FIG. 4.
Functional characterization of MusD and its variants with modified myristoylation and membrane targeting signals. (A) Infectivity assay. 293T cells were transiently transfected with an expression vector for the indicated MusD constructs and a neoTNF-marked defective MusD reporter (in which a “backward” neomycin resistance gene [with the neo ORF interrupted by a “forward” TNF intron] becomes functional only after a complete replicative cycle) (30), plus an expression vector for the VSV-G envelope protein. Supernatants from the transfected cells were collected 48 h posttransfection and used to infect naive target HeLa cells. After a 3-day growth period, infection events were detected upon G418 selection of the cells and quantitated by counting the number of G418r clones. No clone was detected in the absence of the VSV-G expression vector. (B) Transposition assay. HeLa cells were transfected with the same MusD plasmids as in panel A but without the VSV-G envelope protein expression vector; after a 6-day growth period, the cells were seeded (5 × 105 cells per plate) and subjected to G418 selection to detect retrotransposition events (number of G418r clones per cells in selection [see reference 30]).