immobilized replication complexes. (A) Movement of a double-stranded template past an immobilized, tracking replication complex (vertical column) creates positive supercoils in front of the replication fork and negative supercoils behind it. These can only be relaxed by free rotation of the ends (marked with asterisks) or by breaking one of the single strands, as with a nuclease (which would leave a nick or a break), a combination of a nuclease and a ligase, or a topoisomerase. (Modified from reference 80 with permission from the publisher.) (B) Movement of single-stranded templates along immobilized replication complexes does not present topological problems but does require movement of the template and primer strands and some mechanism to prevent their stable annealing.
Effects of various postulated polymerase-polymerase, polymerase-3C, and 3C-3C contacts on viral viability