ABSTRACT
The ability of retroviruses (RVs) to cause neurodegeneration is critically dependent upon two activities of the envelope protein (Env). First, Env facilitates viral genome delivery to CNS target cells through receptor binding and membrane fusion. Second, Env expression within one or more targets indirectly alters the physiology of certain neurons. Although the major Env expressing CNS cell types have been identified for many neurovirulent RVs, it remains unresolved, which targets play a causal role in neuropathogenesis. Moreover, this issue is complicated by the potential for post-infection virus suppression. To address these questions we explored herein, whether and how cryptic neurotropism differences between ecotropic and amphotropic murine leukemia viruses (MLVs) impacted neurovirulence. Neurotropism was first explored ex vivo using (1) acute primary glial cell cultures and (2) neural progenitor cell (NPC)- neural stem cell (NSC) neural sphere (NPH) chimeras. These experiments indicated that primary astrocytes and NPCs acutely restrict amphotropic but not ecotropic virus entry. CNS tropism was investigated using NSC transplant-based Cre-vector pseudotyping wherein mTmG transgenic fluorescent protein reporter mice revealed both productive and suppressed infection. Cre-pseudotyping with FrCasE, a prototypic neurovirulent ecotropic virus, identified glia and endothelia, but not neurons, as targets. Almost two-thirds (62%) of mGFP+ cells failed to show Env expression, suggesting widespread virus suppression. To circumvent RV superinfection interference confounds, targets were also identified using ecotropic packaging NSCs. These experiments identified known ecotropic targets: microglia, oligodendrocyte progenitor cells (OPCs) and endothelia. Additionally, one third of mGFP+ cells were identified as protoplasmic astrocytes, cells that rarely express virus in vivo. A CNS targeting comparison between isogenic ecotropic (FrCasE) and amphotropic (FrAmE) viruses showed a fourfold higher astrocyte targeting by FrCasE. Since ecotropic Env pseudotyping of amphotropic virus in the CNS dramatically exacerbates neurodegeneration, these results strongly suggest that astrocyte infection is a major disease requirement. Moreover, since viral Env protein expression is largely subdetectable in astrocytes, minimal viral protein expression appears sufficient for affecting neuronal physiology. More broadly, these findings raise the specter that subdetectable astrocyte expression of exogenous or endogenous RVs could play a major role in human and animal neurodegenerative diseases.
ABSTRACT
UNLABELLED: Certain murine leukemia viruses (MLVs) are capable of inducing fatal progressive spongiform motor neuron disease in mice that is largely mediated by viral Env glycoprotein expression within central nervous system (CNS) glia. While the etiologic mechanisms and the glial subtypes involved remain unresolved, infection of NG2 glia was recently observed to correlate spatially and temporally with altered neuronal physiology and spongiogenesis. Since one role of NG2 cells is to serve as oligodendrocyte (OL) progenitor cells (OPCs), we examined here whether their infection by neurovirulent (FrCasE) or nonneurovirulent (Fr57E) ecotropic MLVs influenced their viability and/or differentiation. Here, we demonstrate that OPCs, but not OLs, are major CNS targets of both FrCasE and Fr57E. We also show that MLV infection of neural progenitor cells (NPCs) in culture did not affect survival, proliferation, or OPC progenitor marker expression but suppressed certain glial differentiation markers. Assessment of glial differentiation in vivo using transplanted transgenic NPCs showed that, while MLVs did not affect cellular engraftment or survival, they did inhibit OL differentiation, irrespective of MLV neurovirulence. In addition, in chimeric brains, where FrCasE-infected NPC transplants caused neurodegeneration, the transplanted NPCs proliferated. These results suggest that MLV infection is not directly cytotoxic to OPCs but rather acts to interfere with OL differentiation. Since both FrCasE and Fr57E viruses restrict OL differentiation but only FrCasE induces overt neurodegeneration, restriction of OL maturation alone cannot account for neuropathogenesis. Instead neurodegeneration may involve a two-hit scenario where interference with OPC differentiation combined with glial Env-induced neuronal hyperexcitability precipitates disease. IMPORTANCE: A variety of human and animal retroviruses are capable of causing central nervous system (CNS) neurodegeneration manifested as motor and cognitive deficits. These retroviruses infect a variety of CNS cell types; however, the specific role each cell type plays in neuropathogenesis remains to be established. The NG2 glia, whose CNS functions are only now emerging, are a newly appreciated viral target in murine leukemia virus (MLV)-induced neurodegeneration. Since one role of NG2 glia is that of oligodendrocyte progenitor cells (OPCs), we investigated here whether their infection by the neurovirulent MLV FrCasE contributed to neurodegeneration by affecting OPC viability and/or development. Our results show that both neurovirulent and nonneurovirulent MLVs interfere with oligodendrocyte differentiation. Thus, NG2 glial infection could contribute to neurodegeneration by preventing myelin formation and/or repair and by suspending OPCs in a state of persistent susceptibility to excitotoxic insult mediated by neurovirulent virus effects on other glial subtypes.
