Disparate regions of envelope protein regulate syncytium formation versus spongiform encephalopathy in neurological disease induced by murine leukemia virus TR

Abstract

The murine leukemia virus (MLV) TR1.3 provides an excellent model to study the wide range of retrovirus-induced central nervous system (CNS) pathology and disease. TR1.3 rapidly induces thrombotic events in brain microvessels and causes cell-specific syncytium formation of brain capillary endothelial cells (BCEC). A single amino acid substitution, W102G, in the MLV envelope protein (Env) regulates the pathogenic effects. The role of Env in determining this disease phenotype compared to the induction of spongiform encephalomyelitis with a longer latency, as seen in several other MLV and in human retroviruses, was determined by studying in vitro-attenuated TR1.3. Virus cloned from this selection, termed TRM, induced progressive neurological disease characterized by ataxia and paralysis and the appearance of spongiform neurodegeneration throughout the brain stem and spinal cord. This disease was associated with virus replication in both BCEC and highly ramified glial cells. TRM did not induce syncytium formation, either in vivo or in vitro. Sequence and mutational analyses demonstrated that TRM contained a reversion of Env G102W but that neurological disease mapped to the single amino acid substitution Env S159P. The results demonstrate that single nucleotide changes within disparate regions of Env control dramatically different CNS disease patterns.

FIG. 1.

Spongiform encephalopathy in TRM-infected mice. Mice were inoculated with 3 × 10⁵ PFU of TRM at birth, and tissue was extracted for analysis by hematoxylin- and eosin-stained sections (6 μm) fixed in 10% formalin at the indicated times. (A) Spongiform neurodegeneration in the pons and medulla, indicated by arrows, on day40 (magnification, ×8.5); (B) spongiosis in both the deep cerebral cortex (thick arrow) and the molecular layer (thin arrow) on day 40 (magnification, ×8.5); (C) brainstem revealing spongiform changes in both gray matter (thin arrow) and white matter (thick arrow) tracts on day 40 (magnification, ×17); (D) spinal cord with marked pathology in the anterior (thin arrow), but not ventral, horns on day 21 (magnification, ×8.5); (E) spinal cord with vacuolation of neuronal cytoplasm (thin arrow) and hyperchromatic and picnotic nuclei (thick arrow) on day 21 (magnification, ×34).

FIG. 2.

CNS cell tropism of TRM. Fixed thin sections of brain tissue from TRM-infected mice were analyzed for sites of infection by immunohistochemistry. Env and VWF expression in a paraffin section of brain tissue from a TRM-infected mouse on day 21 is shown (magnification, ×34). Virus Env expression appears in green, while VWF expression on endothelial cells appears in red. Colocalization of Env with VWF is indicated by the blue arrow, Env staining adjacent to endothelial cells is indicated by the purple arrow, and Env expression independent of endothelial cells is indicated with a white arrow.

FIG. 3.

Cell fusion potential of TRM Env. Env fusion potential was measured in an in vitro cell-cell fusion assay. Fusion is determined by luciferase activity (relative light units) after coculture of effector cells that express Env with target cells that express either the mCAT1 receptor protein (hatched bars) or no receptor (filled bars). The asterisk indicates statistically significant differences (P < 0.05) in fusion to a common receptor-bearing target, as determined by the unpaired Student's t test.

FIG. 4.

Identification of amino acid variations in MLV Env. The locations of differences in the amino acid sequences of TRM, TR1.3, and FB29 are highlighted on the ribbon model of Friend 57 Env receptor binding domain (9). Amino acids are numbered with the mature form of gp70 lacking a signal peptide.

FIG. 5.

Genetic mapping of TRM disease. TRM disease maps to Env159. The impact of individual TRM site mutants inserted into the backbone of FB29 on neurological disease was determined as described in Table 1. Arrows denote the site of nucleotide changes introduced to generate the mutation.