A targeted approach to identification of vaccinia virus postreplicative transcription elongation factors: genetic evidence for a role of the H5R gene in vaccinia transcription

Abstract

Treatment of wild-type vaccinia virus infected cells with the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) induces the viral postreplicative transcription apparatus to synthesize longer-than-normal mRNAs through an unknown mechanism. Prior studies have shown that virus mutants resistant to or dependent on IBT affect proteins involved in control of viral postreplicative transcription elongation, including G2, J3, and the viral RNA polymerase. Prior studies also suggest that there exist additional unidentified vaccinia genes that influence transcription elongation. The present study was undertaken to target candidate transcription elongation factor genes in an error-prone mutagenesis protocol to determine whether IBT-resistant or -dependent alleles could be isolated in those candidate genes. Mutagenesis of genes in which IBT resistance alleles have previously been isolated, namely A24R (encoding the second largest RNA polymerase subunit, rpo132) and G2R (encoding a positive transcription elongation factor), resulted in isolation of novel IBT resistance and dependence alleles therefore providing proof of principle of the targeted mutagenesis technique. The vaccinia H5 protein has been implicated previously in transcription elongation by virtue of its association with the positive elongation factor G2. Mutagenesis of the vaccinia H5R gene resulted in a novel H5R IBT resistance allele, strongly suggesting that H5 is a positive transcription elongation factor.

Fig. 1. Targeted mutagenesis and selection for IBT resistance

A) A diagram showing the three overlapping fragments, A24R-1, A24R-2 and A24R-3, that were amplified from the A24R gene. B) Marker rescue with error prone PCR fragments. Confluent dishes of BSC40 cells were infected with Dts38 and co-transfected with wild-type genomic DNA and the indicated PCR product. Controls are shown in the top row: “Uninfected” was neither infected nor transfected. “Untransfected” was infected with Dts38 but not transfected. “WT genome” was infected with Dts38 and transfected with wild type genomic DNA alone. In each of the bottom three boxed panels, the top row contains negative controls, transfected with the indicated high-fidelity PCR product, while the bottom row contains dishes transfected with the indicated error-prone PCR product. After a 4 day incubation at 37°C in the presence of IBT, dishes were stained with crystal violet.

Fig. 2. G2R and A24R mutants constructed by error-prone PCR mutagenesis map to the targeted gene or gene fragment

Confluent dishes of BSC40 cells were infected with the Dts38 helper virus and co-transfected with wild-type genomic DNA (“WT genome”) and the indicated PCR product. After a 4 day incubation at 37°C in the presence of IBT, dishes were stained with crystal violet.

Fig. 3. Marker rescue of the H5R-R1 mutation

Confluent dishes of BSC40 cells were infected with the Dts38 helper virus and co-transfected with wild-type genomic DNA (“WT Genome”) and the indicated PCR product. After a 4 day incubation at 37°C in the presence of IBT, dishes were stained with crystal violet.

Fig. 4. Plaque phenotypes of mutant viruses

Confluent monolayers of BSC40 cells in 6 cm dishes were infected with appropriate dilutions of WT virus and the indicated mutants. The dishes were incubated at 37°C for 6 days in the presence and absence of IBT prior to staining with crystal violet.

Fig. 5. Northern analysis of IBT-resistant mutant RNA

Confluent BSC40 cells were infected with the indicated virus at m.o.i. = 15 in the presence or absence of IBT and incubated at 37°C for 9 hours. Total cellular RNA was purified, fractionated by gel electrophoresis and transferred to a nylon membrane. The membrane was probed with a riboprobe specific for the late A10L gene and exposed to film. The virus used for infection is indicated at the top of the autoradiogram. The presence of absence of IBT in the infection is indicated with a “+” or a “−” at the top of the autoradiogram. Size markers are indicated at the left of the autoradiagram, in kb.

Fig. 6. Structural modeling of A24-R1 and A24-R2

A model of the vaccinia rpo312 (gene A24R) subunit was constructed based on homology to the known structure of S. cerevisiae RNA polymerase II subunit rpb2 (Prins et al., 2004). Yeast RNA polymerase residues homologous to the mutant vaccinia alleles were then highlighted on the structure of the elongating yeast RNA polymerase. A) Side (cutaway) view of the RNA polymerase II transcribing complex. The template DNA strand is in blue, the nontemplate DNA strand is in green, and the RNA is in red. Additional structural features of the enzyme are indicated. B-D) Close up views of the yeast RNA polymerase II active site, with residues homologous to the A24R mutations highlighted in orange. The figures show distances in angstroms from the mutant residue to the 3′ end of the RNA, and in D, from the catalytic Mg++ to three conserved active site aspartic acid residues and the mutation. B) A24R-R1 (yeast L514). C and D) A24R-R2 (yeast T884). Colors indicate the following: mutant residue, orange; active site, purple; wall, grey; hybrid binding, green; RNA, red; DNA, blue A) is from Klug (2001).