A single nucleotide substitution in the 5'-untranslated region of the vaccinia N2L gene is responsible for both alpha-amanitin-resistant and temperature-sensitive phenotypes.

The locus responsible for encoding resistance to alpha-amanitin was previously mapped to the vaccinia virus (VV) HindIII N fragment by using cloned wild-type VV DNA fragments to rescue the ability of an alpha-amanitin-resistant/temperature-sensitive VV mutant (alpha rts7) to replicate under nonpermissive conditions. DNA sequencing and transcriptional analyses of this region identified two leftward-reading open reading frames (ORFs), N2L and M1L, as candidates to encode the protein responsible for eliciting both phenotypes. In the present study, high-resolution marker rescue mapping and genomic sequencing techniques have been applied to identify the nature of the mutation within the HindIII N region of the alpha rts7 genome. Interestingly, a single G to T transversion mutation was noted at position -10 relative to the initiator ATG of the N2L ORF. Since transcription of the N2L gene starts at position -12/-13, this places the alpha rts7 mutation within the 5'-untranslated leader of the N2L transcript expressed early in infection and suggests that the transcriptional efficiency, mRNA stability, or translational efficiency must be altered in the mutant RNA. These results identify the N2L ORF as the gene responsible for conferring resistance to alpha-amanitin in the alpha rts7 mutant and suggest that the N2L gene product is the viral function that interacts with the host cell nucleus during VV infection.