The Vaccinia Virus Bifunctional Gene J3 (Nucleoside-2′-O-)-methyltransferase and Poly(A) Polymerase Stimulatory Factor Is Implicated as a Positive Transcription Elongation Factor by Two Genetic Approaches

Abstract

Vaccinia virus genes A18 and G2 affect the elongation and termination of postreplicative viral gene transcription in opposite ways. Viruses with mutations in gene A18 produce abnormally long transcripts, indicating that A18 is a negative transcription elongation factor. Viruses containing mutations in gene G2 produce transcripts that are abnormally short, truncated specifically from their 3' ends, indicating that G2 is a positive transcription elongation factor. Despite the fact that both A18 and G2 are essential genes, A18-G2 double-mutant viruses are viable, presumably because the effects of the mutations are mutually compensatory. In addition, the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) seems to enhance elongation during a vaccinia infection: IBT treatment of a wildtype vaccinia infection induces a phenotype identical to an A18 mutant infection, and G2 mutant viruses are dependent on IBT for growth, presumably because IBT restores the G2 mutant truncated transcripts to a normal length. These observations inspire two independent genetic selections that have now been used to identify an additional vaccinia gene, J3, that regulates postreplicative transcription elongation. In the first selection, a single virus that contains an extragenic suppressor of the A18 temperature-sensitive mutant, Cts23, was isolated. In the second selection, several spontaneous IBT-dependent (IBT(d)) mutant viruses were isolated and characterized genetically. Marker rescue mapping and DNA sequence analysis show that the extragenic suppressor of Cts23 contains a point mutation in the J3 gene, while each of seven new IBT(d) mutants contains null mutations in the J3 gene. The J3 protein has previously been identified as a (nucleoside-2'-O-)-methyltransferase and as a processivity subunit for the heterodimeric viral poly(A) polymerase. The nature of the two independent selections used to isolate the J3 mutants strongly suggests that the J3 protein serves as a positive postreplicative transcription elongation factor during a normal virus infection.

Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication

“…To identify viral proteins involved in transcriptional processivity, Condit and colleagues isolated viral mutants resistant to isatin-␤-thiosemicarbazone (IBT) (55)(56)(57)(58)(59)(60). IBT leads to the synthesis of run-on transcripts, which in turn generate excessive dsRNA that leads to the arrest of infection by the antiviral response.…”

Vaccinia virus transcription

“…About half of the virion-derived RNA Latner et al (2000) polymerase contains this subunit. Heparin-agarose chromatography is capable of separating RNA polymerase into a population containing, and one deficient in, the H4L subunit (Ahn et al, 1994).…”

Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus

“…Post-replicative transcription termination results in heterogenous length transcripts, 1-4-kb-long, that are 5Ј co-terminal (10). Although several factors have been shown to modulate postreplicative transcription termination, no cis-acting sequence similar to early transcription termination has been identified (11)(12)(13). However, a pyrimidine-rich sequence in the coding strand has been identified immediately preceding the 3Ј ends of these post-replicative transcripts (14).…”