Characterisation of the RNA-dependent RNA polymerase from Rabbit hemorrhagic disease virus produced in Escherichia coli

Abstract

All positive-strand RNA viruses encode a RNA-dependent RNA polymerase which in most cases has been only identified on the basis of its sequence conservation. Catalytic activity has been experimentally demonstrated in only a handful of these viral proteins, including that from Rabbit hemorrhagic disease virus. Studies from our laboratory have reported that RHDV RNA polymerase produced in Escherichia coli was enzymatically active showing poly(A)-dependent poly(U) polymerase as well as RNA polymerase activity on heteropolymeric substrates. In this work, we have investigated the in vitro activity of the recombinant 3Dpol from RHDV, including ion requirements, resistance to inhibitors, substrate specificity as well as data on the initiation mechanism of the template-linked products derived from heteropolymeric RNA substrates. Our study demonstrates that in an in vitro reaction recombinant RHDV RNA polymerase generated the minus strand of the heteropolymeric RNA substrates by a "copy-back" mechanism that initiated at the template 3'-terminal OH.

Characterization of a Nodavirus Replicase Revealed a de Novo Initiation Mechanism of RNA Synthesis and Terminal Nucleotidyltransferase Activity

“…To further exclude the possibility that a copy-back mechanism is used by protein A, we designed templates, (Ϫ)-RNA1(1-100) and (Ϫ)-RNA1(1-100)pA, in which the 3Ј-OHs were oxidized. The oxidized 3Ј-OH cannot be used to add extra nucleotides for oligonucleotide elongation and thus would completely block RNA synthesis via a copy-back mechanism (10,11,43). Our results showed that the templates with oxidized 3Ј-OH exhibited the same ability to initiate RNA synthesis (Fig.…”

“…10D, lanes 6 -8). On the other hand, (Ϫ)-RNA1(3-30), but not (Ϫ)-RNA1 (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) or (Ϫ)-RNA1(3-10), was required for the TNTase activity (Fig. 10D, lanes 3-5).…”

“…The blocking of RNA template was conducted by oxidating the 3Ј-hydroxyl group (3Ј-OH), using sodium metaperiodate (NaIO 4 ) (10,11,43). In brief, the RNA templates were placed in 100 mM sodium acetate (pH 5.0) and 100 mM NaIO 4 .…”

Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

“…The de novo initiation mechanism is primer-independent, and RdRPs do not require primer, but normally synthesize dinucleotides as the starting nucleotides on the 3′-end of RNA templates for adding next nucleotides [60], [61]. On the other hand, the primer-dependent initiation mechanism requires the involvement of a primer, which could be an oligonucleotide or a protein [33], [34], [35], [36]. For example, rabbit hemorrhagic disease virus and hepatitis C virus, both of which are (+)-RNA viruses, use RNA primers for the synthesis initiation of their RNA genome [33], [34], [35], while picornaviruses use a uridylylated form of the VPg protein (also named as nonstructural protein 3B) as primer for the synthesis of both (+)- and (−)-RNAs [36].…”

“…So far, viral RdRPs have been reported to employ two principally distinct mechanisms for RNA synthesis initiation [31], [32], including primer-dependent and primer-independent ( de novo ) initiation. The primer-dependent initiation of RNA synthesis requires the involvement of either an oligonucleotide or a protein primer [33], [34], [35], [36], while the de novo initiation does not require primer, but uses the starting nucleotide to provide the 3′ hydroxyl group (3′-OH) for adding the next nucleotide. In the case of de novo initiation, RNA synthesis can be initiated at the terminal or an internal site of the template RNA [28], [29], [31], [37], [38].…”