Intramolecular and Intermolecular Uridylylation by Poliovirus RNA-Dependent RNA Polymerase

Abstract

The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3 end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65-to 70-kDa host protein, in lysates of virus-infected human cells.Many positive-sense single-stranded RNA viral genomes are relatively small templates that encode information for complex viral replication cycles and thus require highly efficient utilization of limited coding capacity. The number of functional activities expressed from any genome can be expanded by utilizing both precursor polypeptides and their processed cleavage products. For example, poliovirus protein 3D is an RNA-dependent RNA polymerase, while its presumed precursor, 3CD, which is a fusion between the 3C protease and 3D polymerase, manifests no polymerase activity but functions as a specific protease with substrate recognition properties different from its cleavage product, 3C protease (29, 68). Another mechanism that expands coding capacity is the utilization of the same polypeptide for multiple functions. For example, in addition to proteolytic activity, 3CD also functions as a specific RNAbinding protein with crucial roles in viral RNA replication. It binds the 5Ј-terminal RNA cloverleaf structure (2, 3, 23, 49) as well as an internal stem-loop structure in the 2C coding region (66, 67); both interactions are required for the initiation of RNA replication. Finally, posttranslational modifications can further modify the function of viral proteins; for example, the covalent myristoylation of viral capsid protein VP0 facilitates its transition from a precursor protein to a component of an assembled capsid (4, 42).The poliovirus genome contains a single open reading frame that c...

Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro

“…As mentioned above, the RNA-dependent RNA polymerase of poliovirus also uses the protein primer VPg, which is not covalently linked to the polymerase, to initiate viral RNA synthesis (32). Similar to what we found for the DHBV RT protein, the poliovirus RNA polymerase could also use priming site(s) on the polymerase protein itself to initiate RNA synthesis, i.e., self uridylylation or the covalent attachment of UMP to the RNA polymerase (37). Although uridylylation of the poliovirus RNA polymerase was found to be dispensable for viral replication, the viral RNA polymerase was found to uridylylate also cellular proteins.…”

The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

“…Only in the presence of Mn 2ϩ , poly(rA), and VPg, did CVB3 3D pol generate two bands. The faster-migrating double-band can be attributed to VPg-pU(pU) (4,48,53). The slower-migrating band might correspond to VPg-poly(U) produced by primer elongation on poly(rA), 3D…”

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

“…Interestingly, a very recent study by Richards et al describes the uridylylation of 3AB in detergent-containing solutions by 3D pol on an oligo A 15 template but not on longer poly(A) templates (53). (This template length-dependent difference has been attributed to a lack of oligomerization of the polymerase on the small RNA template (53). In contrast, VPg itself and minor cleavage intermediates in polyprotein processing (3BC and 3BCD; not shown in Fig.…”

“…3AB has been suggested to be the precursor for uridylylation (52), but under standard in vitro reaction conditions purified 3AB (in detergent) does not serve as substrate for uridylylation (A. Paul and E. Wimmer, unpublished results). Interestingly, a very recent study by Richards et al describes the uridylylation of 3AB in detergent-containing solutions by 3D pol on an oligo A 15 template but not on longer poly(A) templates (53). (This template length-dependent difference has been attributed to a lack of oligomerization of the polymerase on the small RNA template (53).…”