Activation of the Interferon Response by Human Cytomegalovirus Occurs via Cytoplasmic Double-Stranded DNA but Not Glycoprotein B

DNA-dependent activator of interferon regulatory factor (DAI) acts as a cytosolic B-form DNA sensor that induces type I interferons. However, DAI is not required for DNA sensing in certain cell types due to redundancy of the DNA sensing system. Here, we investigated the effect of DAI on herpes simplex virus 1 (HSV-1) infection in HepG2 hepatocellular carcinoma cells. DAI transcription was induced after gamma interferon (IFN-␥) treatment or HSV-1 infection. HSV-1 replication was enhanced by DAI knockdown, and ectopic DAI expression repressed viral replication in a manner requiring the Z␤ and D3 domains, but not the Z␣ domain. This activity of DAI was more prominent at low multiplicity of infection (MOI) and correlated with the reduced expression of viral immediate-early genes. Consistently, DAI repressed the activation of ICP0 promoter in reporter gene assays. DNA-dependent activator of interferon (IFN) regulatory factor (DAI), which is also referred to as Z-DNA binding protein 1 (ZBP1) or DLM-1, was initially identified as a highly upregulated protein in mouse tumor stromal cells and in macrophages treated by gamma IFN (IFN-␥) or lipopolysaccharide (1). Structural analyses have revealed that DAI/ZBP1/DLM-1 (referred to as DAI hereafter) contains the amino-terminal Z-form DNA-binding domains, Z␣ and Z␤, which are homologous to those of adenosine deaminase that acts on RNA (ADAR1), an RNA editing enzyme (2-5). Since Z-DNA is located near the transcription start sites of certain genes in the genome, a role of DAI in transcriptional regulation has been suggested (6, 7). Induction of DAI was also observed in mouse hepatocytes infected with hepatitis B virus (HBV) (8) and in mouse embryonic fibroblasts (MEFs) stimulated by B-form DNA (9).Recently, DAI was shown to act as a cytosolic B-form DNA sensor that initiates IFN responses via activation of the nuclear factor-B (NF-B) and interferon regulatory transcription factor 3 (IRF3) pathways in mice (10). In addition to the Z-DNA-binding domains, a region termed the D3 domain was demonstrated to primarily contribute to the recognition of B-DNA in vitro (10). However, all of the Z␣, Z␤, and D3 domains were required for efficient B-DNA binding in vivo and DAI was suggested to undergo DNA-mediated multimerization to evoke activation of IFN responses (11). The carboxyl-terminal region of DAI was responsible for recruitment of both IRF3 and TANK-binding kinase 1 (TBK1), an IB kinase that activates IRF3 (10). The mechanism by which DAI activates the NF-B pathway was shown to involve recruitment of receptor-interacting protein kinase 1 (RIP1) and RIP3 through a RIP homotypic interaction motif (RHIM)-dependent interaction with DAI (12, 13). Recently, the binding of DAI with RIP3 was shown to mediate virus-induced programmed necrosis (14).The requirement of DAI in induction of IFN response by cytosolic stimulation of B-DNA is dependent on cell type. DAI played a role in the DNA-mediated IFN production in mouse fibroblast L929 (10, 12, 15) and mouse SVEC4-10 endothelial cells (12)...

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confidence: 89%

DNA Sensing-Independent Inhibition of Herpes Simplex Virus 1 Replication by DAI/ZBP1

Pham

Kwon

Kim

et al. 2013

“…Productive HCMV infection induces genes involved in innate immune activation and inflammation (51). This induction occurs in the absence of virus replication and is triggered by recognition of glycoprotein B (gB) and viral double-strand DNA (dsDNA) (52,53). HCMV infection of total PBMCs suggests that Toll-like receptor 2 (TLR2) recognizes virion components, triggering inflammatory cytokine secretion (54).…”

Section: Hcmv-infected Cd14mentioning

confidence: 99%

Human Cytomegalovirus Modulates Monocyte-Mediated Innate Immune Responses during Short-Term Experimental LatencyIn Vitro

Noriega

Haye

Kraus

et al. 2014

The ability of human cytomegalovirus (HCMV) to establish lifelong persistence and reactivate from latency is critical to its success as a pathogen. Here we describe a short-term in vitro model representing the events surrounding HCMV latency and reactivation in circulating peripheral blood monocytes that was developed in order to study the immunological consequence of latent virus carriage. IMPORTANCEHCMV has the ability to establish a lifelong infection within the host, a phenomenon termed latency. We have established a short-term model system in human peripheral blood monocytes to study the immunological relevance of latent virus carriage. Infection of CD14؉ monocytes by HCMV results in the generation of latency-specific transcripts, maintenance of viral genomes, and the capacity to reenter the lytic cycle. During short-term latency in monocytes the virus initiates a program of differentiation to inflammatory macrophages that coincides with the modulation of cytokine secretion and specific cellular processes. HCMVinfected monocytes are hindered in their capacity to exert normal immunoprotective mechanisms. Additionally, latent virus disrupts type I and II interferon signaling at the level of STAT1 phosphorylation. This in vitro model system can significantly contribute to our understanding of the molecular and inflammatory factors that initiate HCMV reactivation in the host and allow the development of strategies to eradicate virus persistence.

