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

Abstract

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 by 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.Rabies remains one of the most dreadful neurotropic infectious diseases affecting humans and animals. Rabies virus (RV) multiplies first at the site of infection and is transported by retrograde flow through peripheral nerves to the central nervous system (CNS), where it causes encephalomyelitis. The mechanism by which RV is transported retrograde from peripheral sites to the neurons is poorly understood. What has recently been described is that the cytoplasmic dynein light chain (LC8), which is involved in the retrograde intracellular transport of organelles, interacts with the phosphoprotein (P) of RV and is incorporated into mature virions (13, 25). The interaction of RV P with LC8 was shown by coimmunoprecipitation analysis, and mapping studies have demonstrated that LC8 interacts in the region between residues 138 and 172 of the P protein. Using biochemical and mutational analysis of selected LC8 binding proteins, Lo et al. (18) identified a consensus sequence containing a K/RXTQT motif that interacts with LC8. The involvement of LC8 in various intracellular transport mechanisms and other cellula...

The dynein light chain 8 binding motif of rabies virus phosphoprotein promotes efficient viral transcription

“…In contrast, Mebatsion (27) had previously demonstrated that deletion of the dynein LC8-BD did not affect viral replication in nonneuronal BHK-1 cells. However, when murine neuroblastoma (NA) cells were infected synchronously at a multiplicity of infection (MOI) of 10, r⌬LC8-N2c grew to 5-to 10-fold lower titers than rN2c (SI Fig.…”

“…In fact, LC8 is incorporated into mature RV virions (26), providing additional evidence that LC8 might be involved in the attachment of the viral particle to the dynein motor complex on entry into the host cell. However, subsequent experiments in 7-day-old mice demonstrated that removing the LC8-BD simply delayed onset of disease, still allowing the transport of RV into the CNS (27,28).…”

Perspectives in Diagnosis and Treatment of Rabies Viral Encephalitis: Insights from Pathogenesis

“…However, mutant viruses with deletion in phosphoprotein encompassing LC8-interacting motif retained their neuroivasiveness and virulence [72]. Neuroattenuation required simultaneous substitution of arginine at position 333 of viral glycoprotein with deletion of LC8, reinforcing that rabies viral glycoprotein plays a more important role than phosphoprotein [72]. Several viruses such as HIV, poliovirus, herpes simplex virus, African swine flu, and adenovirus use dynein for intracellular transport of the virus prior to viral replication in the perinuclear area in the microtubule organizing center, where the new virions are assembled.…”

“…Interaction between the phosphoprotein of rabies virus, and LC8-the cytoplasmic dynein light chain that regulates microtubule-mediated transport-is implicated in viral transport [69][70][71]. However, mutant viruses with deletion in phosphoprotein encompassing LC8-interacting motif retained their neuroivasiveness and virulence [72]. Neuroattenuation required simultaneous substitution of arginine at position 333 of viral glycoprotein with deletion of LC8, reinforcing that rabies viral glycoprotein plays a more important role than phosphoprotein [72].…”

Filogenia de vírus da raiva isolados de morcegos frugívoros do gênero Artibeus e relacionados a morcegos hematófagos com base nos genes codificadores da nucleoproteína N e glicoproteína G

“…O sítio II é descontínuo e está localizado nas posições 34-42 e 198-200 ligadas por pontes dissulfeto (TORDO, 1996). Além disso, a arginina na posição 333 da glicoproteína G tem sido descrita como responsável pela patogenicidade do RABV (TORDO, 1996;MEBATSION, 2001).…”