FIG 3.

Productive infection of primary NPCs by ecotropic MLVs does not affect neural-sphere formation or OPC marker expression but reduces markers of OL and astrocyte differentiation. (A) Examples of NPHs derived from PLP-EGFP transgenic mice with and without infection with FrCasE or Fr57E virus. These NPHs express low levels of EGFP (green) in a subpopulation of cells within the spheres. NPHs were immunostained for the neural stem cell marker nestin (red, first column on left) or stained for the OPC marker NG2, Olig2, or PDGFRα (blue) and double stained for viral Env protein (red), and images were captured using epifluorescence optics. Scale bar, 50 μm. No gross morphological or immunostaining differences were noted with virus infection, although the intensity of EGFP staining in individual cells appeared lower in the MLV-infected NPHs (cf. panel C). (B) Quantitation of the percentages of cells in the NPH cultures expressing viral Envs and the NPC and OPC markers, which were determined after dissociation of the NPHs into single cells to allow more accurate assessment of individual cell expression. The only significant difference in marker expression frequency was a small increase in the percentage of cells expressing nestin with FrCasE- versus mock-infected NPHs (*, P = 0.044; ANOVA). (C) (Left) Nonmerged images of NPHs from panel A (far right column) showing EGFP expression (left; green) and Env staining (right; red), indicative of reduced EGFP expression with virus infection. (Middle) Western blot comparison of FrCasE-, Fr57E-, and mock-infected NPHs probed for MLV Env (top), EGFP (middle), and actin (bottom) showing a dramatic reduction in EGFP expression with MLV infection. (Right) Immunoblotting of NPH extracts with antibody specific for the astrocyte-specific marker GFAP for protein equivalents from mock- and MLV-infected NPHs. Apparent molecular masses are indicated by reference protein sizes listed in daltons. (D) Culture supernatants from NPHs taken after ≥5 passages in culture produced infectious virus when assayed by virus titration assay. Titers are given in FFU/ml. The error bars in the graphs represent standard deviations. ND, not detected. Data sets were analyzed by one-way ANOVA, with Tukey's post hoc test for multiple comparisons.