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. 2019 Mar 12;11(3):246.
doi: 10.3390/v11030246.

Interferon-Independent Upregulation of Interferon-Stimulated Genes during Human Cytomegalovirus Infection is Dependent on IRF3 Expression

Caroline L Ashley  1 , Allison Abendroth  2 , Brian P McSharry  3 , Barry Slobedman  4
Affiliations

Interferon-Independent Upregulation of Interferon-Stimulated Genes during Human Cytomegalovirus Infection is Dependent on IRF3 Expression

Caroline L Ashley et al. Viruses. .

Abstract

The antiviral activity of type I interferons (IFNs) is primarily mediated by interferon-stimulated genes (ISGs). Induction of ISG transcription is achieved when type I IFNs bind to their cognate receptor and activate the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways. Recently it has become clear that a number of viruses are capable of directly upregulating a subset of ISGs in the absence of type I IFN production. Using cells engineered to block either the response to, or production of type I IFN, the regulation of IFN-independent ISGs was examined in the context of human cytomegalovirus (HCMV) infection. Several ISGs, including IFIT1, IFIT2, IFIT3, Mx1, Mx2, CXCL10 and ISG15 were found to be upregulated transcriptionally following HCMV infection independently of type I IFN-initiated JAK-STAT signaling, but dependent on intact IRF3 signaling. ISG15 protein regulation mirrored that of its transcript with IFNβ neutralization failing to completely inhibit ISG15 expression post HCMV infection. In addition, no detectable ISG15 protein expression was observed following HCMV infection in IRF3 knockdown CRISPR/Cas-9 clones indicating that IFN-independent control of ISG expression during HCMV infection of human fibroblasts is absolutely dependent on IRF3 expression.

Keywords: IRF3; ISG15; human cytomegalovirus; interferon; interferon stimulated genes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Regulation of interferon-stimulated gene (ISG) transcript levels following infection with intact or ultraviolet (UV)-irradiated human cytomegalovirus (HCMV) in cells with an abrogated interferon (IFN) response. Primary human foreskin fibroblasts (HFs), nPro expressing HFs (nPro/HFs) and V protein-expressing HFs (V/HFs) were infected in parallel with HCMV or UV-HCMV at an MOI of 3. 6 h.p.i. RNA was extracted, converted to cDNA and the relative levels of various ISG transcripts (normalised to the housekeeping gene GAPDH) were calculated. Individual bars represent the average fold change in transcript level compared to the mock infection for each cell type (set to 1). Error bars indicate the SEM and statistical significance was determined using a Student’s two-tailed t-test, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 2
Figure 2
IRF3-dependent, STAT1-independent regulation of ISG15 following HCMV infection (a) Primary HFs, nPro/HFs and V/HFs were infected at an MOI of 3 with HCMV or UV-HCMV and levels of ISG15 transcript were analyzed 6 h.p.i as in Figure 1. (b) Primary HFs, nPro/HFs and V/HFs were infected at an MOI of 3 with HCMV or UV-HCMV. Protein lysates were harvested at 24 h.p.i. and analyzed by immunoblot, staining for ISG15 and GAPDH. (c) Primary HFs, nPro/HFs and V/HFs were treated with filtered (0.1 μm pore size) supernatant from mock, HCMV or UV-HCMV infected primary HFs. RNA harvested at 6 h post-treatment was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) as in Figure 1. RNA was extracted at 6 h.p.i. from primary HFs treated with 100 U of IFNβ or infected at a multiplicity of infection (MOI) of 3 with HCMV or UV-HCMV in the presence of (d) a control antibody or (e) IFNβ-neutralizing antibody and analyzed by qRT-PCR. Error bars indicate the standard error of the mean (SEM) and statistical significance was calculated using a Student’s two-tailed T-test. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 3
Figure 3
IFN-independent, IRF3-dependent regulation of ISG15 recapitulated in IRF3 knockout (KO) CRISPR/Cas-9 engineered telomerase immortalized (hTERT) HFs. (a) IRF3 KO clones generated by CRISPR/Cas-9 were screened by immunoblot for IRF3 alongside the parental Cas-9 hTERT HFs with GAPDH as a loading control. The dashed boxes denote which clones were chosen for further experiments. (b) Supernatants were collected from the Cas-9 hTERT HFs and individual clones after 24 h of HCMV infection (MOI 3) before IFNβ was quantified by an enzyme-linked immunosorbent assay (ELISA) (N.D: not detected indicates IFNβ levels less than the lower limit of quantification 5 pg/mL). (c) Successful IRF3 KO clones 7, 17 and 20 were infected with HCMV (MOI of 3) or treated with recombinant IFNβ (100U) in parallel with unsuccessful clone 6, and the parental Cas-9 hTERT HFs. Protein lysates were extracted at 24 h.p.i. before immunoblotting for ISG15 and GAPDH. (d) Individual clones and parental Cas-9 hTERT HFs were treated with supernatant from HCMV infected cells for 24 h before immunoblotting for ISG15 and GAPDH.
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