De novo initiation of RNA synthesis by the arterivirus RNA-dependent RNA polymerase

Abstract

All plus-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that functions as the catalytic subunit of the viral replication/transcription complex, directing viral RNA synthesis in concert with other viral proteins and, sometimes, host proteins. RNA synthesis essentially can be initiated by two different mechanisms, de novo initiation and primer-dependent initiation. Most viral RdRps have been identified solely on the basis of comparative sequence analysis, and for many viruses the mechanism of initiation is unknown. In this study, using the family prototype equine arteritis virus (EAV), we address the mechanism of initiation of RNA synthesis in arteriviruses. The RdRp domains of the members of the arterivirus family, which are part of replicase subunit nsp9, were compared to coronavirus RdRps that belong to the same order of Nidovirales, as well as to other RdRps with known initiation mechanisms and three-dimensional structures. We report here the first successful expression and purification of an arterivirus RdRp that is catalytically active in the absence of other viral or cellular proteins. The EAV nsp9/RdRp initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-specific manner. In addition, the requirements for initiation of RNA synthesis from the 3' end of the viral genome were studied in vivo using a reverse genetics approach. These studies suggest that the 3'-terminal nucleotides of the EAV genome play a critical role in viral RNA synthesis.

FIG. 1.

Structure-based sequence alignment of RdRp domains of four arteriviruses, three CoVs, poliovirus (Picornaviridae), rabbit hemorrhagic disease virus (RHDV; Caliciviridae), hepatitis C virus (HCV; Flaviviridae), bovine viral diarrhea virus (BVDV; Flaviviridae), bacteriophage Φ6 (Cystoviridae), and mammalian orthoreovirus (Reoviridae). The picornavirus and calicivirus enzymes are VPg-primer-dependent RdRps, whereas the flavivirus, cystovirus, and reovirus RdRps initiate RNA synthesis de novo. Details on the alignment are given in Material and Methods. The complete alignment is available from the authors upon request. (A) Alignment of motifs G and F3 (finger domain) as well as A, B, and C (palm domain). The predicted secondary structure for the EAV RdRp is depicted above the sequence. Secondary-structure elements of proteins with known structures are given below the alignment. Residues conserved in all RdRps are in white on a dark gray background. Residues conserved in more than 75% of the sequences are boxed and have a light gray background. Black boxes highlight motif G (T/SX_1-2GX_0-1P), which is conserved in Coronaviridae, Picornaviridae, and Caliciviridae, and the SDD signature sequence of motif C that is typical of nidovirus RdRps. (B) Alignment of RdRp thumb domains, starting with motif E. Residues near the N-terminal and C-terminal ends of the conserved β-hairpin are boxed in black. The numbering of BVDV and reovirus RdRp sequences starts with the first residue considered to be part of the RdRp core domain; the sequence of the reovirus RdRp ends with the last residue of the RdRp domain. The RdRp domains were aligned, and the alignments were adjusted according to the predicted secondary structure, as depicted. The aligned sequences and NCBI accession numbers are the following: EAV (NP_127506), lactate dehydrogenase-elevating virus neurovirulent type C (LDV_NV; NP_065670), simian hemorrhagic fever virus (SHFV; NP_742092), porcine reproductive and respiratory syndrome virus (PRRSV; NP_066135), mouse hepatitis virus, strain A59 (MHV-A59; NP_068668), human CoV 229E (HCoV-229E; NP_068668), and SARS-CoV, strain Toronto 2 (SARS_Tor2; NP_828849). The RdRps with known three-dimensional structures are poliovirus (Polio; PDB code 1RA6), RHDV (PDB code 1KHW), HCV (PDB code 1C2P), BVDV (PDB code 1S48), Φ6 (PDB code 1HHS), and mammalian orthoreovirus (PDB code 1MUK).

FIG. 2.

Expression and purification of EAV nsp9/RdRp-His. Coomassie brilliant blue staining of a sodium dodecyl sulfate-polyacrylamide gel showing the two-step purification of the enzyme by Ni-NTA affinity chromatography (Ni-NTA) and S200 gel filtation (GF). C, total protein in the cell lysate; FT, flowthrough; S, soluble fraction; W, waste fraction; P, peak fraction; 26, fraction 26 of the Ni-NTA eluate; M, molecular size marker. On the right side of the gel, different fractions of the run on the S200 gel filtration column are shown. Fractions 32 to 36 were pooled to obtain the nsp9/RdRp-His preparation used in this study.

FIG. 3.

