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. 2010 Jan;38(1):203-14.
doi: 10.1093/nar/gkp904. Epub 2009 Oct 29.

The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent

Aartjan J W te Velthuis  1 , Jamie J Arnold, Craig E Cameron, Sjoerd H E van den Worm, Eric J Snijder
Affiliations

The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent

Aartjan J W te Velthuis et al. Nucleic Acids Res. 2010 Jan.

Erratum in

  • Nucleic Acids Res. 2011 Nov;39(21):9458

Abstract

An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the approximately 30-kb severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNA-dependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo- and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for coronavirus nsp8.

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Figures

Figure 1.
Figure 1.
Expression and purification of the SARS-CoV nsp12. (A) Expression of nsp12 was performed in the presence of the ubiquitin (Ub) protease Ubp1, which removed the N-terminal Ub fusion partner by cleaving after the LRGG signature that formed the junction between the Ub and nsp12 moieties. This in vivo cleavage created the native nsp12 amino-terminus with the sequence SADAS. (B) Western blotting using an nsp12-specific polyclonal antiserum identified a single band of the expected mass after purification of the in vivo cleaved expression product. Isolation of fusion proteins with different N-terminal tags resulted in common patterns of degradation. Similar results were reported by Cheng et al. (24) for a GST-nsp12 fusion protein. (C) Purified nsp12-CHis6 was readily visualized by Coomassie staining as a ∼106-kDa protein product, in line with the expected mass. Inductions in the absence of Ubp1 expression, however, resulted in a mixture of full-length fusion protein and degradation products, likely as a result of improper folding. No RdRp activity was observed for these preparations. (D) Gel filtration analysis of purified nsp12 in assay buffer showed a single peak, corresponding to a globular molecular weight of 140 ± 10 kDa, suggesting that only the monomeric form of nsp12 is present under the conditions used.
Figure 2.
Figure 2.
SARS-CoV nsp12 primer extension assay and active-site mutant. (A) Schematic showing the structure of the partially double-stranded RNA template with 3′ U10 stretch that served as template for primer extension in the initial nsp12-based nucleotide incorporation assay. (B) Comparison of the RdRp activity of wild-type nsp12 and a D618A active-site mutant, which displayed minimal activity after a 60-min incubation at 30°C. (C) A primer extension assay using a 5′ 32P-labeled primer and ATP confirmed that wild-type nsp12, in contrast to the D618A mutant, was able to elongate the 20-mer primer. The slight reduction of input primer in the D618A lane probably resulted from degradation by RNase activity. The D618A mutant showed 8 ± 3% residual activity (mean of three independent experiments). In the loading control (Figure 2B and C, lower panels) the two nsp12 variants were visualized by silver staining of an SDS–PAGE gel.
Figure 3.
Figure 3.
Biochemical determinants of SARS-CoV nsp12 activity. (A and B) Tests to determine the influence of temperature indicated that nsp12 incorporates ATP most efficiently at 37°C, although this effect mainly stemmed from increased initiation. Based on these results, 30°C was taken as the standard for subsequent experiments. (C and D) Titration of the Mg2+ concentration showed that activity reaches its maximum at 6 mM. (E and F) The effect of pH on RdRp activity was evident as well, with lower pHs limiting the activity of the enzyme. All reactions were incubated for 60 min at 30°C, unless otherwise indicated. Error bars in A, C and E represent standard error of the mean (n = 3).
Figure 4.
Figure 4.
Nucleotide incorporation fidelity of nsp12. (A) Experimental set-up of pulse-chase experiments with different nucleotides and a primed poly(U) template (see Figure 2A). The reactions were initiated with a limiting concentration of [α-32P]ATP to allow the incorporation of a first nucleotide and the formation of a stable polymerase-template complex. Subsequently, after 10 min, different unlabeled nucleotides were added to a final concentration of 50 μM to allow elongation for another 30 min. (B) SARS-CoV nsp12 allows only limited transversional and transitional misincorporations. Interestingly, also in the presence of dATP no significant activity was observed, implying that the SARS-CoV RdRp is capable of discriminating between ATP and dATP. (C) Pulse-chase experiments in the presence of 6 mM Mn2+ show that manganese ions promote misincorporation of ribonucleotides (both transversions and transitions). The selection against dATP remained unaltered.
Figure 5.
Figure 5.
RNA binding affinity of nsp12. (A) A fixed concentration (0.2 nM) of radiolabeled dsRNA or ssRNA (data not shown) was titrated with purified nsp12 and the complexes formed were separated from unbound RNA on a native 8% polyacrylamide gel. (B) Free and bound RNA were quantified and fit to the Hill equation (see ‘Materials and Methods’ section), resulting in Kd values of 0.13 ± 0.3 μM for dsRNA and 0.10 ± 0.2 μM for ssRNA binding. R2 values of these Hill fits were 0.97 and 0.98 for dsRNA and ssRNA, respectively. Error bars represent standard error of the mean (n = 3).
Figure 6.
Figure 6.
Analysis of nucleotide incorporation by nsp12. (A) Reactions with 0.1 μM nsp12, 1 μM template and 50 μM ATP were followed over time and quenched with 50 mM EDTA at the time points indicated. Gel analysis showed that, similar to the curves in Figure 3, the major accumulating product is the primer (p) extended by 7 nt (i.e. p + 7) and not the expected full-length product of n + 10. (B) To assess whether nsp12 was capable of DNA-templated incorporation of dATP, reactions were performed as in Figure 6A, but now using a DNA template. The time course illustrates robust [α-32P]dAMP incorporation, implying that the SARS-CoV RdRp is able to utilize both RNA and DNA templates. The incorporation of dAMP was 20% lower than that of AMP on an RNA template. (C) Time courses were additionally performed using an RNA template containing a U20 template region. Similar to the shorter 10-nt template used in Figure 6A, the major product here was around 2 nt shorter than the expected full-length product. (D) Changing the single-stranded template from U20 to a heteromeric (CU)10 not only increased the nucleotide incorporation rate by 34%, but also the behavior of nsp12 on the template. Products of the expected full-length size (p + 20) were observed, but also products longer than the template, hinting at, e.g. template switching by nsp12. Future research will address this observation in more detail. In this reaction both 50 μM ATP and GTP were present. The gel image represents the top half of a 20% denaturing PAGE gel.
Figure 7.
Figure 7.
Analysis of the nucleotide incorporation rate of nsp12. Steady-state time courses were performed with 0.1 μM nsp12 as described in Figure 6. Experimental data were subsequently fit to linear regression to obtain NMP incorporation rates. These clearly illustrated an increase of the incorporation velocity with template length (VU10 = 1.2 ± 0.4 nM/min and VU20 = 27 ± 3 nM/min) and from homopolymeric template to copolymeric template (V(CU)10 = 45 ± 2 nM/min). R2 values were 0.97, 0.99 and 0.99, respectively, and error bars indicate standard error of the mean (n = 3).
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