Pseudorabies Virus Infection of Epithelial Cells Leads to Persistent but Aberrant Activation of the NF-κB Pathway, Inhibiting Hallmark NF-κB-Induced Proinflammatory Gene Expression

Abstract

The nuclear factor kappa B (NF-κB) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-κB, illustrated by proteasome-dependent degradation of the inhibitory NF-κB regulator IκB and nuclear translocation and phosphorylation of the NF-κB subunit p65. PRV-induced persistent activation of NF-κB does not result in expression of negative feedback loop genes, like the gene for IκBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-κB activation. Hence, PRV infection triggers persistent NF-κB activation in an unorthodox way and dramatically modulates the NF-κB signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-κB signaling, which may aid the virus in modulating early proinflammatory responses in the infected host. IMPORTANCE The NF-κB transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-κB, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-κB activation shares some mechanistic features with canonical NF-κB activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IκB kinase (IKK) and even renders infected cells resistant to canonical NF-κB activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-κB activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-κB activation.

Pseudorabies Virus Infection Causes Downregulation of Ligands for the Activating NK Cell Receptor NKG2D

“…Untreated cells served as a control. Successful PAA treatment was confirmed by Western blot, as assessed by expression of US3 (early protein) and lack of expression of gE (late protein) as described earlier [28,35,37] (data not shown). Figure 3 shows that addition of PAA did not affect PRV-induced downregulation of NKG2D ligand expression.…”

“…SK cells were either directly or after 8 h inoculated with PRV strain Kaplan [ 27 ] at a multiplicity of infection (MOI) of 10 and put back on the rocking platform at 37 °C for 14 h. To ensure that this inoculation procedure led to a 100% infection efficiency, flow cytometric detection of viral glycoproteins gB and gD was performed in every experiment (illustrated in Figure 1 A). For the experiment using phosphonoacetic acid (PAA, Sigma-Aldrich, St. Louis, MO, USA, catalog number 284270), SK cells were treated with PAA at a concentration of 400 µg/mL from 30 min prior to infection onwards as described before [ 28 ]. For the experiment using cycloheximide (CHX, Sigma-Aldrich, catalog number C1988), SK cells were treated with CHX at a concentration of 5 µg/mL at 8 h of cultivation.…”

“…RNA isolations were done as described before [ 28 ]. An RNeasy minikit (Qiagen, Hilden, Germany, catalog number 74104) was used according to the manufacturer’s protocol to isolate RNA.…”

“…Median fluorescence intensity was determined by subtracting the median fluorescence intensity of the control staining from the median fluorescence intensity of each sample. RNA isolations were done as described before [28]. An RNeasy minikit (Qiagen, Hilden, Germany, catalog number 74104) was used according to the manufacturer's protocol to isolate RNA.…”

“…In a final volume of 20 µL qPCRs were performed on MicroAmp Fast optical 96-well reaction plates (Applied Biosystems, Thermo Fisher Scientific, catalog number 4346906) using a StepOnePlus real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, catalog number 4376600). The relative expression of the target genes (pULBP1 and pMIC2) was analyzed using the double delta threshold cycle method and normalized to the level of expression of the 28S rRNA gene, which has been validated and used as a reference gene in other similar gene expression analyses on PRV-infected cells [28,30]. Table 2.…”