Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase

Abstract

In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro(-)Pol with an inactivated proteinase domain to prevent autocleavage) and recombinant Pol were purified after synthesis in bacteria and shown to be active RdRp enzymes. In addition, the mutant His-E1189A-ProPol protein (with active proteinase but with the natural ProPol cleavage site blocked) was active as an RdRp, confirming that the norovirus ProPol precursor could possess two enzymatic activities simultaneously. The effects of several UTP analogs on the RdRp activity of the norovirus and feline calicivirus Pro(-)Pol enzymes were compared and found to be similar. Our data suggest that the norovirus ProPol is a bifunctional enzyme during virus replication. The availability of this recombinant ProPol enzyme might prove useful in the development of antiviral drugs for control of the noroviruses associated with acute gastroenteritis.

FIG. 1.

Diagram of the plasmid constructions and recombinant proteins used in this study. (A) Map of the cleavage sites for ORF1 of the MD145-12 norovirus (2). Sequences of the dipeptide cleavage sites and their positions are indicated by arrows. The calculated molecular mass (in kilodaltons) of each protein is shown in parentheses. The designation of the cleavage products is indicated inside boxes and is adapted from the work of Liu et al. and Green et al. (12, 23). (B) Mutagenized residues are indicated above each construction by an arrow and amino acid change. The cleavage site of the protease Ulp1 is indicated below the SUMO construction by an arrow. A negative sign in superscript position indicates that the proteinase (Pro) or the polymerase (Pol) enzymatic activity was inactivated by mutagenesis. The locations of the engineered His tag (underlined) and the SUMO peptide (gray box) are indicated. Designations of the constructs and recombinant proteins are indicated on the left and the right, respectively.

FIG. 2.

Purification of recombinant MD145-12 proteins. (A) SDS-PAGE analysis of His-Pro⁻Pol (lanes 2 and 3), His-Pol (lanes 5 and 6), His-SUMO-Pol (lane 9), and rPol (lane 10). Purification steps (Ni-NTA binding and SEC) and Ulp1 proteinase treatment are indicated above each lane. One microgram of His-Pro⁻Pol and His-Pol was separated by SDS-PAGE in a 4 to 12% Bis-Tris polyacrylamide gel (Invitrogen). One microgram of bovine serum albumin (BSA; Pierce) was added as control for the protein estimation (lane 7). Two and one-half micrograms of SUMO-Pol and rPol was resolved by SDS-PAGE in a 10% polyacrylamide gel. Proteins were visualized by staining with Coomassie blue. Lanes 1, 4, and 8 contain protein molecular weight markers (weights are at left in thousands). (B) RdRp assay of His-Pro⁻Pol fractions obtained during SEC. One microliter of each fraction was assayed in a 15-μl reaction mixture that contained 3 mM MgCl₂, 10 μM UTP, and 5 μCi of [α-³²P]UTP (400 Ci/mmol) for 20 min at 30°C. For this experiment and all subsequent experiments 200 μg of rifampin/ml was added. Other components of the RdRp assay mixture are described in Materials and Methods. Fraction numbers are indicated below the graph. UMP incorporation is given in disintegrations per minute (dpm) and corresponds for each fraction to an aliquot of 5 μl spotted on a DE81 filter.

FIG. 3.

Kinetics of norovirus His-Pro⁻Pol, rPol, and His-Pol UMP incorporation. (A) For each time point (1, 5, 10, 15, 20, 25, and 30 min), a 15-μl reaction mixture containing His-Pro⁻Pol (open circles), rPol (solid triangles), or His-Pol (open triangles) at 1 μM was incubated up to 30 min at 30°C. The reaction mixture contained 100 μM UTP, 5 mM MgCl₂, and 5 μCi of [α-³²P]UTP. The reaction was stopped by adding 3 μl of 0.5 M EDTA, and 6 μl was spotted onto a DE81 filter. The amount of incorporated UMP is given in picomoles (ordinate). For both proteins, mean results and standard deviations of three independent experiments are shown (vertical bars).

FIG. 4.

