Expression and intracellular actions of Vpr 77Q and 77R. (A) Mock-transfected CrFK cells exhibited no Vpr immunoreactivity; (B) A non-expressing Vpr plasmid (pVpr(-)) also show weakly Vpr immunoreactivity in transfected CrFK cells; (C) and (D) Vpr immunoreactivity was readily detected in the cytoplasm and nuclei of CrFK cells transfected with (C) pVpr77R-ND and (D) pVpr77Q-HAD; (E) pVpr77R-ND transfection of U937 cells caused an induction of TNF-α/vpr transcript abundance relative to pVpr(-); (F) likewise, pVpr77R-ND activated IFN-α/vpr transcription in U937 cells; (G) pVpr77R-ND also induced expression of MX1/vpr; (H) Supernatants from both pVpr77Q-HAD and pVpr77R-ND transfected U937 cells were neurotoxic to human fetal neurons (HFN), as evidenced by reduced β-tubulin immunoreactivity, although the supernatants from the pVpr77R-ND transfected U937 cells were more cytotoxic. Original magnification 600×. Real time PCR data was normalized against the matched Vpr mRNA levels. Experiments were carried out in triplicate at least two times (E-G, Dunnett test, relative to control; *p < 0.05, **p < 0.01).