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. 2011 Dec 13;6(12):e28677. doi: 10.1371/journal.pone.0028677

Figure 2. VVΔE3L/NS1 blocks eIF-2α phosphorylation and proinflammatory cytokine production in infected HeLa cells.

Figure 2

A. Viral protein expression. HeLa cells were mock-infected (MOCK) or infected with VACV, VVΔE3L or VVΔE3L/NS1 (5 PFU/cell). Cells were collected at the indicated times p.i., and equal amounts of total protein from cell extracts were separated by SDS-PAGE, transferred to nitrocellulose, and treated with a polyclonal antibody against a VACV recombinant virus that expresses the β-galactosidase gene. An anti-VACV HA antibody was used to confirm the lack of expression of this protein by the recombinant viruses. Actin was used as a protein loading control. The position of HA in VACV infected cells is indicated by an asterisk. The position of β-galactosidase protein observed in VVΔE3L/NS1 is denoted by two asterisks. B. eIF-2α phosphorylation levels during VACV, VVΔE3L or VVΔE3L/NS1 infection. HeLa cells were mock-infected (MOCK) or infected with VACV, VVΔE3L or VVΔE3L/NS1 (5 PFU/cell). Cells were collected at the indicated times p.i., and equal amounts of total protein from cell extracts were fractionated by SDS-PAGE, transferred to nitrocellulose and incubated with antibodies to total eIF-2α or eIF-2α phosphorylated at Ser51. C. Relative expression levels of genes involved in the immune response determined by real time RT-PCR. HeLa cells were mock-infected (MOCK) or infected with VACV, VVΔE3L or VVΔE3L/NS1 (5 PFU/cell) and processed for real time RT-PCR at 6 h.p.i. The name of each gene product and the data values obtained in RT-PCR are indicated. X-Fold gene induction (x-axis) was quantified by real time RT-PCR in three independent experiments. Standard deviation is indicated.