H-1 PV exerts its effect on PHA-activated PBMCs in an NS1-dependent manner.

<p>PBMCs were inoculated with increasing amounts of purified H-1 PV or mock-treated. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell; p.i : post-infection. <b>A</b>. <i>H-1 PV alters PHA-activated PBMC morphology</i>. For each condition, pictures of cells were taken in the course of infection but only those taken 48 h post-infection are shown. Scale bar = 100 µm. <b>B</b>. <i>PHA-activated PBMCs are lysed upon H-1 PV infection</i>. H-1 PV cytotoxicity was assessed using a test based on a bioluminescent reaction measuring the leak of a cellular marker from cytoplasm to culture medium, which reveals the loss of plasma membrane integrity. Assays were performed at different times after H-1 PV inoculation but only 48 and 72 h conditions are shown. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in relative light unit (RLU). <b>C</b>. <i>PHA-activated PBMC death upon H-1 PV infection is related to NS1 protein expression but does not depend on caspase 3 activation</i>. Non activated (NA) and activated (A) PBMC total protein extracts were prepared in the course of infection. Equal amounts of proteins were separated by 4–12% SDS-PAGE and analyzed by Western blot for the presence of NS1 protein, cleaved caspase 3 and both full-length and cleaved PARP. β-actin was used as a loading control. <b>D</b>. <i>PBMCs do not support an H-1 PV productive infection</i>. Both PBMCs and culture supernatants were collected and tested for the presence of infectious viral particles using the Tissue Culture Infectious Dose 50 method. The graph represents the time course of both released and non-released virions amounts which were added and expressed in plaque forming unit (pfu). <i>Representative data from 3 independent experiments</i>.