Opposing Effects of Inhibiting Cap Addition and Cap Methylation on Polyadenylation during Vesicular Stomatitis Virus mRNA Synthesis

Abstract

The multifunctional large (L) polymerase protein of vesicular stomatitis virus (VSV) contains enzymatic activities essential for RNA synthesis, including mRNA cap addition and polyadenylation. We previously mapped amino acid residues G1154, T1157, H1227, and R1228, present within conserved region V (CRV) of L, as essential for mRNA cap addition. Here we show that alanine substitutions to these residues also affect 3-end formation. Specifically, the cap-defective polymerases produced truncated transcripts that contained A-rich sequences at their 3 termini and predominantly terminated within the first 500 nucleotides (nt) of the N gene. To examine how the cap-defective polymerases respond to an authentic VSV termination and reinitiation signal present at each gene junction, we reconstituted RNA synthesis using templates that contained genes inserted (I) at the leader-N gene junction. The I genes ranged in size from 382 to 1,098 nt and were typically transcribed into full-length uncapped transcripts. In addition to lacking a cap structure, the full-length I transcripts synthesized by the cap-defective polymerases lacked an authentic polyadenylate tail and instead contained 0 to 24 A residues. Moreover, the cap-defective polymerases were also unable to copy efficiently the downstream gene. Thus, single amino acid substitutions in CRV of L protein that inhibit cap addition also inhibit polyadenylation and sequential transcription of the genome. In contrast, an amino acid substitution, K1651A, in CRVI of L protein that completely inhibits cap methylation results in the hyperpolyadenylation of mRNA. This work reveals that inhibiting cap addition and cap methylation have opposing effects on polyadenylation during VSV mRNA synthesis and provides evidence in support of a link between correct 5 cap formation and 3 polyadenylation.

Ribose 2′-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein

“…An rVSV containing a 132-nucleotide (nt) gene inserted at the leader-N gene junction (32) was used as the source of the 10-, 51-, and 110-nt RNAs containing the conserved elements of a VSV mRNA. The N-RNA template was purified from this virus and resuspended in 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 1 mM EDTA as described previously (18). RNA was synthesized from 10 g of this template by using 3 g of the cap-defective (H1227A) rL mutant as described previously (18,19).…”

“…The N-RNA template was purified from this virus and resuspended in 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 1 mM EDTA as described previously (18). RNA was synthesized from 10 g of this template by using 3 g of the cap-defective (H1227A) rL mutant as described previously (18,19). To eliminate 5Ј-triphosphorylated RNA transcripts derived from the leader and N genes, we used oligonucleotides designed to anneal to nt 2 to 17 and 66 to 81 of the VSV antigenome and cleaved the resulting RNA-DNA hybrids with RNase H. Briefly, DNA primers were annealed at a 3:1 molar excess of the RNA transcripts and digested with 10 U of RNase H (Takara) for 30 min at 37°C in 60-l reaction mixtures, followed by digestion with 1 U of RQ1 DNase (Promega) for 30 min at 37°C.…”

“…Because cap-defective polymerases of VSV terminate transcription prematurely, we used as the template rVSV-132, which contains a short, nonessential gene inserted between the leader and N genes of VSV (32). Transcription of this template by the cap-defective L protein yields the VSV 47-nt leader RNA, the 123-nt RNA, and some N mRNA (18). To eliminate the leader and N transcripts and to precisely define the 3Ј end of those transcripts from the 132-nt gene, we used oligonucleotide-directed RNase H cleavage as described in Materials and Methods.…”

Messenger RNA Cap Methylation in Vesicular Stomatitis Virus, a Prototype of Non‐Segmented Negative‐Sense RNA Virus

“…It was found that rVSV-K1651A, a mutation in MTase active site and completely defective in G-N-7 and 2'-O methylation, synthesized excessively long poly(A) tails, similar to those produced by wild-type L in the presence of SAH [15]. Similarly, the substitution D1762E at the MTase active site, which inhibits both G-N-7 and 2'-O methylation, produces large polyadenylate in the presence or absence of SAH [97].…”

“…Cap formation is essential for mRNA stability, efficient translation, and gene expression [9][10][11]. It is now firmly established that mRNA capping and methylation in NNS RNA viruses evolves in a mechanism distinct to their hosts [12][13][14][15][16][17][18][19]. Thus, mRNA cap formation is an attractive antiviral target for NNS RNA viruses.…”