Vaccinia virus (VACV) encodes a heterodimeric mRNA capping enzyme consisting of the catalytic large subunit D1 and the stimulatory small subunit D12. This is different from many other nucleocytoplasmic large DNA viruses, which encode the tripartite capping activities in one protein. In vitro studies indicate that while D12 lacks catalytic activity, it enhances D1's methyltransferase function. To define the functional requirement of D12 in VACV replication in infected cells, we generated a D12L (ORF encoding the D12 gene)-deleted virus (vΔD12) using rabbit RK13 cells stably expressing D12. While vΔD12 replication was reduced and associated with impaired viral intermediate and late gene expression and altered plaque morphology, it remained permissive in RK13 cells even without D12 complementation. We further revealed that viral early gene expression was preserved in vΔD12-infected cells, whereas viral DNA replication, intermediate and late gene expression was reduced. Interestingly, vΔD12 infection was unpermissive in monkey BS-C-1 cells, with little intermediate and late gene expression. Furthermore, vΔD12 was unpermissive in rhesus macaque gastrointestinal organoids (enteroids), but remained permissive in human enteroids. These findings reveal differential requirements of D12 for VACV replication in host-specific cells and enteroids |
