A Single Amino Acid Change in the L-Polymerase Protein of Vesicular Stomatitis Virus Completely Abolishes Viral mRNA Cap Methylation

Abstract

The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5-cap structures methylated at the guanine-N7 and 2-O-adenosine positions (7mGpppA m ). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the Lpolymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34°C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40°C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-L-methionine-binding domain of the L protein.Vesicular stomatitis virus (VSV, a rhabdovirus) is a prototypic nonsegmented negative-strand (NNS) RNA virus belonging to the order Mononegavirales, whose members share a similar genome organization and common mechanisms of genome replication and gene expression. This order includes many medically important pathogens, including the lethal rabies, Ebola, Marburg, Nipah, and Hendra viruses. The RNA-dependent RNA polymerase (RdRp) of NNS RNA viruses is packaged into mature virions and consists of two viral subunits, the phosphoprotein (P) and the large (L) protein. The RNA genome of NNS viruses is tightly encapsidated by the nucleocapsid (N) protein, and the resulting nucleocapsid serves as the template for the sequential transcription of monocistronic mRNAs and for genome replication. Recent studies on VSV suggest that two separate RdRp complexes, which differ in their protein content, are involved in genome replication versus mRNA transcription (14, 47).The VSV RdRp produces mRNA transcripts modified at their 5Ј end by capping and cap methylation (71). The mechanism of mRNA 5Ј capping in VSV and other NNS RNA viruses is unusual, where, in contrast to cellular cap structures, both the ␣ and ␤ phosphates in the GpppA triphosphate bridge are derived from a GDP donor (2, 5, 23). The cytoplasmic localization of virus transcription and the unusual mechanism of capping suggest that the guanylyltransferas...

Independent Structural Domains in Paramyxovirus Polymerase Protein

“…The previously reported lack of VSV L trans-complementation (3) suggests that this may, in fact, constitute a general theme of Mononegavirales L domain interaction, which stands in stark contrast to the strong inherent domain affinities observed for L proteins of segmented negative strand RNA viruses of the Arenavirus family (4). Successful splitting of all three Paramyxovirus polymerase proteins analyzed in our study at homologous positions and the presence of comparable catalytic motifs in methyltransferase in CR VI (6,17,81,82) and guanolyl-transferase near the C terminus (21,22) in the MeV, NiV, and RSV L C-frag subunits support that the different L C-terminal fragments harbor equivalent enzymatic activities. Clearly, bringing the Paramyxovirus L N-and C-terminal fragments into close physical proximity is a prerequisite for reconstituting RdRp activity.…”

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

“…In contrast, CRIII, which catalyzes ribonucleotide polymerization, shares homology with all other template-dependent polynucleotide polymerases (34). Although there are differences between the MTase domain and other known MTases, it shares significant homology with known 2Ј-O MTases (7,12,15,21). Thus, the polymerase and methylase domains share a relationship to other enzymes that catalyze similar activities, whereas the capping enzyme has no apparent homologues.…”

“…However, it is clear for all NNS RNA virus L proteins that CRIII contains the polymerase active-site motif, and consistent with this, modification of VSV L protein residue D714, which is predicted to coordinate a catalytically essential Mg 2ϩ ion, inhibits all RdRP activity (37). In addition, CRVI of SeV and VSV L protein has been shown to function as the mRNA cap methylase (15,21,22,30). Based on these assignments, we suspected that either CRIV or -V might serve as the mRNA capping enzyme.…”

West Nile Virus 5′-Cap Structure Is Formed by Sequential Guanine N-7 and Ribose 2′-O Methylations by Nonstructural Protein 5

“…Nevertheless, our findings, together with the finding from a recent study that the C-terminal portion of WNV NS5 could function as an RdRp (26), suggest that flavivirus MTase and RdRp domains are functionally separable in vitro. Similarly, viruses in the order Mononegavirales were shown to contain functionally discrete cap-MTase and RdRp domains in a single L protein (7,12,23). In vesicular stomatitis virus, cap formation was found to be required for nonabortive RdRp-mediated viral mRNA transcription (12).…”