Figure 1.

Characterization of FrCasE-, Fr57E- or mock-infected primary microglia. Panel A shows the basic proviral structures of the viruses employed in this study. The env genes from the NV CasBrE (clone 15-1) and NN Friend virus clone Fr57 were introduced into the background of the Friend virus clone FB-29 to generate FrCasE and Fr57E/F43, respectively. These chimeric viruses differ only in their env genes. The two Fr57 env containing constructs (Fr57E and F43) differ by the restriction sites used for env introduction, however, no amino acid coding differences exists between the two clones. SD = splice donor; SA = splice acceptor. Panel B shows immunostaining of microglia isolated from mixed glial cultures to assess cell morphology, the extent of virus infection, and cell purity. The upper two panels show FrCasE-infected (left) and mock-infected (right) cells, stained with the CasBrE Env-specific monoclonal antibody 667. The lower panels show dual staining of Fr57E-infected microglia for Friend Env protein (left, red), and the macrophage/microglial marker Mac-1 (right). While virtually all the isolated cells were positive for virus infection and microglial markers, the level of cell surface staining varied considerably from very high (arrows) to very low (arrowheads) indicating significant cell surface expression heterogeneity. Importantly, no virus specific cell staining was observed in mock-infected controls using either antibodies to CasBrE or Friend Envs. Panel C shows Western blotting for Env (upper panel) and Gag (lower panel) on multiple FrCasE- (n = 3), Fr57E- (n = 4), and mock-infected (n = 3) microglial preparations. The right hand lane in each cell grouping shows immunoreactivity for mock-, Fr57E-, and FrCasE-infected 3T3 fibroblasts as a control. The far right hand grouping for FrCasE-infected samples shows immunoblotting results using the CasBrE Env specific monoclonal antibody 697. Note that the Env protein from microglia (Mg) is primarily a high molecular weight form for both FrCasE and Fr57E, whereas in 3T3 fibroblasts both high and low molecular weight species are observed. Note also that the Fr57 Env has a higher apparent molecular weight than the CasBrE Env protein consistent with known sequence and glycosylation differences. Pr65Gag is similar for Fr57E- and FrCasE-infected microglia; however, microglia expressed pr65 gag and glycoGag while 3T3 fibroblasts expressed pr65gag and gag-pol polyprotein. Panel D shows the virus expression levels generated from infected microglia and mixed glial cultures over a 24 hour period. No significant differences in virus production were noted between FrCasE (black bars) and Fr57E (gray bars) in either microglial or mixed glial cultures, however, the difference in virus production between microglia and mixed glia were highly significant with p values of <0.01 (FrCasE-infected microglia, n = 4; FrCasE-infected mixed glia, n = 6; Fr57E-infected microglia, n = 4; Fr57E-infected mixed glia, n = 5). No virus was detected from mock-infected microglia or mixed glia and thus the data were not shown.