Vesicular Stomatitis Virus mRNA Capping Machinery Requires Specific cis -Acting Signals in the RNA

Abstract

Many viruses of eukaryotes that use mRNA cap-dependent translation strategies have evolved alternate mechanisms to generate the mRNA cap compared to their hosts. The most divergent of these mechanisms are those used by nonsegmented negative-sense (NNS) RNA viruses, which evolved a capping enzyme that transfers RNA onto GDP, rather than GMP onto the 5 end of the RNA. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we show that mRNA cap formation is further distinct, requiring a specific cis-acting signal in the RNA. Using recombinant VSV, we determined the function of the eight conserved positions of the gene-start sequence in mRNA initiation and cap formation. Alterations to this sequence compromised mRNA initiation and separately formation of the GpppA cap structure. These studies provide genetic and biochemical evidence that the mRNA capping apparatus of VSV evolved an RNA capping machinery that functions in a sequence-specific manner.The 7 m GpppN cap structure found at the 5Ј terminus of eukaryotic mRNAs is required for efficient translation (27) and mRNA stability (13). The cap structure is formed through a series of enzymatic reactions. An RNA triphosphatase removes a single phosphate from the 5Ј end of the RNA to yield 5Ј ppN. The diphosphate is then capped by an RNA guanylyltransferase which transfers Gp from a Gppp donor to form the cap structure, GpppN. The cap is further modified by a guanine-N-7 (G-N-7) methylase which transfers a methyl group from S-adenosyl-L-methionine (SAM) to yield 7 m GpppN and S-adenosyl homocysteine (14). This methylated cap can then be subsequently methylated by a ribose-2Ј-O (2Ј-O) methylase to yield 7 m GpppN m . Many viruses of eukaryotes have evolved unusual strategies to generate capped mRNA, including the nonsegmented negative-sense (NNS) RNA viruses. Studies with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, demonstrated that the capping enzyme transfers RNA onto Gpp, rather than Gp onto the 5Ј end of the RNA (1, 28). The mRNA cap methylase activities of VSV are also unusual in that the G-N-7 and 2Ј-O methylase activities use a single SAM binding site (24). Thus, for these viruses, each step of mRNA cap formation is distinct from that of the host.Much understanding of gene expression strategies of NNS RNA viruses comes from studies of VSV. The VSV genome comprises 11,161 nucleotides (nt) of RNA. This is delivered into the cytoplasm of the host cell as a transcription-competent core in which the RNA is completely encapsidated by the nucleocapsid (N) protein and associated with the RNA-dependent RNA polymerase (RdRP). The viral components of the RdRP are a 29-kDa phosphoprotein (P) and a 241-kDa large (L) polymerase subunit (10, 11). In response to a promoter, the VSV RdRP initiates synthesis at the first gene-start (GS) sequence (3Ј UUGUCNNUAC 5Ј) (25,40,41). The nascent mRNA chain is capped and methylated (1, 26), and in response to a specific gene-end (GE) sequence (3Ј-AUACUUUUUUU GA-5Ј), the RdRP polyadenyla...

A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

“…7B, lanes 10 and 11). This is consistent with the reported sequence-specific requirement for cap formation (29,42). Using this assay, we examined whether polymerases with defects in processivity as well as cap formation were able to cap in trans.…”

“…These data suggest a model for RNA synthesis in which addition of the mRNA cap plays a key role in controlling polymerase processivity. Coupled with the fact that the mRNA capping machinery requires specific signals in the RNA (29,42) and that mRNA cap methylation is accomplished by two activities that share a common SAM binding site (22), these data also show that the entire capping apparatus of these viruses is distinct to that employed by other systems. Furthermore, these experiments define a motif, GxxT[n]HR, that is essential for VSV mRNA cap formation.…”

Respiratory Syncytial Virus Polymerase Can Initiate Transcription from Position 3 of the Leader Promoter

“…A plausible explanation for this is that this single nucleotide change could have facilitated capping of some of the RNA initiated at ϩ3. In the case of VSV, it has been shown that initiation of RNA synthesis and capping of the nascent transcript are both directed by the GS signal (11,12). In addition, it has been shown that capping of VSV mRNA occurs when the transcript reaches 31 nt in length (36).…”

“…They are highly conserved in RSV, with 9 of 10 GS sequences being identical and the tenth having only slight differences (9). In related viruses, the GS signals have been shown to be multifunctional, directing the RdRp to cap and methylate the nascent transcript (10)(11)(12). Capping is thought to allow the RdRp to enter into a stable elongation mode in which it can extend the RNA to the GE signal, where the RdRp polyadenylates and then releases the mRNA (13).…”

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

“…RTPase activity of L protein, the first step in viral mRNA capping, was tested in vitro using synthetic c-32 P-labelled RNA substrate containing sequences corresponding to viral genes. Viral mRNA 59 cis sequences were used as the substrate because in many negative-sense RNA viruses, the capping machinery of these viruses requires specific cis-acting signals in the RNA (Ogino et al, 2005;Ogino & Banerjee, 2007;Wang et al, 2007). Under optimal conditions, L protein in the RNP complex, as well as L protein co-expressed with P protein in insect cells, efficiently catalyses the removal of cphosphate from the triphosphate-ended RNA (Fig.…”