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The appearance of spongiform neurodegeneration in the mammalian central nervous system (CNS) represents a unique pathologic picture typically associated with infection by either unconventional protein infectious agents (prions) or retroviruses. While little is known at the cellular level about how prions induce vacuolar lesions, a detailed picture of how retroviruses induce spongiform pathology is emerging from the analysis of murine leukemia virus (MuLV) models. The best studied of the neurovirulent murine retroviruses is the wild mouse ecotropic virus, Cas-Br-E, which was discovered by Gardner and coworkers in a population of feral mice (13). CNS infection by this virus results in vacuolar changes in motor areas from the cortex through the spinal cord and is manifest clinically, first, as tremulous paralysis of the hindlimbs, progressing to the forelimbs, with associated wasting and eventual death. The appearance and severity of clinical signs and lesions correlates with the level of Cas-Br-E virus infection in the CNS (5), although neurodegeneration cannot occur prior to the 2nd postnatal week no matter how significant the viral load (25). Interestingly, the primary degenerating elements, the motor neurons, are not infected, indicating that neurologic disease is mediated by an indirect mechanism (16, 19, 24). While multiple CNS cell populations are infected, it is microglial infection which specifically correlates with regions of motor neuronal degeneration in vivo (1, 2, 16, 24). Furthermore, CNS transplantation of Cas-Br-E-infected microglia alone is sufficient to induce spongiform neuropathology (26).

Since genetic mapping analysis has demonstrated that the primary determinants for neurovirulence reside within the env gene (8, 30, 31, 38), much of the focus on mechanisms of MuLV-induced neuropathogenesis have centered on the viral envelope protein, the membrane-associated surface glycoprotein which mediates virus binding and entry into the cell. Interestingly, however, neither the expression of high levels of Cas-Br-E envelope protein alone nor production of replication-restricted Cas-Br-E virus is capable of precipitating acute pathological changes in the brain, when either protein or virus is expressed from cells of neuroectodermal origin (27). Rather, our results indicate that late Cas-Br-E virus replication events within the bone marrow-derived microglia are required for inducing neurodegenerative disease. These findings raise the possibility that a unique neurotoxic Env protein is generated upon microglial infection. In this regard, additional Cas-Br-E envelope protein isoforms have been observed when the Cas-Br-E virus spreads to microglia from transplanted Cas-Br-E-infected neural stem cells (27). The unique envelope isoforms observed within the CNS may either be byproducts of the coincident neurodegenerative process or represent Env synthetic events within microglia involved in the precipitation of neuropathogenesis.

How envelope protein synthesis in microglia could be involved in the induction of neurodegeneration is not yet known. Understanding the neuropathogenic process may come from understanding Env biosynthesis. Analysis of MuLV retroviral Env protein synthesis and trafficking in cells in culture (reviewed in reference 10) indicates that envelope is synthesized in the rough endoplasmic reticulum as a precursor protein, where it has its amino-terminal signal sequence cleaved off, undergoes disulfide bonding, obtains multiple asparagine-linked high-mannose sugars, and oligomerizes, prior to transport to the Golgi apparatus. In the Golgi apparatus, the high-mannose sugars are modified to complex type, and the precursor protein polypeptide backbone is cleaved to give rise to the surface-expressed domain (SU) and the transmembrane-associated domain (TM); then the complex is transported to the plasma membrane. SU and TM remain associated by way of noncovalent interactions and in some instances, a disulfide bond (15). Upon assembly into virions and release from the cell, the carboxy-terminal cytoplasmic tail of the TM is cleaved, which renders the SU-TM complex within the virus fusigenic (33). Functional mapping studies have demonstrated that the receptor binding function of envelope resides in the N-terminal half of SU (17), while TM contains the regions responsible for oligomerization, membrane attachment, and fusigenicity.

In an effort to understand whether Env protein synthesis could be involved in neuropathogenesis, Wong and coworkers compared envelope synthesis and processing between nonneurovirulent wild-type Moloney murine leukemia virus (MoMuLV) and a temperature-sensitive MoMuLV mutant, ts1, which is neurovirulent. Interestingly, in a spleen-derived cell line (TB cells), the cleavage of the envelope precursor protein (gpr80) to SU and TM (gp70 plus p15E) was demonstrated to be inefficient for the neurovirulent ts1 virus. Similar results were also observed in primary cultures of CNS glia (36) and, more recently, in immortalized astrocyte cultures (22). Significantly, this inefficient envelope precursor protein processing genetically segregates with the ability to cause clinical neurological disease in vivo (37, 38).

