Differential Induction of Apoptosis, Interferon Signaling, and Phagocytosis in Macrophages Infected with a Panel of Attenuated and Nonattenuated Poxviruses

1. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   FIG 1

   Cellular changes in primary human macrophages following infection with different poxvirus vectors. (A) Morphological changes in human macrophages mock infected or infected with MVA, ANK, WR, NYBH, NYVAC, or CopV (5 PFU/cell) in six-well plates. Changes were examined by phase-contrast microscopy at 2, 6, and 16 h p.i. (hpi). For each group of infections, representative fields are shown at a magnification of ×40. (B) Titration of each viral strain. MVA titers were determined by immunostaining using BHK-21 cells; ANK, WR, NYBH, NYVAC, and CopV titers were determined by plaque assay on BSC40 cells. The images show differential plaque sizes produced by the different poxvirus-derived vectors. (C) VACV growth in infected human macrophages. Cells were infected with MVA, ANK, WR, NYBH, NYVAC, or CopV (0.1 PFU/cell), and at the times indicated, cells were harvested, and virus yields were determined by plaque assay for each virus except MVA, for which the titer was determined by immunostaining. 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 <0.01. T, time.
2. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   FIG 2

   Viral protein expression during MVA, ANK, WR, NYBH, NYVAC, or CopV infection. Primary human macrophages were mock infected or infected with the indicated poxviruses (5 PFU/cell), and at the indicated times p.i., equal amounts of proteins from cell extracts were fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and treated with antibodies to specific virus early (p25) and late proteins (p64 and p14). Molecular weight (MW; in thousands) is indicated based on protein standards. In all blots, detection of actin was used as a loading control.
3. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   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.
4. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   FIG 4

   Number of host genes and pathway analysis of genes differentially regulated by poxvirus infection. (A) Shown are the numbers of cellular genes that exhibited expression changes in our microarray analyses larger than 2-fold or less than 2-fold at 6 h after infection with each virus. (B) Intersections of gene upregulation (fold change [fc] of >2) or downregulation (fold change of <2) in macrophages infected with the indicated viruses are shown in the Venn diagrams. (C) Ingenuity Pathway analysis showing selected canonical pathways significantly modulated in macrophages infected with the indicated poxviruses compared to naive macrophage controls (P < 0.05). The dashed line indicates the threshold set at 1.5 log (P value). ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; GM-CSF, granulocyte-macrophage colony-stimulating factor.
5. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   FIG 5

   Validation of microarray data at the mRNA level. (A) RT-PCR analysis of the relative expression levels of immune response genes. Macrophages from three donors were mock, MVA, NYVAC, WR, CopV, ANK, or NYBH infected and at 6 h p.i. were processed for RT-PCR analysis. The name of each gene product obtained by RT-PCR analysis is indicated. Three independent experiments were carried out, and a representative experiment is shown. Error bars indicate the standard deviations of the means. (B). Macrophages from three donors were MVA, NYVAC, WR, CopV, ANK, or NYBH infected. Mock inoculum is the uninfected cellular inoculum purified using the same protocol for the purification of vaccinia virus. The purified mock inoculum from cultures of BHK-21 cells was used for comparisons with MVA and NYVAC infections, and the purified mock inoculum from BSC40 cells was used for comparisons with the other infections (WR, CopV, ANK, and NYBH). The name of each gene product, microarray data values at 6 h p.i., and values obtained by RT-PCR analysis are indicated. Three independent experiments were carried out, and a representative experiment is shown. Error bars indicate the standard deviations of the means.
6. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   FIG 6

   Antiviral bioassays confirm that MVA-infected macrophages secreted high levels of biologically active type I IFN. Plaque assay analysis of VSV-infected Vero cells left either untreated or pretreated with either IFN-α (1,000 U ml⁻¹ for 16 h) or with the supernatant (S) obtained from MVA-infected macrophages from three independent donors. Results represent the means ± the standard deviations of three independent experiments.
7. 

   View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   FIG 7

   MVA infection increases the phagocytosis of GFP-labeled latex beads in human macrophages. (A) Phagocytosis of GFP-latex beads by human macrophages. Cells were seeded in eight-well tissue culture plates and treated with supernatant (sup) from mock-infected (Uninfected) or MVA-infected macrophages for 16 h. After that, the cells were incubated with 1-μm-diameter latex beads conjugated to GFP in a ratio of 10 latex beads per cell. Phagocytized beads and cells were visualized 2 h after latex bead incubation by phase-contrast microscopy. Representative fields are shown at a magnification of ×40. (B) Cells having phagocytized GFP-labeled latex beads were quantified by immunofluorescence microscopy. Representative phase-contrast images are shown. The graph shows the quantification of phagocytic cells in macrophage cultures treated with supernatant (sup) from mock-infected (Uninfected) or MVA-infected macrophages. 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 cases, P is <0.05.

• Copyright © 2014, American Society for Microbiology. All Rights Reserved.