A Role for 3AB Protein in Poliovirus Genome Replication

Abstract

The poliovirus polypeptide 3AB, the precursor of the genome-bound VPg protein, stimulates in vitro the synthesis of poly(U) directed by the viral polymerase 3Dpol (Lama, J., Paul, A., Harris, K., and Wimmer, E. (1994) J. Biol. Chem. 269, 66-70), suggesting that 3AB could be modulating the activity of the viral polymerase in poliovirus-infected cells. To address the exact function of 3AB in the viral replication cycle, a biochemical and molecular genetic analysis of 3AB has been carried out. 3AB protein bound RNA probes in two different assays, and amino acid positions implicated in the RNA binding activity of 3AB were determined. Mutant proteins with reduced RNA binding activity were unable to stimulate 3Dpol polymerase activity. Purified protein 3A showed no RNA binding or 3Dpol stimulatory activity, but 3A and VPg mutations conferred a synergistic effect on the 3AB functions. Polioviruses encoding for these mutant 3ABs were constructed. These mutant viruses translated their RNA genomes in vitro and processed their polyproteins as wild type virus did. Cells infected with 3AB mutant viruses showed over 90% inhibition in the accumulation of plus and minus viral RNA strands and more than 100-fold reduction of virus yield at 4 h postinfection. Our results suggest that 3AB protein functions in vivo as a co-factor of the viral polymerase and that the activity of 3AB may be regulated by proteolytic processing.

Biochemical and Genetic Studies of the VPg Uridylylation Reaction Catalyzed by the RNA Polymerase of Poliovirus

“…This shortened version exhibited only about one-half of the wt VPg activity, indicating either that the overall length of the peptide cannot be changed or that one or more of the deleted amino acids are important for function. The latter possibility is supported by the observation that a K20E substitution in VPg results in a virus with defective growth properties (24).…”

In vitro interactions between a potyvirus-encoded, genome-linked protein and RNA-dependent RNA polymerase.

“…Moreover, the NIa and VPg polypeptides are capable of stimulating the inherent RNA polymerase activity of the NIb protein. This latter property is similar to that of the poliovirus 3AB protein, which contains the VPg of this virus and is a stimulatory co-factor for RNA synthesis catalysed by 3D pol (Lama et al, 1994(Lama et al, , 1995Molla et al, 1994 ;Plotch & Palant, 1995). Our studies suggest that a plant virus VPg can play an active role in viral RNA replication, which is in addition to its role as the genome-linked protein.…”

“…3AB and 3B) can stimulate the activity of purified poliovirus 3D pol (Lama et al, 1994(Lama et al, , 1995Molla et al, 1994 ;Plotch & Palant, 1995). With poliovirus, the 3AB protein, which includes the VPg, is much more effective in stimulating the polymerase activity of 3D pol than is the VPg by itself (Lama et al, 1994(Lama et al, , 1995Molla et al, 1994 ;Plotch & Palant, 1995). In contrast, the TVMV VPg is itself an effective activator of NIb (Fig.…”

Selective Stimulation of Hepatitis C Virus and Pestivirus NS5B RNA Polymerase Activity by GTP

“…Similar to what we describe here for NS5B, RNA synthesis stimulated by 3AB is primer-dependent. 3AB has a dual function; it can interact with 3D pol (52), and it is an RNA-binding protein able to interact with the template-primer (53). Mutational ablation of RNA binding also reduces 3D pol stimulation, suggesting that 3AB enhances interaction between the template and 3D pol (51,53).…”

“…3AB has a dual function; it can interact with 3D pol (52), and it is an RNA-binding protein able to interact with the template-primer (53). Mutational ablation of RNA binding also reduces 3D pol stimulation, suggesting that 3AB enhances interaction between the template and 3D pol (51,53). An additional function of VPg is to act as an acceptor of UMP added by 3D pol to the OH group of a tyrosine residue within VPg in a template-dependent reaction (54).…”