Analysis of Cas-Br-E envelope synthesis in primary CNS glial cultures indicates normal Cas-Br-E Env processing and virus production. Interestingly, however, infected microglia isolated from the mixed glial cultures appear incapable of processing the Cas-Br-E envelope precursor (gpr85) to SU (gp70) and TM (p15E) (23). This defect is associated with intracellular budding of viral particles, similar to that seen in human immunodeficiency virus-infected monocytes in vitro (14). In contrast, defective budding of virus into microglia in vivo has yet to be reported. However, what has been observed is the appearance of a unique CNS-specific envelope protein isoform which has a smaller apparent molecular size (65 kDa) than that noted in infected spleen (70 kDa) (6, 23, 25). Whether this unique envelope protein species is related to the defective envelope processing noted in vitro and/or to neuropathogenesis in vivo has not been addressed.

Therefore, to understand how envelope protein synthesis might be involved in the induction of neurodegeneration, we examined the expression of envelope protein in the brains of mice infected with the highly neurovirulent chimeric Cas-Br-E retrovirus, FrCas^E. This virus contains the Cas-Br-E env gene in the background of Friend leukemia virus, clone FB29 (30). It was used because it infects the CNS at high levels and induces neurodegenerative disease with a rapid and stereotypic progression (6, 24, 30). Our results revealed the presence of both regional and temporal Cas-Br-E envelope protein isoform heterogeneity. This envelope heterogeneity could be accounted for by unique N-linked Env glycosylation events as a result of the regional and temporal infection of different CNS cell types. Furthermore, our analysis specifically implicates non-virion gp70 envelope isoforms as being associated with the appearance of spongiform neuropathology.

Differential expression of Cas-Br-E envelope protein in a region- and time-specific manner. We have previously reported that envelope protein expression in mice infected with the highly neurovirulent chimeric Cas-Br-E retrovirus, FrCas^E, differs qualitatively between the brain and spleen (6, 23, 25). Extracts from both brain and spleen show significant levels of the envelope precursor protein, gpr85; however, the two tissues differ in the nature of the proteolytically processed envelope proteins expressed. Splenic infection by FrCas^E is characterized by expression of a single proteolytically processed envelope isoform referred to as gp70. In contrast, brain extracts are characterized by the appearance of multiple proteolytically processed envelope protein isoforms, with the dominant species migrating with an apparent molecular size of 65 kDa. Because FrCas^E infection of the brain occurs in diverse areas and in a variety of cell types (6, 24), our prior analyses of whole-brain homogenates constituted a "biochemical average" of the envelope proteins being expressed (6, 23, 25). This is a significant issue because the neuronal infection noted in the FrCas^E disease model is not accompanied by cytopathic changes and occupies physically distinct regions of the brain. We reasoned, then, that dissection of regions of high neuronal infection away from the regions of high microglial infection (areas with extensive pathology) should reveal features of Cas-Br-E Env expression which are relevant to neuropathogenesis.

View larger version (54K): [in this window] [in a new window]   FIG. 1.   Regional and temporal expression of Cas-Br-E envelope protein in the brain. (A) Saggital diagram of the mouse brain. Gray shading indicates regions where pathology arises after infection with the FrCasE virus. Heavy dark lines approximate where brains were dissected for regional Cas-Br-E envelope protein analysis by Western immunoblotting. (B) Cas-Br-E envelope protein immunoblot on dissected brain regions, compared with spleen and purified virus made in dunni fibroblasts. Spleen and CNS samples were generated from FrCasE-infected mice at 14 dpi, and protein sample loads and blot exposure times were adjusted to provide for resolution of the multiple envelope protein isoforms. (C) Time course of CNS expression of the different Cas-Br-E envelope protein isoforms. Samples from two different brains were run for each time point to show the limited variability that occurs during CNS infection. Equivalent sample loads were used for each time course; however, different exposures were used for the different regions to reveal the details of envelope expression. Note that no virus control was loaded for NCX or BS. Abbreviations: OB, olfactory bulb; NCX, neocortex; COL, colliculus; THAL, thalamus; PONS, pons region of the brainstem; MED, medulla oblongata; CB, cerebellum; SC, spinal cord; SPL, spleen; U, uninfected total brain; V, virus from cultured dunni cells; BS, brainstem (pons and medulla together).

