Figure 6. Inefficiency of H5Anh-pp production is independent of the lentiviral backbone used but can be rescued by co-transfection with viral N1 or in sialylation-deficient Lec2 cells.

(A) Comparison of H5pp production using HIV and MLV pseudotyping systems. 293T cells were transfected with a plasmid coding for either H5Cam or H5Anh (empty pcDNA vector was used as the negative control, NC), together with either an HIV or MLV lentiviral backbone, as described in Material and Methods. Viral N1 plasmid was included in the transfection mixture where indicated to produce pseudoparticles containing both HA and NA. Culture supernatant containing H5pp was harvested at 48 hr post transfection and luciferase activity in MDCK cells was measured at 72 hr post H5pp transduction. Results are presented as means ± SD (n = 3 independent experiments). No significant differences were found between MLV- and HIV-backbone at all conditions tested. (B) Production of H5Anh-pp is enhanced in sialylation-deficient Lec2 cells. Cells were transfected with HIV gag/pol containing luciferase reporter gene and a plasmid coding for either H5Cam or H5Anh. NAvb was added 16 hr post transfection. At 48 hr post transfection, supernatant containing H5pp were harvested from CHO or Lec2 cells and used to transduce MDCK target cells. H5anh-pp production in Lec2 cells was restored to a level similar to that of H5Cam-pp as shown by similar luciferase activity detected in MDCK cells. Results are presented as means ± SD (n = 4 independent experiments); *p<0.01 by the unpaired Student's t-test.