Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant

Abstract

Prior biochemical analysis of the heterodimeric vaccinia virus mRNA capping enzyme suggests roles not only in mRNA capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. We report a biochemical analysis of the capping enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA capping enzyme functions not only in mRNA capping but also early gene transcription termination and intermediate gene transcription initiation in vivo.

An Engineered T7 RNA Polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

“…Two dinucleotide cap analogs were selected for capping evaluation studies using T7-68 and WT T7 RNA polymerases. Unlike m 7 GpppG and other dinucleotide analogs, the anti-reverse cap analog m2 7,3'-O GpppG (ARCA) (S1) incorporates in only the forward orientation, by virtue of the 3'-O methylation on the methylated guanosine. This analog is widely used because higher overall capping efficiencies are achieved because of this directional incorporation [16].…”

“…An alternative process, often referred to as enzymatic capping, uses the vaccinia capping enzyme (VCE) complex to add a cap structure to mRNAs post-IVT. However, this process does not always reach completion [6] [7]. As it uses an additional enzyme with unique reaction condition tolerances, it also requires an mRNA isolation step between the IVT and capping reactions, resulting in yield losses, increased complexity and ultimately expense.…”