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. 2013 Jan;87(2):798-806.
doi: 10.1128/JVI.01851-12. Epub 2012 Oct 31.

The viral interferon regulatory factors of kaposi's sarcoma-associated herpesvirus differ in their inhibition of interferon activation mediated by toll-like receptor 3

Sarah R Jacobs  1 , Sean M Gregory, John A West, Amy C Wollish, Christopher L Bennett, David J Blackbourn, Mark T Heise, Blossom Damania
Affiliations

The viral interferon regulatory factors of kaposi's sarcoma-associated herpesvirus differ in their inhibition of interferon activation mediated by toll-like receptor 3

Sarah R Jacobs et al. J Virol. 2013 Jan.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is correlated with three human malignancies and can establish lifelong latent infection in multiple cell types within its human host. In order to establish and maintain infection, KSHV utilizes multiple mechanisms to evade the host immune response. One such mechanism is the expression of a family of genes with homology to cellular interferon (IFN) regulatory factors (IRFs), known as viral IRFs (vIRFs). We demonstrate here that KSHV vIRF1, -2, and -3 have a differential ability to block type I interferon signaling mediated by Toll-like receptor 3 (TLR3), a receptor we have previously shown to be activated upon KSHV infection. vIRF1, -2, and -3 inhibited TLR3-driven activation of IFN transcription reporters. However, only vIRF1 and vIRF2 inhibited increases in both IFN-β message and protein levels following TLR3 activation. The expression of vIRF1 and vIRF2 also allowed for increased replication of a virus known to activate TLR3 signaling. Furthermore, vIRF1 and vIRF2 may block TLR3-mediated signaling via different mechanisms. Altogether, this report indicates that vIRFs are able to block IFN mediated by TLRs but that each vIRF has a unique function and mechanism for blocking antiviral IFN responses.

