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. 2006 Oct;80(20):10083-95.
doi: 10.1128/JVI.00607-06.

Suppression of proinflammatory signal transduction and gene expression by the dual nucleic acid binding domains of the vaccinia virus E3L proteins

Jeffrey O Langland  1 , John C Kash, Victoria Carter, Matthew J Thomas, Michael G Katze, Bertram L Jacobs
Affiliations

Suppression of proinflammatory signal transduction and gene expression by the dual nucleic acid binding domains of the vaccinia virus E3L proteins

Jeffrey O Langland et al. J Virol. 2006 Oct.

Abstract

Cells have evolved elaborate mechanisms to counteract the onslaught of viral infections. To activate these defenses, the viral threat must be recognized. Danger signals, or pathogen-associated molecular patterns, that are induced by pathogens include double-stranded RNA (dsRNA), viral single-stranded RNA, glycolipids, and CpG DNA. Understanding the signal transduction pathways activated and host gene expression induced by these danger signals is vital to understanding virus-host interactions. The vaccinia virus E3L protein is involved in blocking the host antiviral response and increasing pathogenesis, functions that map to separate C-terminal dsRNA- and N-terminal Z-DNA-binding domains. Viruses containing mutations in these domains allow modeling of the role of dsRNA and Z-form nucleic acid in the host response to virus infection. Deletions in the Z-DNA- or dsRNA-binding domains led to activation of signal transduction cascades and up-regulation of host gene expression, with many genes involved in the inflammatory response. These data suggest that poxviruses actively inhibit cellular recognition of viral danger signals and the subsequent cellular response to the viral threat.

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Figures

FIG. 1.
FIG. 1.
Host gene expression regulated by VV. Host genes which were differentially induced during infection with all VV constructs are shown. Genes were sorted based on a threefold (P < 0.01) or greater level of induction for wtVV at 6 hpi and were also significantly induced by VVE3LΔ83N, VVE3LΔ26C, and VVΔE3L. Red boxes represent genes which were significantly induced. Changes (n-fold) in expression levels relative to those of mock-infected cells are shown within each box. TGFB, transforming growth factor β.
FIG. 2.
FIG. 2.
Host gene expression regulated by the C-terminal dsRNA-binding domain of E3L. Host genes which were differentially expressed during infection with VV constructs lacking the C-terminal dsRNA-binding domain of E3L (VVE3LΔ26C and VVΔE3L) are shown. In the left panel, genes were sorted based on a threefold (P < 0.01) or greater level of induction for VVE3LΔ26C at 6 hpi and were unaltered during wtVV infection. The right panel represents genes which were induced during infection with all VV constructs (yellow boxes) but much more highly induced during infection with VVE3LΔ26C (red boxes). Changes (n-fold) in expression levels relative to those of mock-infected cells are shown within each box.
FIG. 3.
FIG. 3.
Host gene expression regulated by the N-terminal Z-DNA-binding domain of E3L. Host genes which were differentially expressed during infection with VV constructs lacking the N-terminal Z-DNA-binding domain of E3L (VVE3LΔ83N and VVΔE3L) are shown. Genes were sorted based on a twofold (P ≤ 0.01) or greater level of induction or repression for VVE3LΔ83N at 2 hpi and were unaltered during wtVV infection. Red boxes represent genes which were significantly induced. Changes (n-fold) in expression levels relative to those of mock-infected cells are shown within each box.
FIG. 4.
FIG. 4.
VV regulation of the host inflammatory response. Cells were infected with wtVV, VVE3LΔ83N, VVE3LΔ26C, or VVΔE3L, and the differential expression levels of host genes involved in several proinflammatory responses are shown. Shades of red indicate host gene induction, and shades of green indicate host gene repression. Gray represents cDNA sequences that were not present on all arrays used.
FIG. 5.
FIG. 5.
VV regulation of signal transduction. Cells were mock infected or infected with wtVV, VVE3LΔ83N, VVE3LΔ26C, or VVΔE3L. Extracts were prepared at 6 hpi and 12 hpi and analyzed for the presence of phosphorylation, nuclear translocation, or degradation of key components of several host signal transduction cascades by Western blot analysis. During gel electrophoresis, equal protein amounts were loaded into each lane. Chemiluminescent bands corresponding to each protein were quantified using ImageQuant software and graphed. Extracts were assayed in duplicate, and graphs illustrate representative results. Nuclear accum., nuclear accumulation.
FIG. 6.
FIG. 6.
VV regulation of the host IFN response. (A) Cells were infected with wtVV, VVE3LΔ83N, VVE3LΔ26C, or VVΔE3L, and the differential expression levels of host genes involved in the IFN response are shown. Shades of red indicate host gene induction, and shades of green indicate host gene repression. Gray represents samples which were eliminated due to chip errors. (B) Cells were mock infected or infected with wtVV or VVΔE3L. At 3 hpi, cells were treated with 100 IU/ml IFN-α for 15 min. Extracts were prepared and analyzed for activation (phosphorylation) of STAT1 by Western blot analysis. During gel electrophoresis, equal protein amounts were loaded into each lane. Chemiluminescent bands corresponding to STAT1 were quantified using ImageQuant software and graphed. ISRE, interferon-stimulated response element; GAS, gamma interferon-activated site.
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