Rabies virus (RABV) phosphoprotein (P) gene
includes five in-frame start codons, resulting in expression of five P protein isoforms,
full-length P1 and P2-P5, with different lengths of N-terminal truncation. These
start codons are highly conserved among RABV strains, strongly suggesting important
roles of the isoforms in the biological properties of RABV. However, while P1
is known as a cofactor of RNA-dependent RNA polymerase, little is known about
functions of P2-P5, including their roles in viral pathogenicity. Our previous
examination of two fixed RABV strains, Nishigahara (Ni) and the derivative
Ni-CE, which cause lethal and non-lethal infections in mice after intracerebral
inoculation, respectively, revealed that Ni-CE strain lacks P protein functions
that are important for viral pathogenicity (Shimizu et al., 2007). In this
study, to evaluate the importance of P3-P5 in viral pathogenicity, we generated
a recombinant Ni-CE strain expressing Ni P3-P5 (Ni-CE+NiP3) by insertion of the
P3 gene region into the Ni-CE genome and checked the pathogenicity in mice. We
found that both Ni-CE+NiP3 strain and Ni-CE+CEP3 strain, which is a control
virus overexpressing Ni-CE P3-P5, grow in mouse neuroblastoma NA cells similarly
to the parental Ni-CE strain, indicating that these recombinant strains retain
fundamental replication ability. Next, we compared the pathogenicity of
Ni-CE+NiP3 strain in mice with the pathogenicities of Ni-CE and Ni-CE+CEP3
strains by intracerebral inoculation of six-week-old ddY mice with 104 FFU
of each strain. While Ni-CE and Ni-CE+CEP3 strains caused non-lethal mild
infection (transient body weight loss) in all of the inoculated mice,
Ni-CE+NiP3 strain caused lethal infection accompanied by severe neurological
symptoms in 60% of the mice. Based on the fact that Ni-CE+NiP3 strain is more
virulent than Ni-CE+CEP3 strain, we conclude that P protein isoforms (P3-P5) of
RABV (Ni strain) play important roles in viral pathogenicity.