Molecular genetic analysis of revertants from a poliovirus mutant that is specifically adapted to the mouse spinal cord

Abstract

SA virus, a mutant of the Mahoney strain of type 1 poliovirus (PV1/Mahoney), replicates specifically in the spinal cords of mice and causes paralysis, although the PV1/Mahoney strain does not show any mouse neurovirulence (Q. Jia, S. Ohka, K. Iwasaki, K. Tohyama, and A. Nomoto, J. Virol. 73:6041-6047, 1999). The key mutation site for the mouse neurovirulence of SA was mapped to nucleotide (nt) 928 of the genome (A to G), resulting in the amino acid substitution of Met for Ile at residue 62 within the capsid protein VP4 (VP4062). A small-plaque phenotype of SA appears to be indicative of its mouse-neurovirulent phenotype. To identify additional amino acid residues involved in the host range determination of PV, a total of 14 large-plaque (LP) variants were isolated from a single point mutant, Mah/I4062M, that showed the SA phenotype. All the LP variants no longer showed any mouse neurovirulence when delivered via an intraspinal inoculation route. Of these, 11 isolates had a back mutation at nt 928 (G to A) that restored the nucleotide of the PV1/Mahoney type. The reversions of the remaining three isolates (LP8, LP9, and LP14) were mediated by a second site mutation. Molecular genetic analysis involving recombinants between Mah/I4062M and the LP variants revealed that the mere substitution of an amino acid residue at position 107 in VP1 (Val to Leu) (LP9), position 33 in VP2 (Val to Ile) (LP14), or position 231 in VP3 (Ile to Thr) (LP8) was sufficient to restore the PV1/Mahoney phenotype. These amino acid residues are located either on the surface or inside of the virus particle. Our results indicate that the mouse neurovirulence of PV is determined by the virion surface structure, which is formed by all four capsid proteins.

FIG. 1

Plaque phenotypes of PV1. Monolayers of confluent AGMK cells grown in 60-mm-diameter dishes were infected with PV1/Mahoney, Mah/I4062M, or LP14. After the dishes were incubated at 37°C for 3 days, the cells were fixed and stained with 1% crystal violet.

FIG. 2

Nucleotide and amino acid substitutions in the genomes of LP variants. The PV genome is shown at the top of the panel. The genome structure of Mah/I4062M is shown as a combination of PV1/Mahoney (black box) and SA (horizontally striped box) sequences. Nucleotide and amino acid (a.a.) substitutions in the genome of Mah/I4062M and LP variants are shown as open boxes. Genome regions shown by diagonal lines were not sequenced. The 50% lethal dose (LD50 [PFU]) value of each virus obtained from the mouse neurovirulence tests following an intraspinal inoculation is shown on the right. Plaque size was measured following a 3-day incubation in AGMK cells, and the results are shown on the right. L, >3 mm; S, <1 mm.

FIG. 3

Recombinants between Mah/I4062M and LP variants. The genome structures of PV1 and Mah/I4062M and the amino acid (a.a.) substitutions in the genomes of the recombinants are as defined in the legend for Fig. 2. Cleavage sites of the restriction enzymes used for the allele replacement are shown by the name of the enzyme with the relevant nucleotide position. The plaque size was defined as in the legend for Fig. 2. The mouse neurovirulence of each virus was tested following an intraspinal inoculation of 10⁶ PFU of each virus. The results are shown on the right (number paralyzed/number infected).

FIG. 4

Kinetics of PV replication in the CNS of mice. (A) A total of 10⁵ PFU of PV1/Mahoney, Mah/I4062M, or LP14 virus was injected into non-Tg IQI mice via an intraspinal inoculation route. (B) A total of 10⁴ PFU of each virus was injected into Tg mice via an intracerebral inoculation route. The brains and spinal cords of the mice were removed at the indicated times and homogenized as described in Materials and Methods. Virus titers were determined by plaque assay.

FIG. 5

One-step growth curves of LP variants. Monolayers of AGMK cells grown on 60-mm-diameter dishes were infected with each virus at an MOI of 10. Cells were scraped at the indicated times and frozen at −80°C. The mixture was prepared for titration by three rounds of freezing and thawing, and the cell debris was removed by low-speed centrifugation. Viral titers were determined by plaque assay in AGMK cells.

FIG. 6

Locations of the amino acid substitutions on the three-dimensional structure of PV1/Mahoney capsid protomer. Single capsid protomer viewed from the side (left) or the outside (right) of the particle, 60 of which form the particle. VP1 is blue, VP2 is light blue, VP3 is green, and VP4 is yellow. Amino acid substitutions for LP phenotype are shown as white spheres. The amino acid residues are given (e.g., 3231, residue 231 of VP3). 5 ×, fivefold axis of symmetry.