Expression of Inducible Nitric Oxide Synthase and Elevation of Tyrosine Nitration of a 32-Kilodalton Cellular Protein in Brain Capillary Endothelial Cells from Rats Infected with a Neuropathogenic Murine Leukemia Virus

A number of murine leukemia viruses (MuLVs) have been shown to induce diseases of the central nervous system (CNS) that are characterized by progressive loss of neuronal function (35, 39). The major cell types within the CNS that are prominently infected with the MuLVs are glial and endothelial cells, with neurons being infrequently infected. The most commonly observed pathological changes are gliosis, neuronal loss, and demyelination. The mechanism(s) by which the MuLVs induce neurological diseases remains to be elucidated. PVC-211 MuLV is a neuropathogenic variant of the leukemia-inducing Friend MuLV (F-MuLV) (21). Infection of susceptible rats with PVC-211 MuLV causes a rapidly progressive neurodegenerative disease characterized by tremor, spasticity, ataxia, and hind limb paralysis. Neuropathological changes include widespread perivascular gliosis, neuropil vacuolation without inflammation, and neuronal degeneration in the brain stem, cerebellum, and spinal cord (19, 28). The primary target of PVC-211 MuLV infection in the CNS is the brain capillary endothelial cell (BCEC), which is resistant to F-MuLV infection (19). The determinant of the BCEC tropism of PVC-211 MuLV was mapped to two amino acids (G116 and K129) which lie within the putative receptor binding domain of the envelope surface glycoprotein (SU) (30). Within the CNS, reactive astrocytes and degenerating neurons showed no evidence of virus infection (19). BCEC tropism of the virus has been shown to be necessary for neuropathogenesis (29), suggesting that CNS injury is indirect and that molecular events in virus-infected BCECs play a very important role in neurological disease induction. Nitric oxide (NO) is an important messenger and effector molecule involved in a number of biological functions (31). NO is synthesized from l-arginine by three isoforms of NO synthases (NOS). Endothelial cell NOS (eNOS) and neuronal NOS are constitutively expressed, and their activities are regulated by Ca2+. In contrast, inducible NOS (iNOS) is inducible and Ca2+ independent (13). In the CNS, NO may play important roles in neurotransmitter release, neurotransmitter reuptake, neurodevelopment, synaptic plasticity, and regulation of gene expression, although excessive production of NO can lead to neurotoxicity (9, 27). iNOS is an attractive candidate for mediating NO-associated neurotoxicities, because long bursts of large amounts of NO are produced by iNOS (7, 9, 32). Indeed, elevated iNOS expression has been demonstrated in such human neurological diseases as Alzheimer's disease (24) and Parkinson's disease (26). Recently, the spongiform vacuolation observed in PVC-211 MuLV-infected brains was reported to be associated with oxidative damage as detected by increased immunoreactivity for 3-nitrotyrosine (NTyr) in infected brains (43). NTyr is widely used as an indicator of NO formation, because nitration of tyrosine is mediated by reactive nitrogen species derived from NO (2, 11, 15). Elevated expression of NTyr has also been reported in human neurodegenerative diseases such as familial amyotrophic lateral sclerosis (41), Alzheimer's disease (17), Parkinson's disease (12), and human immunodeficiency virus type 1 dementia complex (5). In this study, we examined expression of iNOS and elevated expression of NTyr in PVC-211 MuLV-infected BCECs to evaluate the contribution of NO produced by infected BCECs to the neuropathogenicity induced by PVC-211 MuLV infection.