Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity

Abstract

During mRNA synthesis, the polymerase of vesicular stomatitis virus (VSV) copies the genomic RNA to produce five capped and polyadenylated mRNAs with the 5'-terminal structure 7mGpppA(m)pApCpApGpNpNpApUpCp. The 5' mRNA processing events are poorly understood but presumably require triphosphatase, guanylyltransferase, [guanine-N-7]- and [ribose-2'-O]-methyltransferase (MTase) activities. Consistent with a role in mRNA methylation, conserved domain VI of the 241-kDa large (L) polymerase protein shares sequence homology with a bacterial [ribose-2'-O]-MTase, FtsJ/RrmJ. In this report, we generated six L gene mutations to test this homology. Individual substitutions to the predicted MTase active-site residues K1651, D1762, K1795, and E1833 yielded viruses with pinpoint plaque morphologies and 10- to 1,000-fold replication defects in single-step growth assays. Consistent with these defects, viral RNA and protein synthesis was diminished. In contrast, alteration of residue G1674 predicted to bind the methyl donor S-adenosylmethionine did not significantly perturb viral growth and gene expression. Analysis of the mRNA cap structure revealed that alterations to the predicted active site residues decreased [guanine-N-7]- and [ribose-2'-O]-MTase activity below the limit of detection of our assay. In contrast, the alanine substitution at G1674 had no apparent consequence. These data show that the predicted MTase active-site residues K1651, D1762, K1795, and E1833 within domain VI of the VSV L protein are essential for mRNA cap methylation. A model of mRNA processing consistent with these data is presented.

FIG. 1.

Amino acid sequence alignments of a region encompassing domain VI of nsNS RNA virus L proteins with the RrmJ heat shock 2′-O-methyltransferase of E. coli. The primary amino acid sequences are shown. The conserved motifs (X and I to VIII) correspond to the SAM-dependent MTase superfamily (53). Residues modified in the present study are boxed as follows: catalytic (shaded) and SAM binding (unshaded). Predicted or known alpha-helical regions are shown by the cylinders and the β-sheet regions by the arrows. STR, structure of RrmJ and predicted structure for the nsNS RNA viruses; EBOM, Ebola virus; BEFV, bovine ephemeral fever virus; VSIV, VSV (Indiana); RABV, rabies virus; HRSV, human RS virus; SEV, Sendai virus; RRMJ, E. coli heat shock methyltransferase.

FIG. 2.

Recombinant VSV with mutations in the L gene. The plaque morphology of each of the recombinant viruses is shown compared to rVSV. Note that plaques of K1651A, D1762A, K1795A, E1833A, E1833Q*, and E1833Q were developed after 48 h of incubation compared to rVSV and G1674A, which were developed after 24 h. Differing dilutions of the small plaque viruses were plated to emphasize the plaque morphology. The sequence of the modified region for each mutant virus is shown. Note that the sequence trace shown is negative sense for K1651A, G1674A, D1762A, and K1795A and positive sense for E1833A and E1833Q.

FIG. 3.

Single-step growth assay of recombinant VSV in BHK-21 cells. Confluent BHK-21 cells were infected with individual viruses at an MOI of 3. After a 1-h adsorption, the inoculum was removed, the cells were washed with DMEM, and fresh medium (containing 2% fetal bovine serum) was added, followed by incubation at 37°C. Samples of supernatant were harvested at the indicated intervals over a 48-h time period, and the virus titer was determined by plaque assay on Vero cells. Titers are reported as the mean ± the standard deviation among three independent single-step growth experiments.

FIG. 4.

Transcription of viral mRNAs in vitro. (A) Transcription reactions were performed in vitro in the presence of [α-³²P]GTP, the RNA was purified and analyzed by electrophoresis on acid-agarose gels as described in Materials and Methods. The products were detected by using a phosphorimager. The source of the virus used in the in vitro transcription reactions is indicated above the gel, and the identity of the mRNAs is shown on the left. (B) Three independent experiments were used to generate the quantitative analysis shown. For each mRNA the mean ± the standard deviation was expressed as a percentage of that observed for rVSV.

FIG. 5.

Effect of L gene mutations on cap methyltransferase activity. (A) Viral mRNA was synthesized in vitro as described in the text in the presence of either 1 mM SAM or SAH and 15 μCi of [α-³²P]GTP. Purified mRNAs were digested with 2 U of TAP, and the products were analyzed by TLC on PEI-F cellulose sheets. The plates were dried, and the spots were visualized by using a phosphorimager. The identity of the virus is shown at the top of the plate, and the migration of the markers 7^mGp and Gp is shown in the center. (B) Quantitative analysis was performed on five independent experiments. For each virus the released 7^mGp (mean ± the standard deviation) was expressed as a percentage of the total released cap structure.

FIG. 6.

Effect of L gene mutations on [ribose-2′-O] methylation. (A) Viral mRNA was synthesized in vitro as described in the text in the presence of 15 μCi of [³H]SAM. Purified mRNAs were digested with 10 U of RNase T2 and/or 2 U of TAP, and the products were analyzed by TLC on PEI-F cellulose sheets. The plates were dried, and the spots were visualized by using a phosphorimager. The identity of the virus is shown at the top of the plate, and the migration of the markers 7^mGp and Gp is shown on the right. (B) Quantitative analysis was performed on three independent experiments. For each virus the released 7^mG and A^m (mean ± the standard deviation) was expressed as a percentage of that observed for rVSV.

FIG. 7.

Effect of L gene mutations on viral RNA synthesis in BHK-21 cells. (A) BHK-21 cells were infected with the wild-type and mutant viruses at an MOI of 3. Viral RNAs were labeled with [³H]uridine as described in Materials and Methods, resolved by electrophoresis on acid-agarose gels, and visualized by fluorography. RNA extracted from an equivalent number of cells was loaded in each lane. For K1795A, viral RNA was labeled with [³H]uridine at 3, 6, 9, and 12 h postinfection. The infecting virus is indicated above the lanes, along with the time postinfection at which the labeling commenced. The identity of the RNAs is shown on the left. V, genomic and antigenomic replication products; L, G, N, and P/M, mRNA. (B) Autoradiographs of five independent experiments were scanned and analyzed as described in Materials and Methods. For each of the resolved RNA products, the mean ± the standard deviation was expressed as a percentage of that observed for rVSV.

FIG. 8.

Effect of L gene mutations on viral protein synthesis in BHK-21 cells. (A) BHK-21 cells were infected with the wild-type and mutant viruses at an MOI of 3. Proteins were labeled by incorporation of [³⁵S]methionine-cysteine in the presence of actinomycin D as described in Materials and Methods. Cytoplasmic extracts were prepared, and proteins were analyzed by SDS-PAGE and detected by using a phosphorimager. Extract from equivalent numbers of cells was loaded in each lane. For K1795A, viral proteins were labeled either at 3, 6, 9, or 12 h postinfection. The infecting virus is indicated above the lanes, along with the time postinfection at which the labeling commenced. The identity of the proteins is shown on the left. (B) Three independent experiments were used to generate the quantitative analysis shown. For each protein the mean ± the standard deviation was expressed as a percentage of that observed for rVSV.