Multiple sclerosis (MS) is a central nervous system (CNS) disease characterized by patches of demyelination and infiltration of inflammatory cells (21). The etiology of this disabling diseasehas not yet been determined, but both genetic factors, such as genes encoding human leukocyte antigens, T-cell receptors, and myelin basic protein (MBP) (18,19,26), and environmental factors such as viruses have been implicated (31). At least four human demyelinating diseases have a known viral etiology: subacute sclerosing panencephalitis as a late complication of measles virus infection of childhood (35), progressive multifocal leukoencephalopathy caused by the JC papovavirus (57), encephalopathy and myelopathy (neuro-AIDS) caused by human immunodeficiency virus (43), and human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis (28). Over the last decades, several viruses have been associated with MS, based on detection of virions, viral nucleic acids, or viral proteins in CNS or the presence of antiviral antibodies in serum and/or cerebrospinal fluid. A confirmed association with MS is awaited but may involve more than one virus.Several studies have associated human coronaviruses (HCoV) with MS. Coronavirus-like particles were detected in autopsied brain tissue from an MS patient (56). Two coronaviruses that are molecularly related to murine neurotropic coronaviruses were isolated from brain material obtained at autopsy from two MS patients (9). Intrathecal anti-HCoV antibody synthesis indicative of a CNS infection was reported in MS patients (45). HCoV RNA was detected in MS patient brains (37, 51) and in cerebrospinal fluid of MS and other neurological disease (OND) patients (15). Coronavirus antigens were also detected in MS patient brains (37). Moreover, we have shown that HCoV can infect human astrocytes and microglia in primary cultures (8) and can acutely and persistently infect immortalized human glial cells (4, 5). Thus, accumulating evidence suggests that these viruses, first isolated as pathogens of the respiratory tract and now associated with up to one-third of human common colds (39), might be neurotropic, neuroinvasive, and neurovirulent in humans, as is the case for their murine counterpart, the coronavirus mouse hepatitis virus (MHV). Interestingly, upper respiratory infections of viral origin were shown to be an important trigger of MS attacks (2,40,48). Moreover, coronavirus seasonal patterns fit the observed occurrence of MS exacerbations (48).MHV-induced demyelinating disease involving coronaviruses is used as an animal model for elucidating the complex pathogenesis of MS. As for MS, MHV pathogenesis is multifactorial (27). Its outcome is influenced by genetics of the host and virus, dose and route of inoculation, and host age and immunological status at the time of infection (58). Neurotropic MHV strains could invade the CNS following an intranasal inoculation in mice (34) and could also gain access to the CNS via the hematogenous and/or lymphatic systems in mice (7) and i...

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A recombinant vaccinia virus vector was used to coexpress the two candidate mouse prohormone convertases, PC1 and PC2, together with mouse proopiomelanocortin (POMC) in the constitutively secreting cell line BSC-40 and in the endocrine tissue-derived cell lines PC12 and AtT-20, which exhibit regulated secretion. Monitoring of POMC processing demonstrated the distinct cleavage specificities of PCi and PC2, since in the cell lines analyzed (i) PCi cleaves POMC into corticotropin and fB-lipotropin, (ii) PC2 cleaves POMC into (3-endorphin, an N-terminally extended corticotropin containing the joining peptide, and either aMSH or desacetyl-aMSH, and (iii) PC2 cleaves POMC at the five pairs of basic residues analyzed, whereas PC1 cleaves two of them preferentially, suggesting that PC2 has a broader spectrum of activity than PC1. These data are consistent with our hypothesis on the physiological role of PCi and PC2 as distinct proprotein convertases acting alone or together to produce a set of tissuespecific maturation products in the brain and in peripheral tissues.Limited proteolysis of inactive precursors at pairs of basic residues or, less frequently, at single basic amino acids (1, 2) is a general mechanism by which the cell produces a variety of active proteins and peptides. Until recently, the molecular nature of the processing enzyme(s) was not identified except for the yeast pro-a mating factor precursor, where a subtilisin-like serine proteinase called KEX2 was proven to be the physiological convertase (3). In the last year, different groups obtained the complete cDNA structures of three distinct candidate mammalian convertases, furin (4), PC1 (5, 6), and PC2 (5, 7). The putative convertase function of these proteins was largely based on sequence homology with KEX2 (8, 9) and on their tissue and cellular distribution, especially for PC1 and PC2 (5, 6). Here we show that the expression of PC1 or PC2 by vaccinia virus (VV) recombinants results in the production of distinct proteolytic enzymes in three cell lines: the mouse pituitary corticotroph line AtT-20 and two that do not express these enzymes endogenously, the African green monkey kidney epithelial cell line BSC-40 and the rat pheochromocytoma cell line PC12. These proteinases cleave proopiomelanocortin (POMC) into the same set of distinct peptides known to be produced in vivo in the hypothalamus, adenohypophysis (AP), and pars intermedia of the pituitary, including adrenocorticotropin (ACTH), p-lipotropin (,8LPH), ,f-endorphin (f3End), and a-melanotropin (aMSH) or desacetyl-aMSH.MATERIALS AND METHODS Ws. Purified recombinant VVs using the full-length mouse (i) PC1 and mPC2 cDNA inserts (5, 6) (VV:mPC1 and VV:mPC2) were prepared as described (10). VV:POMC (mouse) was a gift of G. Thomas (Vollum Institute, Portland, OR).Northern Blot Analysis. Total RNA was prepared as described (11). The [a-32P]UTP-labeled cRNA probes were generated from cDNA sequences of mPC1 (6) and mPC2 (5) inserted in plasmid Rc/CMV vector (Invitrogen, San Diego) and linearized...

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