Activity of the purified EAV nsp9/RdRp on a poly(A)/oligo(dT) primer-template complex. (A) Time course of [³H]UTP incorporation (in cpm) on poly(A)/oligo(dT) using two concentrations of EAV RdRp (2 and 4 μM). As a specificity control, the reaction with 4 μM of enzyme was also carried out in the presence of rifampin (rif.), an inhibitor of E. coli DNA-dependent RNA polymerase. RNA synthesis was monitored in a filter-binding assay, followed by liquid scintillation counting. The data set on the right (C-) shows the results of a control reaction without the enzyme. (B) Optimization of the concentration of the catalytic ions Mg²⁺ and Mn²⁺ for RNA synthesis on poly(A)/oligo(dT). [³H]UTP incorporation (in cpm) after 30 min in the presence of increasing concentrations of Mg²⁺ and Mn²⁺ was measured by a filter-binding assay and liquid scintillation counting.

FIG. 4.

De novo and primer-dependent activity of the purified EAV nsp9/RdRp on homopolymeric RNA templates. [³H]UTP incorporation was measured by a filter-binding assay and liquid scintillation counting after a 30-min reaction on the homopolymeric templates poly(A) (pA), poly(C) (pC), and poly(U) (pU). Besides de novo-initiated RNA synthesis (the left bar of each set), primer-dependent activity was tested using 18-nucleotide DNA or RNA oligonucleotides complementary to the template. Activity was measured in three independent experiments, and incorporation on poly(A)/oligo(dT) was arbitrarily set at 1.

FIG. 5.

Visualization of RNA products derived from EAV nsp9/RdRp activity. (A) De novo-initiated synthesis and primer-dependent RNA synthesis were tested on the homopolymeric templates poly(A) (pA), poly(C) (pC), and poly(U) (pU) and on viral RNA templates representing the 3′-proximal domain of the EAV genome either with (EAV-A) or without (EAV) a poly(A)₂₀ tail. Reaction products were analyzed on a 6% polyacrylamide-7 M urea gel. For the reactions analyzed in lanes 1 to 5 and 11 to 15, α-³²P-labeled nucleotides were used. For lanes 6 to 10 and 16 to 20, complementary γ-³²P-labeled 18-nucleotide DNA oligonucleotides were annealed onto the templates poly(A)/oligo(dT), poly(U)/oligo(dA), poly(C)/oligo(dG), EAV-A/oligo(dT), and EAV/oligo(dT) [although oligo(dT) is not complementary to the EAV template]. Subsequently, these RdRp assays were performed in the presence of unlabeled nucleotides only. The full-length product is marked FL, and for better visualization of this product a shorter exposure of the top part of the gel is shown below. (B) Demonstration of de novo initiation using a poly(U) template (lanes 7 to 12) and a poly(A)/oligo(dT) primer-template combination (lanes 1 to 6). The reactions were stopped at different time points (0, 5, 10, 30, 60, and 120 min) and analyzed on a 14% polyacrylamide-7 M urea gel. Several short reaction products were detected, corresponding to dinucleotides (U2 and A2), trinucleotides (U3 and A3), and tetranucleotides (U4 and A4). In addition, some diphosphate products were detected, migrating slower than their corresponding triphosphate products (compare ATP and UTP to ADP and UDP).

FIG. 6.

Time course analysis of de novo and primer-dependent initiation of RNA synthesis by the purified EAV nsp9/RdRp. [³H]UTP incorporation was measured using the homopolymeric templates poly(A) (pA) and poly(U) (pU) and viral RNA templates encompassing the EAV 3′ genome terminus with (EAV-A) or without (EAV) a poly(A)₂₀ tail. De novo-initiated activity and activity stimulated by the presence of an oligo(dT) DNA primer were monitored using filter-binding assays and liquid scintillation counting.

FIG. 7.

Analysis of EAV mutants carrying mutations in the 3′ terminus of the genome. (A) BHK-21 cells were transfected with a series of mutants in which the two terminal C residues were replaced with either G (cg, gc, and gg) or U (cu, uc, uu). Also, a mutant lacking the poly(A) tail (noA) was included. The production of viral proteins was monitored by immunofluorescence microscopy using antisera recognizing replicase subunit nsp3 and nucleocapsid protein (N). In addition, supernatants harvested from the transfected cell cultures at 24 h posttransfection were tested for the presence of progeny virus using plaque assays. (B) Viral RNA synthesis of EAV mutants in the 3′ genome terminus. Infectious RNA was transfected into BHK-21 cells, and intracellular RNA was isolated at 14 h posttransfection. The RNA was separated in a denaturing agarose gel and analyzed by hybridization to an oligonucleotide detecting all plus-strand viral RNAs. The positions of the genome (RNA1) and sg mRNAs (RNA2 to RNA7) are indicated. wt, wild type; hpt, hours posttransfection.