FIG 3.

Protein synthesis inhibition, apoptosis induction, and rRNA breakdown during poxvirus infection of primary human macrophages. (A) Metabolic labeling of proteins during MVA, ANK, WR, NYBH, NYVAC, or CopV infection. Human macrophages were mock infected or infected with the indicated poxviruses (5 PFU/cell); at the times indicated, cells were labeled (30 min) with [³⁵S]Met-Cys Promix (50 μCi/ml), and equal amounts of proteins were subsequently analyzed by SDS-PAGE (10%) and autoradiography. Molecular weight (MW; in thousands) is indicated based on protein standards. (B) Time course of PARP-1 cleavage during MVA, ANK, WR, NYBH, NYVAC, or CopV infection. Human macrophages were mock infected or infected (5 PFU/cell) with the indicated poxviruses, and at the times indicated, total protein (100 μg) was fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-PARP-1. An 89-kDa PARP-1 cleavage product was observed at 16 h p.i. Molecular weight standards (MW; in thousands) are indicated. eIF-2α or phosphorylated eIF-2α (eIF-2α-P) at Ser51 was measured using specific antibodies. (C) Quantification of cells undergoing apoptosis after infection with MVA, ANK, WR, NYBH, NYVAC, or CopV. At 24 h hours after mock infection or infection with the indicated viruses (5 PFU/cell), cells were fixed, and apoptosis was measured using a Caspase-Glo 3/7 assay kit. Results represent the means ± the standard deviations of three independent experiments. P values from a two-tailed t test assuming nonequal variance were determined. In all the cases, P is <0.05. DL, detection limit. (D) MVA or NYVAC infection of primary human macrophages causes rRNA breakdown. Total rRNA was isolated from uninfected (mock) or poxvirus-infected macrophages (5 PFU/cell). At indicated times p.i., 2 μg of total RNA was separated by electrophoresis and subsequently stained with ethidium bromide. RNAs were also analyzed using an Agilent 2100 Bioanalyzer. RNA integrity numbers are listed under each corresponding sample.