Figure 5

IFITMs silencing in HIV-1 producing cells results in virions of increased infectivity. HIV-1 vectors coding for control (Luciferase) or IFITMs specific target sequences were obtained by DNA transfection of HEK293T cells, normalized and used on target cells. A) HeLaP4 were shortly selected with Puromycin (present in the vector) and then challenged with an MOI of 1 of replication competent HIV-1 virus (NL4-3) to obtain a large proportion of virus producing cells. After cell washing and trypsin treatment, newly produced viruses were recovered 2-3 days afterwards and virions and cell lysates were examined by WB at this time. The infectivity of exo-RT normalized virions was determined on HeLaP4 cells by MAGI assay. B) To improve silencing efficiency in MDM, vectors were used along with an MOI-equivalent of 0,5 of VLPs-Vpx. Three days after, MDM were challenged with an MOI of 1 of replication competent ADA virus, prior to extensive cell washing and trypsin treatment. Newly produced viral particles were retrieved 4 to 6 days after and their infectivity determined after exo-RT normalization on HeLaP5 cells. C) Knockdown Jurkat cells were obtained as in A. Cells were then challenged with an MOI of 0,1 of replication competent NL4-3 and spreading infections were analyzed by harvesting aliquots of the culture supernatant at different times post infection. Viral spread was determined by exo-RT activity. D) The intrinsic infectivity of viral particles was determined as above on the supernatants of cells obtained 9 days post infection. WB panels present knockdowns obtained for the different cell types and the graph presents averages and SEM obtained in 3 to 4 independent experiments and donors. *; statistically significant difference after a Student t test: p ≤ 0,05. The replication curve shown in C depicts a typical result obtained out of 3.