Distinct attenuation phenotypes caused by mutations in the translational starting window of Theiler's murine encephalomyelitis virus

Abstract

Upon initiation of translation of picornavirus RNA, the ribosome is believed to bind the internal ribosome entry site of the template and then to form a productive complex with a downstream RNA segment, the starting window. The presence or absence of an AUG triplet within the starting window of the RNA of Theiler's murine encephalomyelitis virus (a picornavirus) is known to modulate its neurovirulence. In this study, mutants of this virus in which the starting windows, lying upstream of the viral polyprotein reading frame, had AUGs with different nonoptimal contexts were engineered. Upon intracerebral inoculation of mice, the mutants proved to be partially attenuated, as judged by a significant increase in the dose causing paralysis in 50% of the animals (PD50). Mutants with similar PD50s might differ from one another by eliciting either a severe, fatal tetraplegy or only mild, recoverable neurologic lesions. Some of the mutants triggered a chronic inflammatory reaction in the white matter of the spinal cord in the absence of detectable viral RNA or antigen. Thus, point mutations changing the context of an AUG within the starting window outside the polyprotein reading frame may differently affect the morbidity and mortality caused by a viral infection and may result in distinct attenuation phenotypes.

FIG. 1

Genome structures of GDVII and its derivatives. Dots in the GDVII sequence are for alignment. AUGs are underlined. For the mutants with the 27-nt inserts, the AUG and its variable contexts within the starting window are in boldface and the stop codon that follows is in italics.

FIG. 2

Time course of clinical signs after intracerebral inoculation of mice with mutants 43 and 63 at 10⁸ and 10^8.5 TCD₅₀, respectively. Twenty-eight and 24 animals were infected simultaneously with mutants 43 and 63, respectively, and monitored individually. The numbers of mice that were paralyzed, died, and developed the second wave of paralytic symptoms are shown as gray, black, and white areas, respectively. Numbers on the x axis are days p.i.

FIG. 3

Dependence of the outcome of infection by 7 to 10 weeks p.i. on the time of the appearance of the first clinical signs. The proportions of dead (black bars), irrecoverably paralyzed (gray), and fully recovered (white) mice are shown. Data were collected from several experiments. The numbers of animals that got sick during the first (1 w), second (2 w), and third (3 w) weeks, respectively, were as follows: 72, 70, and 26 (mutant 43); 66, 34, and 8 (mutant 63); 38, 35 and 10 (mutant 64); and 29, 9, 0 (mutant 65).

FIG. 4

Histological findings for SJL/J mice after inoculation with 10⁵ PFU of wt and mutant viruses. Longitudinal sections of the spinal cords of the infected animals were prepared on day 5 for GDVII (A) on day 6 for mutants 63 (B), 64 (C), and 43 (D), and on day 22 for mutant 43 (E and F). Viral antigens were detected by the immunoperoxidase assay with a polyclonal anti-TMEV rabbit serum (brown clusters). Virus-infected cells were localized in the gray matter and were more abundant in the GDVII- and mutant 63-infected animals than in the mutant 43 and 64 infections. In the case of mutants 43 and 64 (but not in the case of GDVII and mutant 63), there were marked signs of inflammation in the white matter (arrows) even at day 22 p.i., when no viral antigen was detectable (E and F). Magnification, ×312.

FIG. 5

Viral RNA in the CNSs of infected mice as assayed by dot blot hybridization. GDVII- and mutant-infected mice were sacrificed on days 5 and 6 p.i., respectively. Total cytoplasmic RNA was extracted from the brains and spinal cords of infected mice, loaded on the membrane (in the amounts [in micrograms] indicated at the top) and hybridized with ³²CTP-labeled PCR-amplified, virus-specific DNA. The numbers on the left correspond to individual mice.