Lyssaviruses, including Australian bat lyssavirus (ABLV), rabies virus (RABV), and Mokola virus, are a globally distributed genus of zoonotic pathogens that cause rabies disease with a remarkable case-fatality rate of 100% in humans. Human infection results in >55,000 deaths/year worldwide, and the spread of ABLV to humans and horses in Qld., coupled with recent outbreaks of RABV in the Indonesian archipelago, have highlighted to the risk of emergence and invasion by these viruses in Australasia.
We found previously that the phospho(P-)proteins of lyssaviruses interact with signal transducers and activators of transcription (STATs) 1-3. This is thought to effect viral evasion of interferon (IFN)-mediated innate immune responses as a major pathogenicity factor. However,
the significance of these interactions to in vivo infection and disease progression is unknown. Through an approach combining next-generation sequencing/bioinformatics,
site-directed mutagenesis, single live-cell imaging, immune signalling assays,
protein-protein interaction analysis, viral reverse genetics, and an in vivo
disease model, we have identified the P-protein-STAT interaction site for the first time. We further developed mutations able to specifically inhibit P-protein’s function in antagonising IFN/STAT responses without impairing other critical functions of P-protein in genome replication and inhibition of viral induction of IFN.
Using these mutations, we generated the first fully viable “STAT-blind” lyssavirus. Growth of the mutant virus in IFN-deficient cells was indistinguishable
from that of parental wild-type virus, but the mutant virus lacked the capacity to inhibit STAT responses and was severely impaired in its resistance to IFN. Intracerebral
inoculation of mice indicated that the mutant virus was profoundly
attenuated, showing strongly impaired growth in the CNS and
causing no neurological symptoms or deaths. By contrast, the wild-type strain was invariably lethal.
These findings provide the first direct evidence that STAT interactions of P-protein are vital to lyssavirus infection of the CNS and the development of lethal rabies, with significance to
efforts to generate new attenuated vaccine strains and/or novel therapeutics against this incurable disease.