Similar Structural Basis for Membrane Localization and Protein Priming by an RNA-dependent RNA Polymerase

Abstract

Protein primers are used to initiate genomic synthesis of several RNA and DNA viruses, although the structural details of the primer-polymerase interactions are not yet known. Poliovirus polymerase binds with high affinity to the membrane-bound viral protein 3AB but uridylylates only the smaller peptide 3B in vitro. Mutational analysis of the polymerase identified four surface residues on the three-dimensional structure of poliovirus polymerase whose wild-type identity is required for 3AB binding. These mutants also decreased 3B uridylylation, arguing that the binding sites for the membrane tether and the protein primer overlap. Mutation of flanking residues between the 3AB binding site and the polymerase active site specifically decreased 3B uridylylation, likely affecting steps subsequent to binding. The physical overlap of sites for protein priming and membrane association should facilitate replication initiation in the membrane-associated complex.The replication of linear nucleic acids without loss of coding information from the ends is a problem that has been solved in several ways by cellular and viral genomes. Solutions include those used by vaccinia virus, which primes DNA replication from hairpins that can be refolded to regenerate the terminal sequences (1, 2), by Drosophila, which repair damaged chromosomal termini by recombination (3), by many eukaryotic cells, which add end-specific telomeric sequences postreplicatively, and by viruses such as adenovirus and ⌽29, which use protein primers that are covalently linked to the initiating nucleotides (reviewed in Ref. 4). For mammalian positive-strand RNA viruses such as poliovirus and hepatitis C, two mechanisms are suspected: de novo initiation (5-8) and protein priming (9) from the genomic ends.The protein primer for the synthesis of poliovirus RNA includes, at a minimum, the 22-amino acid viral peptide 3B (also called VPg), which is found covalently linked to the 5Ј ends of all newly synthesized positive and negative strands. Poliovirus translates its proteins as a single, large polyprotein that is cleaved into the proteins required for virion formation, host modification, and RNA replication. In many cases, proteolytic precursors have functions distinct from those of the limit digestion products. Evidence for the use of 3B. As a protein primer comes from in vitro experiments in which it was demonstrated that 3B is uridylylated in the presence of UTP, the poliovirus RNA-dependent RNA polymerase (3D), and an RNA template (9). The RNA used to template the uridylylation of 3B can either be poly(A), poliovirus RNA, or the small cis replication enhancer RNA (10, 11), an internal sequence required for RNA replication in infected cells (12). Whether 3B itself serves as the primer within infected cells and how it is brought into the RNA replication complex are not yet known.Evidence for direct binding between polymerase and 3B was observed in the two-hybrid system (13), although a stronger signal was observed between the polymerase and a larger polypeptide th...

Ultrastructural and biochemical analyses of hepatitis C virus-associated host cell membranes

“…Depending on the virus, replication may occur on rearranged convoluted membranes, single or double membrane vesicles of various sizes derived from the endoplasmic reticulum (ER), the intermediate compartment between the ER and the Golgi apparatus, the trans-Golgi network, mitochondria or even lysosomes (Miller & KrijnseLocker, 2008). These membrane structures induced by positive-strand RNA viruses probably serve as a scaffold for the assembly of viral replication complexes by providing an organization and environment facilitating viral replication (Lyle et al, 2002;Schwartz et al, 2002). Some positivestranded RNA viruses including some strains of poliovirus (Jackson et al, 2005), coxsackievirus B3 (Wong et al, 2008), dengue virus (Lee et al, 2008) and mouse hepatitis coronavirus (Prentice et al, 2004) may take over the host autophagy machinery to facilitate their own replication.…”

Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication

“…Yeast two hybrid and biochemical analyses have indicated that the 3AB protein strongly interacts with 3D pol and the sequences primarily responsible for this interaction are located in the 3B domain of the protein (9,11). Four amino acids in a hydrophobic patch on the surface of 3D pol were recently identified as binding partners of 3AB (8). Protein 3AB also has the propensity to dimerize and form higher oligomers in solution in the absence of detergent.…”

“…Some of the viral nonstructural proteins (2B, 2C ATPase and 3A) associate with the replication complex via their own membrane binding domains while others are recruited to the complex by protein/protein or protein/RNA interactions (1,7). The PV RNA polymerase 3D pol is an example of a soluble protein that interacts with another protein, 3AB, whose hydrophobic domain, located in 3A, anchors the complex to membranes (8,9). 3AB is a small basic protein that contains a hydrophobic and membrane-binding region near the C-terminus of its 3A domain.…”

Formation of Higher-Order Foot-and-Mouth Disease Virus 3D pol Complexes Is Dependent on Elongation Activity

“…This is particularly evident with the less-active serotype O enzyme. The addition of ZnCl 2 (60 M) to enhance the activity according to previously reported PV protocols (2,8,9,12,21) had little effect. Using a similar assay, we showed that the D388A mutant was unable to incorporate [ 32 P]UTP, as expected (Fig.…”