“…As a member of the ISGs, viperin was effectively induced by SeV ( Fig. 1) (15). With a high-throughput screen assay of all 84 proteins carried by HSV-1, dual-luciferase reporter gene assays were performed in HEK293T cells cotransfected with viperin-luciferase reporter plasmid and individual HSV-1 protein expression plasmid for 20 h and infected with SeV (17).…”

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confidence: 99%

“…To investigate whether HSV-1 could induce the expression of viperin, HEK293T cells were infected with wild-type (WT) HSV-1 at different multiplicities of infection (MOI) or with Sendai virus (SeV) (15). Infection with SeV induced a significant amount of viperin; however, infection with a low MOI (0.2) of HSV-1 induced only a trace amount of viperin, and infection with a moderate MOI (2) abrogated the expression of viperin (Fig.…”

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confidence: 99%

Herpes Simplex Virus 1 Counteracts Viperin via Its Virion Host Shutoff Protein UL41

Shen

Wang

et al. 2014

bThe interferon (IFN)-inducible viperin protein restricts a broad range of viruses. However, whether viperin plays a role during herpes simplex virus 1 (HSV-1) infection is poorly understood. In the present study, it was shown for the first time that wild-type (WT) HSV-1 infection couldn't induce viperin production, and ectopically expressed viperin inhibited the replication of UL41-null HSV-1 but not WT viruses. The underlying molecular mechanism is that UL41 counteracts viperin's antiviral activity by reducing its mRNA accumulation. Viperin is a highly conserved, 361-amino-acid protein. It was first identified as a gamma interferon (IFN-␥)-inducible protein which is directly induced by human cytomegalovirus (HCMV), and its constitutive expression is low (1). The viperin gene (also known as cig5 or RASD2) can also be categorized as an antiviral interferon-stimulated gene (ISG) which limits the replication of many DNA and RNA viruses (1-14). However, whether viperin plays a role during herpes simplex virus 1 (HSV-1) infection is unknown.To investigate whether HSV-1 could induce the expression of viperin, HEK293T cells were infected with wild-type (WT) HSV-1 at different multiplicities of infection (MOI) or with Sendai virus (SeV) (15). Infection with SeV induced a significant amount of viperin; however, infection with a low MOI (0.2) of HSV-1 induced only a trace amount of viperin, and infection with a moderate MOI (2) abrogated the expression of viperin (Fig. 1A).To further explore whether viperin could inhibit the replication of WT HSV-1, HEK293T cells with ectopic expression of viperin-Flag were infected with HSV-1 at an MOI of 0.2. Then cells were harvested at the time points indicated in the figures, and viral plaque assay was performed to determine viral replication (16). As a result, ectopically expressed viperin did not affect the replication of WT HSV-1 (Fig. 1B). The data from Western blot (WB) analysis also showed that viperin did not affect viral protein expression (Fig. 1C). These results demonstrated that ectopic expression of viperin failed to inhibit the replication of WT HSV-1.The aforementioned data led us to hypothesize that at least one of the HSV-1 proteins could counteract the expression of viperin. As a member of the ISGs, viperin was effectively induced by SeV ( Fig. 1) (15). With a high-throughput screen assay of all 84 proteins carried by HSV-1, dual-luciferase reporter gene assays were performed in HEK293T cells cotransfected with viperin-luciferase reporter plasmid and individual HSV-1 protein expression plasmid for 20 h and infected with SeV (17). As a result, ectopically expressed UL41 abrogated the expression of viperin; however, other HSV-1 proteins did not (data not shown). UL41 has been reported to degrade both viral and cellular mRNAs (18)(19)(20)(21)(22)(23)(24)(25)(26). Recently, mRNA of tetherin has been reported to be degraded by UL41 (27). Meanwhile, ICP0, an E3 ubiquitin ligase, promotes degradation of many cellular antiviral proteins, such as IRF3, IRF7, IFI16, and ATRX ...