Effect of various conditions on the activity of norovirus His-Pro⁻Pol. (A) Effect of the enzyme concentration on the amount of incorporated UMP. One, 2, and 3 μM concentrations of His-Pro⁻Pol were assayed in a 15-μl reaction mixture containing 3 mM MgCl₂, 100 μM UTP, and 5 μCi of [α-³²P]UTP. The mixture was incubated for 20 min at 30°C and stopped with 0.5 M EDTA. Six microliters of the final mixture was spotted onto a DE81 membrane for counting. Final concentrations of the recombinant polymerase are indicated below the graph. The incorporated UMP is normalized against the value of 3 μM His-Pro⁻Pol (100%). (B) The polymerase activity of His-Pro⁻Pol (1 μM) was analyzed following incubation for 30 min at various temperatures (T_inc) (indicated on abscissa). The RdRp assay was performed in a 30-μl reaction mixture which contained 10 μM UTP, 10 μCi of [α-³²P]UTP, 3.3 mM MgCl₂, and the other components described in Materials and Methods. The UMP incorporation rates are given in percentages of the value obtained at 30°C (100%). (C) Effect of NaCl on the His-Pro⁻Pol activity. The 30-μl reaction mixture was incubated for 20 min at 30°C and contained 1 μM His-Pro⁻Pol, 10 μM UTP, 10 μCi of [α-³²P]UTP, 3.3 mM MgCl₂, and increasing concentrations of NaCl (0 to 50 mM), indicated below the gel. The negative control was an RdRp assay of His-Pro⁻Pol without MgCl₂ and NaCl. The UMP incorporation rates (ordinate) are normalized against the amount of incorporated UMP in an RdRp assay containing 3.3 MgCl₂ and no NaCl (100%). (D) Effect of divalent (magnesium and calcium) and monovalent (potassium) cations on the polymerase activity. His-Pro⁻Pol (1 μM) was incubated at 30°C for 20 min with 3.3 mM (CH₃COO)₂Mg, MgSO₄, MgCl₂, CaCl₂, KCl, or H₂O (negative control) in a 15-μl reaction mixture containing 10 μM UTP and 5 μCi of [α-³²P]UTP. The amount of incorporated UMP (ordinate) is normalized against values obtained with magnesium acetate (100%).

FIG. 5.

Synthesis of RNA from a heteropolymeric template by recombinant norovirus polymerases. Recombinant enzyme (1 μM) was incubated with 1 μg of the 830-nt green fluorescent protein RNA template for 1 h at 30°C in the presence of [α-³²P]UTP; 3 mM MgCl₂ was included in each reaction except that shown in lane 4 (no magnesium). The recombinant protein used in each assay is indicated above the gel (lanes 1 to 6). The synthesized RNA was purified, precipitated in ethanol, dried, heated in glyoxyl sample buffer, and analyzed in a 1% agarose gel. The gel was dried and exposed to film. In lane 7, 1 μg of the green fluorescent protein DNA template was linearized by MluI and transcribed by T7 polymerase in the presence of 10 μCi of [α-³²P]UTP. The sizes of the green fluorescent protein RNA transcript (830 nt) and the RNA dimer (approximately 1,660 nt) produced by the norovirus polymerases are indicated.

FIG. 6.

Template switching activity of MD145 His-Pro⁻Pol in absence or presence of RNA (rA₃₀) and DNA (dA₃₀) acceptor templates over a 10-min period. Reactions were initiated by mixing His-Pro⁻Pol (5 μM), 10 μM dT₁₅-rA₃₀, and [α-³²P]UTP (0.67 μM), followed by incubation at 30°C for 3 min. Heparin, an excess of UTP, and either no acceptor template, rA₃₀ acceptor template (100 μM), or dA₃₀ acceptor template (100 μM) were then added to the reactions and incubated at 30°C. Reactions were quenched at the times (in minutes) indicated above the gel by addition of EDTA. Products were resolved by electrophoresis on a denaturing 10% polyacrylamide gel, and signals were detected using a phosphorimager. The sizes corresponding to the length of the acceptor template and primer are indicated by arrows. Larger products consistent with synthesis by template switching are indicated by an asterisk. Sizes (in nucleotides) of the DNA marker are shown on the left.

FIG. 7.

Effect of UTP analogs on norovirus and FCV polymerase activity. (A) The norovirus His-Pro⁻Pol at 1 μM was incubated for 20 min at 30°C with increasing concentrations (1, 5, 50, 100, and 200 μM) of UTP analogs. The modified NTPs are 5-iodo-UTP (compound A), pseudo-UTP (compound B), 5-methyl-UTP (compound C), 2-thio-UTP (compound D), and 6-aza-UTP (compound E). The relative quantities of incorporated UMP (ordinate) are given in percentages. The data (shown as percentages of UMP incorporation) were normalized based upon the UMP incorporated by His-Pro⁻Pol (100%) in the absence of UTP analog. (B) The recombinant FCV Pro^M-Pol at 1 μM was assayed as described in the legend for panel A except that the reaction mixture was incubated for 2 min. Code names of the compounds are identical to those in panel A. The data (shown as percentages of UMP incorporation) are normalized based upon the UMP incorporated by FCV rPro^M-Pol (100%) in the absence of UTP analog.

FIG. 8.

Effect of nucleoside analogs on the growth of FCV. CRFK cells were incubated with 2 mM nucleoside for 1 h prior to infection with FCV in the presence of 2 mM inhibitors. At 8 h postinfection, the effect of each analog was monitored by plaque titration assay. The nucleosides (abscissa) are 5-iodouridine (compound A), pseudouridine (compound B), 5-methyluridine (compound C), 2-thiouridine (compound D), 6-azauridine (compound E), and ribavirin. The virus titers are indicated on the left (ordinate) in PFU per milliliter (PFU/ml). FCV infection in the absence of analog was included as a control.