Incorporation of CNS-derived Cas-Br-E envelope proteins into virions. We previously reported that infection of the CNS by FrCas^E appeared to result in viral particle production by all infected CNS cell types upon examination by electron microscopy (24). Given that multiple isoforms of Cas-Br-E envelope protein were observed in various brain regions of FrCas^E-infected animals, we were interested in determining which, if any, of these species were incorporated into virions. Failure of envelope to be incorporated into virions might suggest specific intracellular modifications which could be important for pathogenesis. Therefore, freshly dissected tissue was homogenized by 20 strokes of a Wheaton homogenizer in 10 volumes of 150 mM NaCl-50 mM Tris-HCl (pH 7.4)-0.1 mM EDTA at 4°C. Homogenates were sedimented at 10,000 × g for 10 min at 4°C to pellet nuclei and large cellular fragments. Supernatants (1-ml samples) were further sedimented over 10 ml of 20 to 60% sucrose gradients at 38,000 rpm in a Beckman SW41 rotor for 3 h. Sucrose gradient fractions were collected (0.5 ml each), measured for density by directly weighing 50-µl samples (n = 3), and tested for Gag and envelope protein by dot immunoblot assays on nitrocellulose membranes with rabbit anti-p30 Gag protein and Cas-Br-E-specific anti-gp70 envelope monoclonal antibody 697, respectively (not shown). Fractions positive for Gag and/or envelope proteins were subjected to SDS-PAGE and Cas-Br-E Env protein immunoblot analysis. The results, shown in Fig. 2A, indicate that the 65-kDa protein is the primary CNS envelope protein found sedimenting at a density consistent with that of virions made in cultured fibroblasts (1.14 to 1.16 g/ml). The gp70 envelope protein isoforms from the brain appeared at lower densities, suggesting that they were not associated with virions but rather were associated with other cellular fractions. Interestingly, the gp70 peak noted for extracts taken from the spleens of FrCas^E-infected animals was rather small compared to the level of total gp70 in the tissue extract. This suggests that most of the spleen-associated gp70 protein was not associated with virions. This contrasts remarkably with the results obtained from the cerebellum, where the vast majority of the 65-kDa protein sediments with virions. Thus, the spleen results suggests that either virus is quickly transported away from the spleen parenchyma into the blood once gp70 is incorporated into virus or the infection of cells in the spleen is not highly productive. Since spleen cells from infected mice readily score positive in infectious-center assays (4), these results suggest that cultured target cells may aid the infectious process. A similar phenomenon was observed for Cas-Br-E-infected microglia in culture, where the cells produce little free virus but score 100% positive by infectious-center assay (23). It will be of interest in the future to compare virus production from the spleen after infection with nonneurovirulent viruses to evaluate whether the envelope protein is the primary determinant responsible for the nonproductive phenotype. It may be that the virus replication process occurring in spleen cells closely mimics that occurring in CNS microglia.

View larger version (39K): [in this window] [in a new window]   FIG. 2.   Sucrose gradient sedimentation analysis of envelope protein in FrCasE-infected tissues. (A) Western blot of sucrose gradient fractions after sedimentation of extracts from different CNS regions and the spleen. Fractions were loaded from left to right, starting with the higher-density fractions on the left. Downward-pointing arrows indicate the fractions with densities of 1.14 to 1.16 g/ml, correlating with fractions expressing the infectivity peak (see panel B). Note that for CNS extracts the 65-kDa protein is the predominant isoform observed associated with virion density. (B) Sedimentation behavior of infectivity in the different CNS regions. Peak infectivity for all regions occurs at 1.14 to 1.16 g/ml, consistent with that noted for virions made in culture and coincident with the 65-kDa protein peaks noted in panel A. The asterisk indicates that virus titers equal to or greater than 300 focus-forming units (FFU)/10 µl could not be distinguished from one another in this analysis. Therefore, these points were placed at 300. Abbreviations: NCX, neocortex; BS, brainstem; CB, cerebellum; SC, spinal cord; SPL, spleen; V, virus from cultured dunni cells.

