Ribose 2'-O methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine-N-7 methylation by the large polymerase protein

Abstract

During conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently at the ribose 2'-O position. For vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses, the two methylase activities share a binding site for the methyl donor S-adenosyl-l-methionine and are inhibited by individual amino acid substitutions within the C-terminal domain of the large (L) polymerase protein. This led to the suggestion that a single methylase domain functions for both 2'-O and G-N-7 methylations. Here we report a trans-methylation assay that recapitulates both ribose 2'-O and G-N-7 modifications by using purified recombinant L and in vitro-synthesized RNA. Using this assay, we demonstrate that VSV L typically modifies the 2'-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2'-O methylation of the substrate RNA. Amino acid substitutions in the C terminus of L that prevent all cap methylation in recombinant VSV (rVSV) partially retain the ability to G-N-7 methylate a pre-2'-O-methylated RNA, therefore uncoupling the effect of substitutions in the C terminus of the L protein on the two methylations. In addition, we show that the 2'-O and G-N-7 methylase activities act specifically on RNA substrates that contain the conserved elements of a VSV mRNA start at the 5' terminus. This study provides new mechanistic insights into the mRNA cap methylase activities of VSV L, demonstrates that 2'-O methylation precedes and facilitates subsequent G-N-7 methylation, and reveals an RNA sequence and length requirement for the two methylase activities. We propose a model of regulation of the activity of the C terminus of L protein in 2'-O and G-N-7 methylation of the cap structure.

FIG. 1.

Reconstitution of the mRNA cap MTase activities of the VSV L protein in vitro. (A) Baculovirus-expressed rL and rP were purified, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized following Coomassie blue staining. Lane 1, 5 μg purified VSV; lane 2, 2 μg rL; lane 3, 1 μg rP. (B) A 5′-triphosphorylated RNA containing the conserved nucleotides of a VSV mRNA start sequence was synthesized in vitro by the cap-defective (H1227A) VSV L protein from template VSV-123 containing a 123-nt gene inserted between the leader and N genes. The transcription products were purified, and the leader and N RNA transcripts were digested by oligonucleotide-directed RNase H cleavage. The transcripts of the 123 gene were similarly digested to produce a 110-nt RNA that was capped by the vaccinia virus capping enzyme in the presence of [α-³²P]GTP. The resulting labeled transcript was analyzed on a 6% polyacrylamide/urea gel and detected with a PhosphorImager. Lane 1 (marker [10-bp double-stranded DNA ladder from Invitrogen]) labeled with [γ-³²P]ATP and T4 polynucleotide kinase (NEB); lane 2, Gp*ppA-RNA. The values on the left are sizes in base pairs. (C) The Gp*ppA-RNA substrate was incubated in the presence of SAM and no enzyme (lane 1), rL (lane 2), rP (lane 3), or rL-rP (lane 4) at 30°C for 3 h. The products of the reactions were digested with nuclease P1 and then separated by TLC on PEI cellulose F sheets. Control reaction mixtures contained vaccinia virus VP39 (VV 2′-O), D1/D12 (VV G-N-7), or both, and the products were used as standards for migration of Gp*ppAm (lane 5), m⁷Gp*ppA (lane 6), and m⁷Gp*ppAm (lane 7), respectively. Spots were visualized with a PhosphorImager.

FIG. 2.

Effect of the methylation status of the cap structure on the kinetics of cap methylation. (A) Twenty femtomoles of Gp*ppA-RNA was incubated with 2 μg of rL in the presence of 100 μM SAM for the indicated times, and the products were digested with nuclease P1 and then separated by TLC on PEI cellulose F as described in Materials and Methods. The Gp*ppA, Gp*ppAm, m⁷Gp*ppA, and m⁷Gp*ppAm spots were quantified with a PhosphorImager. The percentage of each methylated cap species was calculated as follows: (intensity of the spot corresponding to a specific methylated species of Gp*ppA/total sum of intensities of the spots corresponding to all species of Gp*ppA) × 100. The result is shown graphically as the mean of two independent experiments. (B and C) Same as panel A, except that the RNA substrate was pre-2′-O (B) or G-N-7 (C) methylated.

FIG. 3.

Biochemical properties of the 2′-O and G-N-7 MTase activities of VSV L. Twenty femtomoles of Gp*ppA-RNA was incubated with 2 μg of rL in the presence of 100 μM SAM and varied conditions of (A) pH, (B) MgCl₂, (C) NaCl, and (D) temperature for 1.5 h. Each one of these parameters was varied while keeping the other three at constant optimal values. The Gp*ppA, Gp*ppAm, m⁷Gp*ppA, and m⁷Gp*ppAm spots were quantified with a PhosphorImager. The percentage of each methylated cap species was calculated as follows: (intensity of the spot corresponding to a specific methylated species of Gp*ppA/total sum of the intensities of the spots corresponding to all species of Gp*ppA) × 100. The percentages of 2′-O methylation (Gp*ppAm and m⁷Gp*ppAm) (□) and G-N-7 methylation m⁷Gp*ppAm (▪) from a representative experiment are shown.

FIG. 4.

The mRNA cap methylase activities require specific RNA sequence elements. Twenty femtomoles of VSV mRNA (Gp*ppA-RNA) or a T7 transcript (Gp*ppG-RNA) was incubated in methylation buffer containing 100 μM SAM and no enzyme (lanes 1, 3, 5, and 7) or 2 μg rL (lanes 2, 4, 6, and 8) at 30°C for 3 h. The RNA substrate was premethylated at the 2′-O position by using vaccinia virus VP39 (lanes 5 to 8). Standards for migration of the GpppG cap structures were generated using VV-MTases (lanes 9 to 11). The products of the reactions were digested with nuclease P1 and then separated on a TLC plate. Spots were visualized with a PhosphorImager.

FIG. 5.

Length requirement of the mRNA substrate for the MTase activity of VSV L. Twenty femtomoles of Gp*ppA-RNA consisting of 110 nt (lanes 1 and 2), 51 nt (lanes 3 and 4), 10 nt (lanes 5 and 6), or 5 nt (lanes 7 and 8) was incubated in methylation buffer containing 100 μM SAM and no enzyme (lanes 1, 3, 5, and 7) or 2 μg rL (lane 2, 4, 6, and 8) at 30°C for 3 h. The products of the reactions were digested with nuclease P1 and then separated on a TLC plate. Spots were visualized with a PhosphorImager.

FIG. 6.

Single amino acid substitutions in CRVI of L inhibit 2′-O methylation. (A) Twenty femtomoles of Gp*ppA-RNA (lanes 1 to 6) or Gp*ppAm-RNA (lanes 7 to 12) was incubated in methylation buffer containing 100 μM SAM and 2 μg of the indicated L protein at 30°C for 6 h. The products of the reactions were digested with nuclease P1 and then separated on a TLC plate. Spots were visualized with a PhosphorImager. (B) The products of methylation were quantified, and the means of three independent experiments are displayed graphically. The upper part of the panel represents the extent of methylation when the RNA substrate was unmethylated, and the lower part represents the extent of methylation following the pre-2′-O methylation of the substrate RNA. Wt, wild type.