Factors Required for the Uridylylation of the Foot-and-Mouth Disease Virus 3B1, 3B2, and 3B3 Peptides by the RNA-Dependent RNA Polymerase (3D ^pol ) In Vitro

Abstract

The 5 terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D pol . To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D pol in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5 untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.Picornaviruses, including foot-and-mouth disease virus (FMDV), poliovirus (PV), and human rhinoviruses (HRVs), have a positive-sense RNA genome of about 8 kb that is infectious (1, 5). Following virus attachment and entry into the cell, the RNA genome is delivered into the cytoplasm and translation of the genome is required to produce the viral proteins that are necessary for virus assembly and RNA replication. The genome encodes a single large polyprotein that is processed, largely by internal trans-acting proteases, to produce about 12 mature proteins plus various precursors (some of these have distinct functions). The proteins encoded within the P1 region form the capsid, while proteins encoded in the P2 and P3 regions are required for RNA replication. After some rounds of translation, there has to be a switch so that translation of the viral RNA is stopped and RNA replication can commence, since these two processes appear incompatible on the same RNA molecule (11). Most picornaviruses replicate with high efficiency within susceptible cells, and within a few hours, the amount of viral RNA can represent 5% of the total RNA in cells (a level similar to that of all the cellular cytoplasmic mRNAs together). Nearly all of the FMDV RNA generated by replication is infectious; indeed, microinjection of cells with as little as 1 to 2 molecules of viral RNA is sufficient to initiate an infection (4). Thus, the replication of FMDV genomes within cells is remarkably efficient.To replicate the positive-sense genome, an antisense RNA has to be synthesized which then functions as the template for the production of new positive-sense infectious genomes (32). RNA is synthesized by the viral 3D protein that functions as an RNA-dependent RNA polymerase and will be referred to as 3D pol . Interestingly, 3D pol requires the uridylylated form of the 3B/VPg peptide (VPgpU or VPgpUpU) to act as the primer for both positive-and negative-strand synthesis. In recent...

Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus

“…The RNA-dependent RNA polymerase (RdRp), known as 3Dpol, responsible for replication of the genome (via negative strand intermediates), is located at the C-terminus of the polyprotein. Synthesis of the viral RNA is initiated by VPg, which undergoes post-translational uridylation by 3Dpol in conjunction with the precursor protein, 3CD, in the presence of cre (Nayak et al 2005). cre is known to contain an essential AAACA motif; however, the details of the uridylation and priming mechanisms are poorly understood, and although these are common to all picornaviruses, there are a number of details that are unique to FMDV.…”

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

“…Over the past 8 years much has been learned about oriItemplated VPg uridylylation in vitro for a variety of picornaviruses (28,49,53,77,92). However, it is still unclear whether or not VPg, 3BC(D), or 3AB is used in vivo to initiate genome replication.…”

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

“…Some strains of FMDV complete the entire life cycle in only 3 h in vitro (20). Uridylation of VPg to form VPgpUpU is carried out by 3D pol but requires the presence of 3CD in an ancillary catalytic role to work efficiently (15). Although synthetic versions of VPg peptide can be uridylated in vitro, the true precursor in vivo (3AB or 3BCD) is unknown and controversial.…”

“…The 5= UTR is exceptionally long (ϳ1,200 nucleotides), constituting oneseventh of the viral genome. It includes a 400-nucleotide region predicted to be double stranded in structure (3), a poly(C) tract of ϳ200 nucleotides (20), a series of pseudoknots (3,6), a cis-acting replication element (cre) necessary for the uridylation of the peptide primer of replication (VPg) (13,15), and an internal ribosome entry site (IRES) necessary for the initiation of protein synthesis (18). The 3= UTR is shorter but includes two stem-loop structures and a poly(A) tract.…”

“…MW, molecular weights in thousands. (B) Samples of 3D pol (C) and 3D pol (O) were assayed for elongation activity using the incorporation of [ 32 P]UTP in a MOPS (morpholinepropanesulfonic acid)/NaCl buffer (with 5 mM MgCl 2 ) with a poly(A) template (0.1 g/l) and an oligo(U) 15 primer (1 M) as described previously (5). Samples were assayed after 20 or 25 min, respectively.…”