Differential glycosylation accounts for the complex envelope protein expression patterns in the CNS. In order to understand what accounted for the appearance of the multiple CNS Env isoforms, protein extracts from the spleen and representative brain regions (6) were treated with recombinant peptide N-glycosidase F (PNGase F; peptide-N⁴-(N-acetyl -glucosaminyl)asparagine amidase; EC 3.5.1.52 and EC 3.2.2.18; Genzyme) to remove all asparagine-linked (N-linked) sugars and leave the protein backbone. Because the Cas-Br-E envelope protein has 7 potential N-linked glycosylation sites (32), this approach could determine whether the multiple Cas-Br-E Env isoforms observed were due to alterations in the protein backbone or to posttranslational sugar modifications. The deglycosylation reactions were carried out on 10% tissue homogenates from which nuclei had been removed. Samples were denatured by boiling in the presence of 0.1% SDS-1 mM dithiothreitol-200 mM Tris-HCl (pH 8.0) for 5 min and then were incubated with PNGase F (1.3 U/50 µl of extract) for 16 h at 37°C after addition of Triton X-100 to a final concentration of 0.5%. Reactions were terminated by the addition of SDS-PAGE sample buffer and boiling. Immunoblotting of the deglycosylated tissue extracts after separation by SDS-10% PAGE (Fig. 3A) showed that the envelope protein profiles from different brain regions and the spleen were rendered indistinguishable by PNGase F treatment. These results suggest that the multiple gp70 and gp65 isoforms of Cas-Br-E envelope appeared as a result of differential N-linked glycosylation.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Glycosylation analysis of Cas-Br-E envelope protein from FrCasE-infected mice 14 dpi. (A) Effect of PNGase F treatment of detergent extracts from spleen and dissected CNS regions. Representative extracts were incubated overnight with (+) or without () PNGase F, followed by Western immunoblot analysis with the Cas-Br-E envelope monoclonal antibody 697 (6). Overnight incubation of envelope extracts in deglycosylation buffer without PNGase F (upper panel) results in reduced envelope isoform resolution by SDS-PAGE and Western blot analysis (compared to Fig. 1A and B). However, despite the extended 37°C incubation, no envelope proteolysis was apparent. The lower panel shows that PNGase F treatment effectively eliminated the envelope isoform heterogeneity observed in the untreated samples. Protein loads were adjusted to give comparable signals for envelope protein. Abbreviations: SPL, spleen; TB, total brain; CB, cerebellum; SC, spinal cord; NCX, neocortex; BS, brainstem. (B) Comparison between treatment of spleen SPL, SC, and CB envelope extracts with endo H and PNGase F as detected by Western blotting. Endo H treatment of extracts from the brain (SC and CB) resulted in Env protein shifts to lower-molecular-weight proteins, whereas endo H treatment of spleen only shifted the gpr85 precursor protein. Because endo H treatment fails to shift any of the Envs to the fully deglycosylated 55-kDa form, the Env proteins all traffic through the Golgi apparatus, where some N-linked sugars are modified to complex type. As shown for SPL and CB, incomplete deglycosylation of envelope protein was sometimes observed in PNGase F-treated samples; however, this could be eliminated by a more prolonged primary digestion or a secondary digestion with PNGase F. V, Cas-Br-E virus supernatant from cultured dunni cells. (C) Endo H (top panel) and PNGase F (bottom panel) treatment of virion-enriched sucrose gradient fractions from either the infected CB or tissue-cultured dunni fibroblasts (TC). Despite the partial purification away from other cellular proteins which could be inhibiting the endo H, both gp65 and gp70 were only partially susceptible to endo H treatment. This result indicates that some high-mannose sugars are processed to the complex form in the Golgi apparatus, while others are not. Treatment of a mixture of cerebellar (gp65) and tissue culture (gp70) virus fractions with PNGase F yields a single band migrating at approximately 55 kDa, further confirming that the protein mobility differences reside in the N-linked sugar modifications.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Schematic representation of the Cas-Br-E glycosylation sites in relation to the functional domains in MuLV Env. Small asterisks, consensus MuLV glycosylation sites; light asterisks, absence of a consensus glycosylation site in the Cas-Br-E Env sequence; large asterisk, additional nonconsensus site specific for Cas-Br-E Env (18). VRA and VRB, variable MuLV Env domains believed to be involved in receptor specificity and thus in binding (7, 17). Arrows indicate sites of proteolytic cleavage which occur, from left to right, in the endoplasmic reticulum, the Golgi apparatus, and the budding virion.