Analysis of a Temperature-Sensitive Vaccinia Virus Mutant in the Viral mRNA Capping Enzyme Isolated by Clustered Charge-to-Alanine Mutagenesis and Transient Dominant Selection

Abstract

We have previously reported the successful development of a targeted genetic method for the creation of temperature-sensitive vaccinia virus mutants [D. E. Hassett and R. C. Condit (1994) Proc. Natl. Acad. Sci. USA 91, 4554-4558]. This method has now been applied to the large subunit of the multifunctional vaccinia virus capping enzyme, encoded by gene D1R. Ten clustered charge-to-alanine mutations were created in a cloned copy of D1R. Four of these mutations were successfully transferred into the viral genome using transient dominant selection, and each of these four mutations yielded viruses with plaque phenotypes different from that of wild-type virus. Two of the mutant viruses, 516 and 793, were temperature sensitive in a plaque assay. Mutant 793 was also temperature sensitive in a one-step growth experiment. Phenotypic characterization of the 793 virus under both permissive and nonpermissive conditions revealed nearly normal patterns of viral protein and mRNA synthesis. Under nonpermissive conditions the 793 virus was defective in telomere resolution and blocked at an intermediate stage of viral morphogenesis. In vitro assays of various capping enzyme activities revealed that in permeabilized virions, enzyme guanylylate intermediate formation was reduced and methyltransferase activity was thermolabile, while in solubilized virion extracts enzyme guanylylate activity was reduced and both guanylyltransferase and methyltransferase activities were absent. Thus, the 793 mutation affects at least two separate enzymatic activities of the capping enzyme, guanylyltransferase and methyltransferase, and when incorporated into the virus genome, the mutation yields a virus that is temperature sensitive for growth, telomere resolution, and virion morphogenesis.

Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus H5 Gene: Isolation of a Dominant, Temperature-Sensitive Mutant with a Profound Defect in Morphogenesis

“…Clustered charge-to-alanine mutagenesis has been used successfully in vaccinia virus and other systems for this very purpose (12,16,21,22,48). The premise of this approach is the likelihood that clusters of charged residues will lie on the surface of a native protein, such that mutations in the region would be unlikely to cause a global disturbance of protein folding.…”

“…Clustered charge-to-alanine mutagenesis has previously been successful in generating vaccinia virus ts mutants with mutations in the G2 protein and the capping enzyme (21,22). The experience of the Condit laboratory and of investigators working with other systems suggests that approximately 30 to 40% of alleles generated by this approach will confer a ts phenotype (12,21,48).…”

Vaccinia Virus Gene A18R DNA Helicase Is a Transcript Release Factor

“…A18R mutants do not affect early viral transcription in vivo (28), but this is not atypical for mutations in vaccinia virion enzymes. For example, temperature-sensitive mutants in the virion early transcription initiation factor VETF (45), the RNA helicase NPH-2 (46), the mRNA capping enzyme (47), and the RNA polymerase (48,49) have shown no pronounced effect on early transcription in vivo. Although the mechanism of early transcription termination is reasonably well understood, a role for A18R as an early transcript release factor has not been ruled out.…”

A Yeast-Based Genetic System for Functional Analysis of Viral mRNA Capping Enzymes

“…The enzyme is a heterodimer of 95-and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The vaccinia virus D1 and D12 genes are essential for virus replication, insofar as mutations that elicit temperature-sensitive virus growth phenotypes have been mapped to the two capping enzyme subunits (6,18). However, the genetic landscape is complicated, because vaccinia virus capping enzyme plays a larger role in viral gene expression; it serves as a transcription termination factor during the synthesis of viral early mRNAs (29,50) and as an initiation factor during the transcription of intermediate genes (55).…”

“…However, the genetic landscape is complicated, because vaccinia virus capping enzyme plays a larger role in viral gene expression; it serves as a transcription termination factor during the synthesis of viral early mRNAs (29,50) and as an initiation factor during the transcription of intermediate genes (55). Amazingly, the D1 and D12 temperature-sensitive mutant viruses display no gross defect in viral gene expression at the restrictive temperature but are instead defective in resolving concatemeric DNA replication intermediates into the hairpin telomeres of the mature viral genome (6,18). This mysterious phenotype has no obvious connection to the known mRNA-processing or transcription functions of the D1 and D12 proteins.…”