Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression

Abstract

Previous studies indicated that the vaccinia virus D10 protein, which is conserved in all sequenced poxviruses, participates in the rapid turnover of host and viral mRNAs. D10 contains a motif present in the family of Nudix/MutT enzymes, a subset of which has been shown to enhance mRNA turnover in eukaryotic cells through cleavage of the 5' cap (m7GpppNm-). Here, we demonstrate that a purified recombinant D10 fusion protein possesses an intrinsic activity that liberates m7GDP from capped RNA substrates. Furthermore, point mutations in the Nudix/MutT motif abolished decapping activity. D10 has a strong affinity for capped RNA substrates (Km approximately 3 nm). RNAs of 24-309 nt were decapped to comparable extents, whereas the cap of a 12-nt RNA was uncleaved. At large molar ratios relative to capped RNA substrate, competitor m7GpppG, m7GTP, or m7GDP inhibited decapping, whereas even higher concentrations of unmethylated analogs did not. High concentrations of uncapped RNA were also inhibitory, suggesting that D10 recognizes its substrate through interaction with both cap and RNA moieties. Thus far, poxviruses represent the only virus family shown to encode a Nudix hydrolase-decapping enzyme. Although it may seem self-destructive for a virus to encode a decapping and a capping enzyme, accelerated mRNA turnover helps eliminate competing host mRNAs and allows stage-specific synthesis of viral proteins.

Fig. 1.

Recombinant VACV D10 has RNA-decapping activity. (A) VACV D10 was expressed in E. coli as an MBP–D10 fusion protein and purified sequentially over amylose and heparin columns. Purified protein was resolved by SDS/PAGE and stained with Coomassie blue. Protein mass standards (in kDa) are indicated at the left. (B) MBP–D10 (30 ng) was incubated with 20 fmol of ³²P-cap-labeled actin RNA (309 nt) in decapping buffer for 30 min at 37°C. A portion of the reaction was treated with two units of nucleoside diphosphate kinase in the presence of 1 mM ATP for 30 min at 37°C. Aliquots of the reaction were resolved by polyethyleneimine-cellulose TLC in 0.75 M LiCl. Unlabeled standards were run in parallel and visualized by UV shadowing. Radioactivity was detected by autoradiography. NDPK, nucleoside diphosphate kinase.

Fig. 2.

The Nudix/MutT motif of D10 is essential for RNA decapping. (A) Two mutated versions of MBP–D10 were expressed in E. coli and purified in parallel with wild-type MBP–D10 over an amylose column. One mutant, E144Q/E145Q, contains two point mutations in which glutamic acid residues at positions 144 and 145 were converted to glutamine. The second mutant, E141Q, contains a point mutation at position 141 that converts glutamic acid to glutamine. One microgram each of MBP-D10, E144Q/E145Q, and E141Q was used for decapping assays, and the products were resolved by TLC as in Fig. 1. (B) Equivalent amounts of wild-type MBP–D10 and the two mutant MBP–D10 proteins (E144Q/E145Q and E141Q) were resolved by SDS/PAGE electrophoresis and stained with Coomassie blue. Protein mass standards (in kilodaltons) are indicated at the left.

Fig. 3.

Determination of kinetic parameters. (A) Time course of RNA decapping. In this reaction, 40 fmol of ³²P-cap-labeled RNA was incubated with 10 ng of MPB–D10; at indicated times, the percent product released as m⁷GDP was determined by TLC as in Fig. 1 and quantified with a PhosphorImager (GE Healthcare, Piscataway, NJ). (B) Effect of D10 concentration on decapping. The percent product released in 30 min was determined with the indicated amounts of D10 as in A. (C) Determination of K_m. Decapping reactions were performed in triplicate with 10 ng of MBP–D10 and 45, 90, 224, 449, or 673 fmol of ³²P-cap-labeled RNA for 30 min and quantified as in A. A K_m of 3.4 nM was determined with a Hanes plot by using arithmetic mean substrate concentrations (S̄) as described in ref. .

Fig. 4.

Effect of RNA length on decapping and competition with uncapped RNA. (A) Decapping of defined-length capped RNAs. ³²P cap-labeled RNAs of 12-, 24-, 36-, 48-, or 309-nt and 30 ng of MBP–D10 were mixed in the decapping assay, and the products were resolved by TLC as in Fig. 1. (B) Uncapped RNA inhibits decapping. Uncapped, unlabeled 309-nt actin RNA was added in increasing amounts to the decapping reaction, and the products were resolved by TLC. The percentage of product released was calculated with a PhosphorImager.

Fig. 5.

Methylated nucleotide derivatives inhibit D10 decapping. (A) m⁷GpppG or GpppG at the inhibitor concentrations indicated was added to the decapping reaction containing 20 fmol of capped RNA and 30 ng of MBP–D10 protein, and the products were resolved by TLC. The percent of product released was quantified with a PhosphorImager. (B) m⁷GTP or GTP was added to the decapping reaction and analyzed as in A. (C) m⁷GDP or GDP was included in the decapping reaction and analyzed as in A. (D) m⁷GpppG and m⁷GpppA were compared for their ability to inhibit D10 cap cleavage. The points are the averages of two experiments.