FIG. 2.

(A) Chimeric GPs. Schematic representations of chimeric GPs and titers of the corresponding pseudotyped retroviral vectors on HEK293A cells. Functional chimeras are indicated by a # symbol before the chimera. The boundaries used to generate the chimeras are based on the signal peptide SSP (S) and conserved cysteine residues in GP1 that delineate segments 1 through 5. TCRV and AMAV sequences (white) and JUNV and GTOV sequences (black) are indicated. Only the N-terminal part of the GP2 sequence is represented. In addition, the boundaries of the following partial segments are as indicated: Jm4 extends to JUNV residue 207, Jn4 extends to JUNV residue 184, Jc4 extends from JUNV residue 208, + indicates extension into JUNV GP2 to residue 280, An4 extends to AMAV residue 183, and Ac4 extends from AMAV residue 184. Titers were determined as previously described (27, 31), and values shown are the means plus standard deviations (SD) (error bars) for three to six independent experiments. The titers are shown for both unconcentrated vector stocks (black) and 10× concentrated stocks (gray); an asterisk indicates no detectable titer. VSV, vesicular stomatitis virus. (B) Western blotting of GP-pseudotyped vector particles. Vectors were generated in HEK293T cells, pelleted by ultracentrifugation, deglycosylated, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (31). Functional chimeric GPs are boxed. Depending on PAGE conditions, GPC could be seen as both the full-length GPC, as well as the SSP cleaved form comprising only GP1-GP2. TCRV and JUNV proteins were detected using anti-GP2 antiserum, while GTOV and AMAV proteins were constructed as C-terminal Flag-tagged proteins and detected using anti-Flag antibody.