Figure 2

TULV-induced ERK1/2 inactivation correlates with replication efficiency. To confirm that TULV-mediated ERK1/2 inactivation is replication-dependent, we employed UV-inactivated TULV as a replication-incompetent negative control in ERK1/2 phosphorylation assay. Vero E6 cells were infected with a 0.1 multiplicity of infection of TULV or mock-infected with UV-inactivated virus (UV). Cells were collected at 4 and 10 days post infection and 100 μg of protein lysate immunoblotted to detect phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 and hantavirus nucleocapsid protein N. Bands were subjected to intensity analysis (ImageJ software; http://rsb.info.nih.gov/ij) and the amount of p-ERK1/2 related to the amount of total ERK1/2 in individual samples. Fold change was calculated in relation to mock-infected sample at the respective day post infection (p.i.). Virus titers were determined as focus forming units (FFU) from conditioned media of cell cultures. Error bars for virus titer-measurements represent standard deviation.