A determinant for central nervous system persistence localized in the capsid of Theiler's murine encephalomyelitis virus by using recombinant viruses

Abstract

The demyelinating process in Theiler's murine encephalomyelitis virus (TMEV) infection in mice requires virus persistence in the central nervous system. Using recombinant TMEV assembled between the virulent GDVII and less virulent BeAn virus cDNAs, we now provide additional evidence supporting the localization of a persistence determinant to the leader P1 (capsid) sequences. Further, recombinant viruses in which BeAn sequences progressively replaced those of GDVII within the capsid starting at the leader NH2 terminus suggest that a conformational determinant requiring homologous sequences in both the VP2 puff and VP1 loop regions, which are in close contact on the virion surface, might underlie persistence.

FIG. 1

The genome structures of parental and recombinant TMEV. The organization of the TMEV RNA genome, showing the 5′ untranslated region (UTR), leader protein, and capsid proteins, is depicted at the top. Filled bars, the highly virulent GDVII virus sequences; open bars, the less virulent BeAn virus sequences. Recombinant cDNA clones were assembled in pGEM-4 downstream of the T7 RNA polymerase promoter by exchanging equivalent sequences from full-length parental clones (2, 16). Leader P1 (capsid) exchanges were in subgenomic clones with the restriction endonucleases shown here. Subgenomic constructs were assembled into full-length clones by using the unique XhoI site at 1D/2A and the ScaI site in pGEM-4. An XhoI site, which was absent in wild-type GDVII, was introduced by a T-to-C mutation by site-directed mutagenesis as described elsewhere (16). Recombinant 33 was made by PCR amplification and splicing by overlap extension to yield BeAn sequences between nucleotides 1523 and 2219 (6). The 10% difference in the nucleotide sequences of the two parental viruses enabled confirmation of the origin of all exchanged genomic parts by cleavage of cDNA clones with restriction endonucleases. In addition, the capsid sequences across splice sites in the recombinants and the 2376 BeAn nucleotides between the SspI and MluI sites in recombinant 41 were verified by PCR amplification and dideoxynucleotide sequencing of RNA isolated from viral stocks. (A) Parental viruses, recombinant viruses in which the GDVII and BeAn leader capsid regions were exchanged, and recombinant viruses that progressively replaced GDVII with BeAn sequences within the capsid starting at the SspI restriction endonuclease site in the NH2 terminus of the leader; (B) TMEV recombinants that progressively replaced GDVII with BeAn sequences within the capsid starting at the XhoI restriction endonuclease site at the 1D/2A cleavage site.

FIG. 2

One-step growth kinetics of GDVII and BeAn parental viruses and recombinant viruses in which the GDVII and BeAn leader-capsid regions were exchanged (A) and TMEV recombinants that progressively replaced GDVII with BeAn sequences within the capsid starting at the SspI site at the NH2 terminus of the leader protein (B). BHK-21 cell monolayers were infected at a multiplicity of infection of 10 and incubated at 33°C until the indicated times, when virus lysates were prepared.

FIG. 3

Virus titers in spinal cord homogenates from individual mice killed at approximately 90 days after i.c. inoculation with BeAn or recombinant viruses. The minimum detectable virus level of the assay is 50 PFU (dashed line).