Fig. 7

Protease inhibitors block replication of HERV-K viral particles: HeLa cells were transfected with pCD-HK/Rev and HIV-1 Tat plasmids. a HIV protease inhibitors were added to Hela cells 6 h post transfection and the reverse transcriptase (RT) activity in the culture supernatant was determined by PERT assay at 24 h post-treatment. b Darunavir, Lopinavir, Indinavir, Amprenavir or Atazanavir were added to HeLa cells 6 h post transfection in a twofold serial dilution ranging from 31.25 nM to 1 µM and RT activity in the culture supernatant was determined by PERT assay at 48 h post-treatment. c Darunavir and Lopinavir were further tested using tenfold serial dilution of the compounds, ranging from 100 nM to 100 µM. Viral supernatant was collected 48 h post-treatment and analyzed by PERT assay. Any change in Ct (threshold cycle) was compared to vehicle control and reported as percent inhibition. Data represent mean ± SEM of at least three different experiments. d, e 293 cells were transfected with pCD-HK/Rev in the presence or absence of 1 µM Darunavir or Lopinavir. After 48 h, HERV-K viral particles were harvested from the culture medium. The expression of Gag in the cell lysate and viral particles was determined by Western blot analysis. Darunavir and Lopinavir effectively blocked the processing of Gag (70 kDa) into mature capsid (CA) protein (27 kDa) both in the cell lysate and in the viral particles (d). Viral supernatant was analyzed by both PERT assay and viral RNA PCR, indicator of numbers of viral particles. The viral supernatant was also used to infect HeLa cells. After 6 days post-infection, HERV-K Gag mRNA in the infected cells was measured by PCR. Values from HERV-K with PI treatment were expressed as percentage of that without PI treatment