Characterization of a baculovirus-encoded RNA 5'-triphosphatase

Abstract

Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5'-triphosphatase that hydrolyzes the gamma-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 microM gamma-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min-1 and converts ATP to ADP with Vmax of 8 min-1 and Km of 25 microM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5' ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase.

FIG. 1

AcNPV phosphatase resembles the RNA triphosphatase domains of metazoan capping enzymes. The amino acid sequence of the 168-amino-acid AcNPV-encoded phosphatase (Baculo) is aligned with the N-terminal RNA triphosphatase domains of mouse capping enzyme (Mus CE) and C. elegans capping enzyme (Cel CE). Gaps in the sequences are indicated by dashes; amino acids conserved in all three proteins are denoted by asterisks. The protein phosphatase signature motif is highlighted in the shaded box. The active-site cysteine is in boldface. The presumptive reaction pathway involving formation of a cysteinyl phosphate intermediate is shown.

FIG. 2

Phosphocellulose chromatography. Aliquots of the Ni-agarose 250 mM imidazole eluate fraction (lane Ni), the phosphocellulose flowthrough fraction (lane F), and the phosphocellulose NaCl eluate fractions (lanes 1 and 2, 50 mM; lane 3, 100 mM; lane 4, 200 mM; lane 5, 300 mM, lane 6, 400 mM; lane 7, 500 mM; lane 8, 600 mM; lane 9, 700 mM; lane 10, 1.0 M NaCl) were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (in kilodaltons) of marker proteins (lane M) are indicated at the left. The asterisk indicates the position of the baculovirus polypeptide.

FIG. 3

RNA triphosphatase activity. (A) Aliquots (3 μg of protein) of the 300 mM NaCl phosphocellulose eluate fraction of wild-type (WT) AcNPV phosphatase and the C119A mutant were electrophoresed through a 10% polyacrylamide gel containing 0.1% SDS. Polypeptides were visualized by staining with Coomassie blue dye. The positions and sizes (in kilodaltons) of marker proteins are indicated at the left. (B) RNA triphosphatase reaction mixtures containing 10 pmol of [γ-³²P]poly(A) and either wild-type (WT) or C119A protein as specified were incubated for 15 min at 30°C. The extent of ³²P_i release from poly(A) is plotted as function of input enzyme.

FIG. 4

Sedimentation analysis. A sample of the phosphocellulose preparation of wild-type AcNPV phosphatase was sedimented in a 15 to 30% glycerol gradient containing 0.3 M NaCl as described in Materials and Methods. Fractions were collected from the bottoms of the tubes. (A) Aliquots (20 μl) of the indicated glycerol gradient fractions were analyzed by SDS-PAGE. A Coomassie blue-stained gel is shown. The positions and sizes (in kilodaltons) of coelectrophoresed marker polypeptides are indicated at the left. (B) RNA triphosphatase reaction mixtures containing 10 pmol of [γ-³²P]poly(A) and 1 μl of the indicated glycerol gradient fractions were incubated for 15 min at 30°C. The peaks of marker proteins catalase, BSA, and cytochrome c (Cyt C), which were centrifuged in a parallel gradient, are indicated by arrows.

FIG. 5

Specific activity of the 9.5S and 2.5S forms of AcNPV RNA triphosphatase. Reaction mixtures containing 10 pmol of [γ-³²P]poly(A) and glycerol gradient fraction 6 or 16 as specified were incubated for 15 min at 30°C. The extent of ³²P_i release from poly(A) is plotted as function of input enzyme (expressed as femtomoles of the 23-kDa protomer).

FIG. 6

Kinetics of ATP hydrolysis by the 9.5S phosphatase. (A) Reaction mixtures containing (per 10 μl) 10 μM [γ-³²P]ATP and either 2 or 4 pmol of 23-kDa protein from glycerol gradient fraction 6 were incubated at 30°C. Aliquots (10 μl) were withdrawn at 1, 2, 5, 10, 15, and 30 min and quenched immediately by mixing with 1 μl of 1 M formic acid. The reaction products were analyzed by PEI-cellulose TLC. The extent of ³²P_i formation is plotted as function of reaction time. (B) Reaction mixtures containing (per 10 μl) 10 μM [α-³²P]ATP and either 2 or 4 pmol of 23-kDa protein from fraction 6 were incubated at 30°C. The extents of ADP and AMP formation are plotted as function of reaction time.

FIG. 7

Characterization of the NTPase activity of the 2.5S phosphatase. (A) ATP and GTP hydrolysis. Reaction mixtures containing (per 10 μl) 10 μM [α-³²P]ATP or [α-³²P]GTP and 1 pmol of 23-kDa protein from glycerol gradient fraction 16 were incubated at 30°C. Aliquots (10 μl) were withdrawn at the times specified and quenched immediately by mixing with 1 μl of 1 M formic acid. The reaction products were analyzed by PEI-cellulose TLC. The extents of ADP or GDP formation and AMP or GMP formation are plotted as function of reaction time. (B) Steady-state kinetic parameters of ATP hydrolysis. Reaction mixtures (10 μl) containing 1 pmol of 23-kDa protein from glycerol gradient fraction 16 and either 1, 2, 4, 6, 10, 20, 50, 100, or 200 μM [α³²P]ATP were incubated at 30°C for 15 min. The extent of ADP formation (in picomoles) was determined by TLC analysis of the reaction products. A double-reciprocal plot of the rate of ADP formation (min⁻¹ = pmol of ADP formed/15) versus [ATP] is shown.