Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation

Abstract

The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap methylation.

Identification of Sendai Virus L Protein Amino Acid Residues Affecting Viral mRNA Cap Methylation

“…Our recent analysis of the VSV hr1 mutant showed that a single-amino-acid substitution (D1671V) in this putative AdoMet-binding glycine-rich motif completely eliminated viral mRNA cap methylation at both the guanine-N7 and 2Ј-O-adenosine positions (17), thus experimentally supporting the above-described predictions (5,13). In addition, other authors demonstrated that substitutions at other positions within the VSV L protein domain VI (in-cluding an invariant lysine 1651 and aa 1670 and 1672 within the glycine-rich motif) also resulted in various defects in mRNA cap methylation (16,17,29,30). The importance of domain VI in cap methylation was biochemically demonstrated for Sendai virus (SeV) (family Paramyxoviridae) by Ogino et al (40) using an in vitro assay with a fragment of the L protein that contained domain VI.…”

“…Specifically, VSV mutants defective in cap methylation were temperature-sensitive (ts) and, more interestingly, host range restricted (hr), as manifested by their inability to grow in certain nonpermissive cell lines (e.g., HEp-2 cells) while retaining their ability to grow to high titers in permissive cells (e.g., BHK-21 cells) (16,17,24,25). Previous studies linked the inability of VSV cap methylation-defective mutants to grow in HEp-2 cells to the nontranslatability of primary VSV transcripts (24,25) and showed that host cells methylate viral mRNA in permissive cell lines through an unknown mechanism (24).…”

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

“…The amino acid change responsible for this, F1488S, is located in the variable region of L between CRV and CRVI. Other single amino acid substitutions in this region inhibit mRNA cap methylation (13), most likely by indirectly affecting the function of CRVI of L protein. The effect of the F1488S substitution on polyadenylation is consistent with the observation of giant heterogenous polyadenylate on VSV mRNA synthesized in the presence of the methylation inhibitor SAH (33).…”

“…5), providing further support that failing to methylate the mRNA cap structure can result in the synthesis of large polyadenylate. However, the mRNA cap structures synthesized by viruses containing the F1488 substitutions are in fact methylated (12,13), illustrating that there is no clear-cut relationship between cap methylation and hyperpolyadenylation.…”

Identification of genetic mutations associated with attenuation and changes in tropism of Urabe mumps virus

“…We believe that the mutations in the polymerase of Gw7 have not been generated by passage in culture since purification, as we have confirmed these mutations in a second ''G'' virus isolated independently from the same vaccine lot (unpublished results). None of the differences in L are within motifs associated with RNAbinding, with GDNQ template recognition/phosphodiester bond-forming, or with purine nucleotide binding [Poch et al, 1990;Sidhu et al, 1993], nor are they in sites associated with methylation of mRNA caps [Grdzelishvili et al, 2006]. Three of the amino acids are Equal numbers of Vero and A549 cells were incubated with virus at MOI ¼ 0.1 for 1 hr on ice.…”