Extensive attenuation of rabies virus by simultaneously modifying the dynein light chain binding site in the P protein and replacing Arg333 in the G protein

Rabies virus (RV) is a highly neurotropic virus that migrates from the portal of entry to the central nervous system (CNS). The cytoplasmic dynein light chain (LC8), which is involved in a variety of intracellular motile events, was shown to interact with RV phosphoprotein (P). In order to determine the functional significance of this interaction, P residues 143 to 149 or 139 to 149 encompassing a conserved LC8-interacting motif (K/RXTQT) were deleted from recombinant viruses SAD-L16 and SAD-D29. These viruses are identical except for a replacement of the arginine at position 333 (R333) of the RV glycoprotein ky an aspartic acid in SAD-D29. SAD-L16 virus is fully pathogenic for mice, whereas SAD-D29 is nonpathogenic for adult mice but retained pathogenicity for suckling mice. The deletions introduced into the LC8 binding site abolished the P-LC8 interaction and blocked LC8 incorporation into virions. All the mutants propagated in cell culture as efficiently as the parent strains. The pathogenicity of the mutants was then compared with that of the parent viruses by inoculating suckling mice. SAD-L16 derivatives were as pathogenic as their parent virus after Intramuscular inoculation, indicating that LC8 is dispensable for the spread of a pathogenic RV from a peripheral site to the CNS. In contrast, SAD-D29-derived deletion mutants were attenuated by as much as 30-fold after intramuscular inoculation but remained as pathogenic as the parent virus when inoculated directly into the brain. This remarkable attenuation after intramuscular but not after intracranial inoculation suggested that abolishing the P-LC8 interaction reduces the efficiency of peripheral spread of the more attenuated SAD-D29 strain. These results demonstrate that elimination of the LC8 ligand and simultaneous substitution of R333 considerably attenuate RV pathogenicity and may be helpful in designing and developing highly safe live-RV-based vaccines.