The degree of folding instability of the envelope protein of a neurovirulent murine retrovirus correlates with the severity of the neurological disease.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates a series of responses known collectively as the unfolded protein response (UPR) (33, 43). The UPR consists of three parallel signal transduction pathways activated by transmembrane proteins projecting into the ER lumen which act as sensors of misfolded proteins. The three sensors—Ire1α, ATF6, and PERK—are maintained in an inactive state by the binding of the major ER chaperone BiP to their luminal domains. The accumulation of misfolded proteins in the ER lumen causes BiP to be displaced from the sensor molecules and bind to exposed hydrophobic patches on protein-folding intermediates. This leads to activation of the sensors and initiation of the downstream signaling pathways. Ire1α an endoribonuclease, triggers the upregulation of the protein degradative machinery leading to the removal of terminally misfolded proteins. ATF6 is an ER resident transcription factor that upregulates the expression of genes encoding ER chaperones, including BiP, which improve the folding efficiency of the ER. PERK, an ER resident kinase, phosphorylates the translation initiation factor eukaryotic translation initiation factor 2α, leading to global downregulation of protein translation and consequently a decrease in the load of client proteins entering the ER. These are adaptive responses that contribute to the reestablishment of a state of protein folding homeostasis. If homeostasis is not achieved, ER linked cell death pathways are activated (29, 44). Over the last few years, it has become clear that ER stress is associated with a variety of human degenerative diseases. These include disorders as diverse as diabetes, retinitis pigmentosa, brittle bone disease (osteogenesis imperfecta), and a variety of hereditary leukodystrophies (45), as well as Parkinson's and Alzheimer's disease (37). In some of these diseases, ER stress is activated by the accumulation of a mutant protein. In other cases, the ER stress is caused by dysfunction of one of the elements of the UPR, limiting its capacity to respond to demands on the folding machinery. The UPR is also activated by some viruses, including hantavirus, flaviviruses, paramyxoviruses, and Borna virus (17, 29), as well as prion agents (20), although a role for ER stress in disease pathogenesis remains speculative. A possible role for ER stress in the pathogenesis of Borna virus-induced neurodegeneration has been reported (53). However, it is not known whether the insult to the ER is due to misfolding of a viral protein. The evidence is stronger for a role of ER stress in the pathogenesis of a neurodegenerative disease caused by a small group of murine retroviruses. Derivatives of CasBrE (13), a virus originally isolated from feral mice, and Moloney murine leukemia virus ts1 (MoMLVts1) (54), a temperature-sensitive strain of MoMLV, cause a noninflammatory spongiform encephalopathy resembling histopathologically the prion diseases. The neurovirulence of these viruses is determined by the sequence of the envelope protein (7, 39, 49, 55), although the nature of the toxicity has remained elusive. We have studied the role of the envelope protein in disease pathogenesis using two coisogenic viruses, FrCasNC (9) and F43 (2). FrCasNC contains the envelope gene and 3′pol sequences from the neurovirulent virus CasBrE and causes a rapidly progressive and fatal neurological disease characterized by tremor, paralysis, and wasting. F43 carries the envelope gene and 3′ pol sequences of a nonneurovirulent virus, FMLV57 (38), and is apathogenic. Both viruses are highly neuroinvasive and infect the same spectrum of cells in the brain (2). Transcriptional analysis of brain tissue from infected mice revealed that only the neurovirulent virus induced the UPR (8). Activation of the UPR was found to be a consequence of the folding instability of the FrCasNC envelope protein, which was retained in the ER and degraded by the ubiquitin-proteasome system (9). In addition, histopathologic studies revealed that one of the downstream target genes of the UPR, CHOP, is highly expressed in cells in the brain exhibiting signs of cytopathology (4). In the present study, we further explored the association between protein misfolding and neuropathology. A domain swapping strategy was used to construct a series of highly neuroinvasive viruses carrying F43/FrCasNC sequences. These viruses differed in the severity of the neurological disease they induced. Neuropathogenicity was precipitated only by those viruses carrying the receptor-binding domain (RBD) of the envelope protein from FrCasNC. Of particular interest was the observation that the severity of the neurological disease was directly related to the degree of folding instability of the envelope protein in the ER.