Unique N-linked glycosylation of CasBrE Env influences its stability, processing, and viral infectivity but not its neurotoxicity.

The envelope protein (Env) from the CasBrE murine leukemia virus (MLV) can cause acute spongiform neurodegeneration analogous to that induced by prions. Upon central nervous system (CNS) infection, Env is expressed as multiple isoforms owing to differential asparagine (N)-linked glycosylation. Because N-glycosylation can affect protein folding, stability, and quality control, we explored whether unique CasBrE Env glycosylation features could influence neurovirulence. CasBrE Env possesses 6/8 consensus MLV glycosylation sites (gs) but is missing gs3 and gs5 and contains a putative site (gs*). Twenty-nine mutants were generated by modifying these three sites, individually or in combination, to mimic the amino acid sequence in the nonneurovirulent Friend 57 MLV. Three basic viral phenotypes were observed: replication defective (dead; titer < 1 focus-forming unit [FFU]/ml), replication compromised (RC) (titer = 10(2) to 10(5) FFU/ml); and wild-type-like (WTL) (titer > 10(5) FFU/ml). Env protein was undetectable in dead mutants, while RC and WTL mutants showed variations in Env expression, processing, virus incorporation, virus entry, and virus spread. The newly introduced gs3 and gs5 sites were glycosylated, whereas gs* was not. Six WTL mutants tested in mice showed no clear attenuation in disease onset or severity versus controls. Furthermore, three RC viruses tested by neural stem cell (NSC)-mediated brainstem dissemination also induced acute spongiosis. Thus, while unique N-glycosylation affected structural features of Env involved in protein stability, proteolytic processing, and virus assembly and entry, these changes had minimal impact on CasBrE Env neurotoxicity. These findings suggest that the Env protein domains responsible for spongiogenesis represent highly stable elements upon which the more variable viral functional domains have evolved.

Effects of AAV-2-mediated aspartoacylase gene transfer in the tremor rat model of Canavan disease.

The tremor rat is a spontaneous epilepsy model with a seizure phenotype caused by a deletion in the aspartoacylase (ASPA) gene. The absence of ASPA expression in these animals results in undetectable levels of enzyme activity and the accumulation of the substrate N-acetyl-aspartate (NAA) in brain, leading to generalized myelin vacuolation and severe motor and cognitive impairment. In support of human gene therapy for CD, recombinant adeno-associated viral vector (AAV-2) expressing ASPA was stereotactically delivered to the tremor rat brain and effects on the mutant phenotype were measured. AAV-ASPA gene transfer resulted in elevated aspartoacylase bioactivity compared to untreated mutant animals and elicited a significant decrease in the pathologically elevated whole-brain NAA levels. Assessment of motor function via quantitative rotorod testing demonstrated that rats injected with AAV-ASPA significantly improved on tests of balance and coordinated locomotion compared to animals receiving control vectors. This study provides evidence that AAV-2-mediated aspartoacylase gene transfer to the brain improves biochemical and behavioral deficits in tremor rat mutants (tm/tm) and supports the rationale of human gene transfer for Canavan disease.