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

Abstract

Viruses of the order Mononegavirales all encode a large (L) polymerase protein responsible for the replication and transcription of the viral genome as well as all posttranscriptional modifications of viral mRNAs. The L protein is conserved among all members of the Mononegavirales and has six conserved regions ("domains"). Using vesicular stomatitis virus (VSV) (family Rhabdoviridae) experimental system, we and others recently identified several conserved amino acid residues within L protein domain VI which are required for viral mRNA cap methylation. To verify that these critical amino acid residues have a similar function in other members of the Mononegavirales, we examined the Sendai virus (SeV) (family Paramyxoviridae) L protein by targeting homologous amino acid residues important for cap methylation in VSV which are highly conserved among all members of the Mononegavirales and are believed to constitute the L protein catalytic and S-adenosylmethionine-binding sites. In addition, an SeV L protein mutant with a deletion of the entire domain VI was generated. First, L mutants were tested for their abilities to synthesize viral mRNAs. While the domain VI deletion completely inactivated L, most of the amino acid substitutions had minor effects on mRNA synthesis. Using a reverse genetics approach, these mutations were introduced into the SeV genome, and recombinant infectious SeV mutants with single alanine substitutions at L positions 1782, 1804, 1805, and 1806 or a double substitution at positions 1804 and 1806 were generated. The mutant SeV virions were purified, detergent activated, and analyzed for their abilities to synthesize viral mRNAs methylated at their cap structures. In addition, further studies were done to examine these SeV mutants for a possible host range phenotype, which was previously shown for VSV cap methylation-defective mutants. In agreement with a predicted role of the SeV L protein invariant lysine 1782 as a catalytic residue, the recombinant virus with a single K1782A substitution was completely defective in cap methylation and showed a host range phenotype. In addition, the E1805A mutation within the putative S-adenosylmethionine-binding site of L resulted in a 60% reduction in cap methylation. In contrast to the homologous VSV mutants, other recombinant SeV mutants with amino acid substitutions at this site were neither defective in cap methylation nor host range restricted. The results of this initial study using an SeV experimental system demonstrate similarities as well as differences between the L protein cap methylation domains in different members of the Mononegavirales.The order Mononegavirales includes many medically important pathogens including the lethal rabies, Ebola, Marburg, Nipah, and Hendra viruses. All members of this order share a similar genome organization and common mechanisms of genome replication and gene expression (27,32,48). The RNAdependent RNA polymerase of members of the Mononegavirales is packaged into mature virions and consists of two viral subunits, th...

Insertion of Enhanced Green Fluorescent Protein in a Hinge Region of Vesicular Stomatitis Virus L Polymerase Protein Creates a Temperature-Sensitive Virus That Displays No Virion-Associated Polymerase Activity In Vitro

“…It encodes the viral mRNA cap methylase (MTase), first recognized through sequence homology to other RNA methylases (2,10). This has since been confirmed by mutagenesis studies of domain VI motifs predicted to be required for methylase activity for both VSV L protein (13,18) and the Sendai paramyxovirus L protein (22). The methylase domain is so far the only L-protein region with discernible homology to proteins from other organisms and for which we can be fairly confident of its boundaries.…”

Structure-guided design of small-molecule therapeutics against RSV disease

“…The guanylyltransferase domain presents a case in point, since inhibiting the viral capping machinery using guanosine nucleotide analogs constitutes a proven antiviral approach against pathogens of other RNA virus families [71]. In addition, it may also be possible to target the proposed S-adenosyl-l-methionine transferase domain responsible for 5′-cap methylation [69, 72]. The therapeutic potential of S-adenosyl-l-homocysteine derivatives for use as potential antivirals has likewise been proven for other RNA virus families [73].…”

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

“…However, the "trans-methylation" assay used in their study does not allow detection of 2'-O methylation although it is known that Sendai virus encodes two MTases. More recently, recombinant Sendai virus carrying mutations in catalytic KDKE tetrad and SAM binding site were recovered from infectious clones [91,92]. It was found that these mutations affected mRNA cap methylation.…”

“…Our group and others have identified a panel of MTase-defective VSV mutants which are attenuated in cell culture as well as in animal models [14,17,84,107]. In addition, MTase-defective Sendai viruses also showed significant defects in viral growth in cell culture [91,92]. By combining multiple substitutions within the methylation region, it should be possible to generate an attenuated virus that is genetically stable, as reversion to wild type at any single amino acid should not provide a fitness gain.…”

“…Introducing mutations in the MTase may provide a novel approach to generate live attenuated viruses for these viruses. It was reported that recombinant Sendai virus carrying point mutations in the MTase catalytic site (rSeV-K1782A) and the SAM binding site (rSeV-E1805A) were attenuated in cell culture [91]. It will be of interesting to determine whether these Sendai recombinants are attenuated in vitro.…”