S-adenosyl homocysteine-induced hyperpolyadenylation of vesicular stomatitis virus mRNA requires the methyltransferase activity of L protein

Abstract

There are many unique aspects of vesicular stomatitis virus (VSV) transcription. In addition to its unusual mRNA capping and methyltransferase mechanisms, the addition of S-adenosyl homocysteine (SAH), which is the by-product and competitive inhibitor of S-adenosyl methionine (SAM)-mediated methyltransferase reactions, leads to synthesis of poly(A) tails on the 3' end of VSV mRNAs that are 10- or 20-fold longer than normal. The mechanism by which this occurs is not understood, since it has been shown that productive transcription is not dependent on 5' cap methylation and full-length VSV mRNAs can be synthesized in the absence of SAM. To investigate this unusual phenotype, we assayed the effects of SAH on transcription using a panel of recombinant viruses that contained mutations in domain VI of the VSV L protein. The L proteins we investigated displayed a range of 5' cap methyltransferase activities. In the present study, we show that the ability of the VSV L protein to catalyze methyl transfer correlates with its sensitivity to SAH with respect to polyadenylation, thereby indicating an intriguing connection between 5' and 3' end mRNA modifications. We also identified an L protein mutant that hyperpolyadenylates mRNA irrespective of the presence or absence of exogenous SAH. Further, the data presented here show that the wild-type L protein hyperpolyadenylates a percentage of VSV mRNAs in infected cells as well as in vitro.

FIG. 1.

Location of domain VI L-protein residues examined. Spatial arrangement of the domain VI residues examined previously for their role in methyltransferase activity (12) and in the present study for SAH sensitivity. The model is shown as a ribbon diagram except for the highlighted residues, which are shown in space-fill arrangement. Alpha helices are colored purple. Beta strands are colored yellow. Random coils are colored light blue. Carbon atoms are green, and oxygen atoms are red. The SAM molecule is shown as a stick model: carbon atoms are colored light blue, nitrogen atoms are colored dark blue, oxygen atoms are colored red, and sulfur atoms are colored yellow.

FIG. 2.

Sensitivity of domain VI L protein mutants to SAH in vitro. (A) Viral RNAs were transcribed in vitro in the presence of 1 mM SAH or no additional substrate. Transcription reactions were labeled with [³³P]ATP. Labeled RNAs were purified and resolved on 1.5% acid agarose-urea gels. The gels were fluorogrammed, and radiolabeled RNAs were detected by autoradiography. In order to visualize RNAs transcribed by L1716Y and G1672A rVSVs, the films were exposed for 10 times longer (lanes labeled “10X exp”). The identities of VSV mRNAs are denoted on the left. (B) The RNAs shown in panel A were digested with RNase H following annealing with oligo(dT). Digested RNAs were ethanol precipitated and resolved on 1.5% acid agarose-urea gels. The gels were fluorogrammed, and radiolabeled RNAs were detected via autoradiography. The identities of labeled RNAs are denoted on the left.

FIG. 3.

Direct analysis of the poly(A) tails synthesized by domain VI L protein mutants in the presence or absence of exogenous SAH. (A) The L1716T/Y and D1762E/G/N RNAs shown in Fig. 2A were digested with RNase A and resolved on 1.5% acid agarose-urea gels. Representative digests for rVSV (+/−) SAH are shown in each panel: rVSV samples were digested with RNase A or RNase A, followed by dT/RNase H digestion. The gels were fluorogrammed, and radiolabeled RNAs were detected by autoradiography. [³³P]ATP-labeled in vitro-transcribed size markers were generated from RNA Century Plus marker templates (Ambion) and resolved alongside undigested and digested RNAs. The location of each size marker is denoted on the left. The identities of undigested VSV mRNAs are also denoted on the left. The sizes of each undigested VSV mRNA minus their respective poly(A) tails are 1,332 nt (N), 821 nt (P), 837 nt (M), and 1,672 nt (G). (B) Same as in panel A, except the RNAs analyzed were synthesized by G1672A/P, G1674P, and G1675P L proteins.

FIG. 4.

Analysis of poly(A) tail synthesis by the F1488S rVSV. (A) Viral RNAs were transcribed in vitro in the presence of 1 mM SAH or no additional substrate. Transcription reactions were labeled with [³³P]ATP. Labeled RNAs were purified and resolved on 1.5% acid agarose-urea gels. The gels were fluorogrammed, and radiolabeled RNAs were detected by autoradiography. The identities of VSV mRNAs are denoted on the left. The sizes of each undigested VSV mRNA minus their respective poly(A) tails are 1,332 nt (N), 821 nt (P), 837 nt (M), and 1,672 nt (G). (B) The RNAs shown in panel A were digested with RNase H in the presence of oligo(dT). Digested RNAs were ethanol precipitated and resolved on 1.5% acid agarose-urea gels. The gels were fluorogrammed, and radiolabeled RNAs were detected via autoradiography. The dT/RNase H-digested RNA synthesized by the F1488S L protein in the presence of 1 mM SAH after a 2.5-fold exposure of film is shown in lane 6. The identities of labeled RNAs are denoted on the left. (C) The RNAs shown in panel A were digested with RNase A and resolved on 1.5% acid agarose-urea gels. Representative digests for rVSV (+/−) SAH are shown in each panel: rVSV samples were digested with RNase A or RNase A, followed by dT/RNase H digestion. The gels were fluorogrammed, and radiolabeled RNAs were detected by autoradiography. [³³P]ATP-labeled in vitro-transcribed size markers were generated from RNA Century Plus marker templates (Ambion) and resolved alongside undigested and digested RNAs. The location of each size marker is denoted on the left. The identities of undigested VSV mRNAs are also denoted on the left. The sizes of each undigested VSV mRNA minus their respective poly(A) tails are 1,332 nt (N), 821 nt (P), 837 nt (M), and 1,672 nt (G).

FIG. 5.

Effect of AdOX on VSV transcription in cells. BHK cells were infected with wild-type VSV at an MOI of 5 at 37°C. At 2 hpi, cells were treated with 5 μM AdOX and incubated at 37°C. Viral RNAs were labeled with [³H]adenine (lanes 1 to 4) or [³H]uridine (lanes 5 to 8) in the presence of actinomycin D for 1 h. RNAs were harvested, and two-thirds of each sample was digested with RNase A as described in Materials and Methods. Radiolabeled RNAs were visualized by acid agarose-urea gel electrophoresis and fluorography.