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Figures

Fig 1
Fig 1
KSHV infection leads to decreased TLR3 and CXCL10 message levels. THP1 monocytic cells were infected with KSHV and harvested at the indicated time points. Latently infected KSHV-THP1 cells maintained under selection were harvested concurrently as controls. RNA was isolated from cells, and relative message levels were determined by quantitative RT-PCR. TLR3 (A) and CXCL10 (B) message levels were normalized to that of β-actin and are represented as fold increases over those of mock-infected monocytes after normalization. Values represent the means plus or minus the standard deviations of the means from duplicate biological replicates. *, P < 0.02; **, P < 0.005; and ***, P < 0.002 (by Student's t test).
Fig 2
Fig 2
vIRF1, -2, and -3 block activation of IFN-responsive promoters. 293 and 293-TLR3 cells were transfected with the control vector or vectors expressing vIRF1, -2, or -3 and cotransfected with ISRE (A) or pIFN-β (B) luciferase reporters. Twenty-four hours posttransfection, cells were treated with poly(I·C) for 24 h and harvested. Luciferase activity was measured and normalized to total protein content. (C) Lysates harvested 24 h after poly(I·C) treatment were subjected to SDS-PAGE and immunoblotted with the epitope tags Myc (vIRF1), Xpress (vIRF2), and FLAG (vIRF3) to determine the expression levels of vIRFs posttransfection. Values represent the means plus or minus the standard deviations of the means from triplicate samples. *, P < 0.02 (by Student's t test).
Fig 3
Fig 3
vIRF1 and vIRF2 block TLR3-mediated transcription and production of IFN-β. 293-TLR3 cells were transfected with the control vector or vectors expressing vIRF1, -2, or -3. Twenty-four hours posttransfection, cells were treated with poly(I·C) for 14 h, and cells were harvested for analysis by quantitative RT-PCR. Relative CXCL10 (A), IFN-α (B), and IFN-β (C) message levels were normalized to that of β-actin and represented as fold increases over those of untreated cells. (D and E) Cells were transfected with pcDNA3 vIRF-FLAG constructs or empty pcDNA3 vector as the control. Twenty-four hours posttransfection, cells were treated with poly(I·C), and cells and supernatants were harvested 24 h later. (D) Supernatants were quantitated for secreted IFN-β levels by ELISA. (E) Lysed cells were subjected to SDS-PAGE and immunoblotted with anti-FLAG to determine expression levels of vIRFs posttransfection. The asterisks denote nonspecific bands. (F) 293-TLR3 cells were transfected with control vector or vIRF1, -2, or -3. One day posttransfection, cells were infected with EMCV at an MOI of 0.1 for 24 h before harvesting. Quantitative RT-PCR was performed on RNA isolated from EMCV-infected cells to assess IFN-β message levels normalized to that of β-actin, represented as fold increase over control vector mock-infected cells. Values represent the means plus or minus the standard deviations of the means from triplicate samples. *, P < 0.03, and **, P < 0.02 (by Student's t test).
Fig 4
Fig 4
vIRF1 expression inhibits IRF3 downstream of TLR3. (A) 293 and 293-TLR3 cells were transfected with the control vector or vectors expressing vIRF1, -2, or -3. Twenty-four hours posttransfection, cells were treated with poly(I·C) for 16 h before cells were harvested, lysed, and subjected to immunoblotting. (B) 293-TLR3 cells were transfected with the control vector or vectors expressing vIRF1, -2, or -3, and 4 h posttransfection, cells were trypsinized and moved to 6-well dishes containing coverslips. Twenty hours after replating, cells were treated with poly(I·C) for 1 h before coverslips were harvested and stained for IRF3 (shown in green). DAPI staining is shown in blue. Arrows point to vIRF-expressing cells. (C) 293-TLR3 cells were transfected with a control vector or vIRF1, -2, or -3 and cotransfected with control vector or cellular IRF3. One day posttransfection, cells were treated with poly(I·C) for 24 h before supernatants were harvested and subjected to IFN-β ELISA. Values represent the means plus or minus the standard deviations of the means of results from triplicate samples. *, P < 0.05; **, P < 0.04; and ***, P < 0.01 (by Student's t test).
Fig 5
Fig 5
vIRF expression does not alter EMCV infectivity. 293-TLR3 cells were transfected with the control vector or vectors expressing vIRF1, -2, or -3. Twenty-four hours posttransfection, cells were infected with EMCV at an MOI of 0.001 for 24 h before harvesting. (A) Quantitative RT-PCR was performed on RNA to measure EMCV genetic material, normalized to that of β-actin, represented as the fold increase over vector mock-infected cells. (B) Plaque assays were performed on L929 cells with six different 1:10 serial dilutions of viral supernatant. (C) Lysates harvested at the time of EMCV infection were subjected to SDS-PAGE and immunoblotted with the epitope tags Myc (vIRF1), Xpress (vIRF2), and FLAG (vIRF3) to determine the expression levels of vIRFs posttransfection. Values represent the means plus or minus the standard deviations of the means from triplicate biological replicates.
Fig 6
Fig 6
vIRF1 or vIRF2 expression leads to increased EMCV viral production following poly(I·C) pretreatment. 293-TLR3 cells were transfected with the control vector or vectors expressing vIRF1, -2, or -3. Twenty-four hours posttransfection, cells were treated with poly(I·C) for 6 h prior to infection with EMCV at an MOI of 0.001 for 24 h before harvesting. (A) Quantitative RT-PCR was performed on RNA to investigate EMCV genetic material, normalized to that of β-actin, represented as the fold increase over vector mock-infected cells. (B) Plaque assays were performed on L929 cells with six different 1:10 serial dilutions of viral supernatant. (C) Lysates harvested at the time of EMCV infection were subjected to SDS-PAGE and immunoblotted with the epitope tags Myc (vIRF1), Xpress (vIRF2), and FLAG (vIRF3) to determine the expression levels of vIRFs posttransfection. Values represent the means plus or minus the standard deviations of the means from triplicate biological replicates. *, P < 0.02 (Student's t test).
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