Efficient N-glycosylation at position 37, but not at position 146, in the street rabies virus glycoprotein reduces pathogenicity

Most street rabies viruses have two N-glycosylation sites in their glycoproteins (G proteins), i.e., at Asn(37) and Asn(319), but Asn(37) is usually not core-glycosylated in an efficient manner. Previously, we reported the possible roles of single additional N-glycosylations at Asn(194) or Asn(247) in the cell adaptation and reduced pathogenicity of a street rabies virus, which suggest that N-glycosylation is closely related to the evolution of rabies viruses. In this study, we characterized two novel N-glycosylation-modified variants, N5C#7 and N5C#8, which were cloned using the limiting dilution method after serial passaging of the street rabies virus strain 1088 in mouse neuroblastoma-derived NA cells. N5C#7 had an L38R mutation in the G protein, which led to efficient core glycosylation at Asn(37). On the other hand, N5C#8 had a D146N mutation in the G protein, which led to an additional N-glycosylation at position 146. Both variants replicated highly efficiently in NA cells compared with the parental strain. Like the parental strain, both variants caused lethal infections in adult mice after intracerebral inoculation. However, N5C#7 exhibited reduced pathogenicity after intramuscular inoculation, whereas N5C#8 displayed the same level of pathogenicity as the parental strain. In summary, the efficient core glycosylation at position 37 was related to cell adaptation and the reduced pathogenicity of the street rabies virus. By contrast, despite of being related to cell adaptation, the additional N-glycosylation at position 146 did not affect the pathogenicity, which is consistent with a report that street rabies virus strains with N-glycosylation sites at positions 37, 146, and 319 have been isolated from rabid animals. Thus, the results of the present study provide additional evidence that supports the relationship between G protein N-glycosylation and rabies virus evolution. (C) 2013 Elsevier B.V. All rights reserved.