A novel accessory gene product of tick-borne Dhori-Orthomyxovirus, encoded by overlooked spliced transcripts of RNA segment 6

This study identifies and characterizes a previously unrecognized accessory gene product encoded by RNA segment 6 of Dhori virus (DHOV), a tick-borne orthomyxovirus within the genus Thogotovirus. Contrary to the long-held assumption that Dhori-like viruses do not use RNA splicing, the authors demonstrate that DHOV segment 6 undergoes splicing in both mammalian and tick cells. This splicing event generates a novel protein, termed M2-248, which consists of the N-terminal portion of the canonical matrix protein fused to a unique C-terminal sequence translated from a −1 shifted reading frame. The splicing sites and the M2-specific sequence are conserved among Dhori-like viruses. M2-248 expression was confirmed by RT-PCR, mass spectrometry, and western blotting, and the protein was shown to be incorporated into released virions and to interact with the matrix protein. Using reverse genetics, the authors generated recombinant DHOV lacking M2 expression and demonstrated that loss of M2 does not impair viral replication, assembly, or particle morphology in cell culture. However, M2-deficient virus exhibited markedly reduced virulence in mice, with improved survival despite only modest reductions in viral replication and inflammatory cytokine induction. Functional analyses showed that, unlike the related Thogotovirus ML protein, M2-248 does not act as a type I interferon antagonist. These findings reveal an unexpected coding strategy in Dhori-like orthomyxoviruses and identify M2-248 as a novel virulence factor acting through an interferon-independent mechanism.
(TMR)