A complex zinc finger controls the enzymatic activities of nidovirus helicases - PubMed (original) (raw)

A complex zinc finger controls the enzymatic activities of nidovirus helicases

Anja Seybert et al. J Virol. 2005 Jan.

Abstract

Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.

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Figures

FIG. 1.

FIG. 1.

Sequences of the N-terminal ZBDs associated with the arterivirus EAV nsp10 and coronavirus HCoV-229E nsp13 helicases. Amino acid residues are numbered according to their positions in the pp1ab replicase polyproteins of EAV and HCoV-229E, respectively. 3C-like protease cleavage sites that flank the EAV and HCoV-229E helicases in pp1ab are given, and the hinge spacer region of nsp10 (residues Glu2427 to Pro2430) is underlined. Also indicated are the amino acid substitutions characterized in this study.

FIG. 2.

FIG. 2.

Effects of substitutions of conserved ZBD Cys and His residues on the enzymatic activities of EAV nsp10 in vitro and on EAV reproduction in BHK-21 cells. Helicase (duplex-unwinding) activities were determined by using a forked DNA substrate containing a 22-bp duplex region and 30-nucleotide, single-stranded oligo(dT) tails (24). This partial-duplex DNA substrate was incubated with 2 pmol (if not indicated otherwise) of purified MBP-nsp10 or its mutant derivatives (for details, see Materials and Methods). Reaction products were analyzed on nondenaturing polyacrylamide gels. Lane 1, incubation without protein; lane 2, heat-denatured substrate; lane 3, MBP-nsp10 (wild type [WT]); lane 4, MBP-nsp10_C2377H; lane 5, MBP-nsp10_C2395H; lane 6, MBP-nsp10_C2395A; lane 7, MBP-nsp10_H2399C; lane 8, MBP-nsp10_H2399A; lane 9, MBP-nsp10_C2412H; lane 10, MBP-nsp10_H2414C; lane 11, MBP-nsp10_H2414A; lane 12, MBP-nsp10_K2534Q; lane 13, 1 pmol of MBP-nsp10_K2534Q and 1 pmol of MBP-nsp10_C2377H; lane 14, MBP-nsp10_K2534Q and MBP-nsp10_C2377H; lane 15, 1 pmol of MBP-nsp10_K2534Q and 1 pmol of MBP-nsp10_H2399C. Below the gel, the relative ATPase activities of the corresponding proteins are given. The activity of the wild-type protein, MBP-nsp10, was taken to be 1.0, and all other ATPase activities were normalized to this value. Also listed are the phenotypes in tissue culture of EAV mutants containing the respective nsp10 ZBD substitutions (Table 1) (35). Titers of progeny virus were determined from tissue culture supernatants harvested at 24 h posttransfection. RNA replication and sgRNA synthesis, respectively, were assessed by immunofluorescence analysis by using antibodies specific for the replicase gene product, nsp3, and the nucleocapsid protein, respectively (for details, see Materials and Methods). RNA synthesis of the EAV mutants, EAV_C2395H and EAV_H2414C, which had delayed growth kinetics, was also studied by Northern blotting (Fig. 3). −, absent; +, present; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

FIG. 3.

FIG. 3.

Hybridization analysis of the RNA synthesis of EAV nsp10 mutants EAV_C2395H and EAV_H2414C. Intracellular RNA was isolated from similar numbers of infected cells at 12 h postinfection. Both genome replication and sgRNA synthesis of the two mutants were reduced compared to a wild-type (WT) control. EAV RNAs 1 to 7 are indicated to the left.

FIG. 4.

FIG. 4.

Effects of substitutions and deletions in the EAV nsp10 hinge spacer region. Helicase activity assays were carried out under the same conditions as in the experiment shown in Fig. 2. Lane 1, reaction without protein; lane 2, heat-denatured substrate; lane 3, MBP-nsp10 (wild type [WT]); lane 4, MBP-nsp10_K2534Q; lane 5, MBP-nsp10_S2429P; lane 6, MBP-nsp10_S2429G/P2430G; lane 7, MBP-nsp10_S2429P/P2430S; lane 8, MBP-nsp10_ΔE2427; lane 9, MBP-nsp10_ΔG2428. Below the gel, the relative ATPase activities of the respective proteins are given. The ATPase activity of the wild-type protein, MBP-nsp10, was taken to be 1.0, and all other activities were normalized to this value. In the bottom panel, the tissue culture phenotypes are given for the EAV mutants containing the respective substitutions or deletions in the hinge spacer region (35). −, absent; +, present; ±, strongly reduced; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

FIG. 5.

FIG. 5.

Purification of bacterially expressed MBP-EAV nsp10 and MBP-HCoV-229E nsp13 fusion proteins. Fusion proteins (1 μg each) were separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Lane M shows the molecular mass markers. (A) MBP-nsp10 fusion proteins. Lane 1, wild-type (WT) MBP-nsp10; lanes 2 to 15, mutant derivatives of MBP-nsp10, with the respective amino acid substitutions indicated above the gel. (B) MBP-nsp13 fusion proteins. Lane 1, MBP-nsp13; lanes 2 to 9, mutant derivatives of MBP-nsp13, with the respective amino acid substitutions indicated above the gel.

FIG. 6.

FIG. 6.

Zn2+ is an essential structural cofactor for the ATPase activities of arterivirus EAV nsp10 and coronavirus HCoV-229E nsp13. MBP-nsp10 and MBP-nsp13 were denatured in urea-containing buffer and subsequently renatured in buffer containing zinc acetate and EDTA, respectively. The ATPase activities of the renatured MBP-nsp10 (lanes 2 and 3) and MBP-nsp13 (lanes 5 and 6) fusion proteins were analyzed by the [γ-32P]ATP thin-layer chromatography assay described in Materials and Methods. ATPase reactions were done with buffer alone (lanes 1 and 4), with proteins renatured in the presence of 100 μM zinc acetate (ZnOAc, lanes 2 and 5), and with proteins renatured in the presence of 10 mM EDTA (lanes 3 and 6). The positions of ATP and Pi are indicated.

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