Rabies lyssavirus phosphoprotein possesses RNA duplex-unwinding activities

Rabies lyssavirus (RABV) is a prototypical neurotropic virus that belongs to the Rhabdoviridae family and causes one of the most lethal zoonotic diseases. For RNA viruses, RNA-unwinding proteins such as helicases and RNA chaperones are crucial for the replication and/or correct folding of viral RNAs. Although numerous RNA viruses have been found to encode their RNA-unwinding proteins, no RNA-unwinding activity has been identified in the family Rhabdoviridae. Herein, we uncovered that RABV phosphoprotein (RABV-P) possesses both a nucleoside triphosphate (NTP)-dependent helicase-like RNA duplex-unwinding activity and an NTP-independent duplex-unwinding and annealing activity. The C-terminal domain of RABV-P is required for its RNA-unwinding activities, whereas the NTP-dependent helicase-like unwinding activity also relies on its central oligomerization domain (COD). Furthermore, we designed two peptides (named P90 and P110) to target the COD and revealed that these peptides effectively inhibited RABV replication in cells. Overall, the study provides the first demonstration of the RNA helicase-like and NTP-independent RNA-unwinding/annealing activities associated with a rhabdovirus-encoded protein. This study highlights the functional significance of P in the RABV life cycle and probably other rhabdoviruses, and targeting RABV-P represents a promising strategy for developing novel antivirals against this lethal pathogen.