Review

Nuclear sensing of viral DNA, epigenetic regulation of herpes simplexvirus infection, and innate immunity

David M. Knipe n

Harvard Medical School, Department of Microbiology and Immunobiology, 77 Avenue Louis Pasteur, Boston, MA 02115, United States

a r t i c l e i n f o

Article history:Received 23 December 2014Returned to author for revisions4 February 2015Accepted 5 February 2015Available online 3 March 2015

Keywords:EpigeneticsDNA virusIFI16cGASInnate immunity

a b s t r a c t

Herpes simplex virus (HSV) undergoes a lytic infection in epithelial cells and a latent infection inneuronal cells, and epigenetic mechanisms play a major role in the differential gene expression underthe two conditions. HSV viron DNA is not associated with histones but is rapidly loaded withheterochromatin upon entry into the cell. Viral proteins promote reversal of the epigenetic silencingin epithelial cells while the viral latency-associated transcript promotes additional heterochromatin inneuronal cells. The cellular sensors that initiate the chromatinization of foreign DNA have not been fullydefined. IFI16 and cGAS are both essential for innate sensing of HSV DNA, and new evidence shows howthey work together to initiate innate signaling. IFI16 also plays a role in the heterochromatinization ofHSV DNA, and this review will examine how IFI16 integrates epigenetic regulation and innate sensing offoreign viral DNA to show how these two responses are related.

& 2015 Elsevier Inc. All rights reserved.

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153Epigenetic regulation of herpes simplex virus lytic and latent infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154Sensing of foreign DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154Sensing of HSV DNA, innate responses, and epigenetic regulation of foreign DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154Sensing of HSV DNA and epigenetic silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156Viral evasion of IFI16. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157cGAS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157cGAS and IFI16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157Role of IFI16 and cGAS in sensing DNA of other viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157Normal roles of IFI16 in human cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Introduction

When foreign DNA is introduced into mammalian cells, host cellresponses recognize it as a potential “danger” and initiate a series ofresponses that attempt to control its expression and minimize itsdamage of the cell, including epigenetic silencing of the DNA toreduce its expression, induction of DNA damage responses, induction

of innate responses, and recruitment of nuclear domain 10 (ND10) orPML body components that reduce its expression. The mechanism(s)by which foreign DNA is recognized and how it is distinguished from“self” DNA are not known, and the relationships between theseresponses to foreign DNA have not been defined. Herpes simplexvirus (HSV) DNA evokes the same foreign DNA responses in that HSVDNA is not associated with histones in virions but becomes rapidlyassociated with chromatin upon entry into host cells, DNA damageresponse proteins and ND10 proteins localize near HSV genomes,and innate responses are induced in response to HSV DNA. However,little has been known until recently about the cellular sensors that

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/yviro

Virology

http://dx.doi.org/10.1016/j.virol.2015.02.0090042-6822/& 2015 Elsevier Inc. All rights reserved.

n Tel.: þ1 617 432 1934.E-mail address: david_knipe@hms.harvard.edu

Virology 479-480 (2015) 153–159

recognize HSV DNA and initiate innate responses and chromatiniza-tion. Epigenetic mechanisms play a major role in the HSV gene exp-ression during lytic infection of epithelial cells and latent infection ofsensory neurons. Recent studies have shown a potential link betweenthe innate responses to HSV and epigenetic regulation. Therefore, inthis review I will summarize the epigenetic mechanisms regulatingHSV gene expression and discuss the relationships between foreignDNA sensing, epigenetic regulation of that DNA, and innate immuneresponses. I will also examine how the cellular interferon-inducibleprotein 16 (IFI16) plays a role in sensing of herpesviral DNA and inintegrating the induction of innate responses and epigenetic silencingof the viral DNA.

Epigenetic regulation of herpes simplex virus lytic and latentinfection

HSV undergoes a lytic infection at the initial mucosal sites of in-fection followed by spread to sensory neurons where it establishesa latent infection (Roizman et al., 2013). Lytic infection involves theexpression of more than 80 viral genes while latent infection involvesthe abundant expression of only the latency-associated transcript(LAT) and miRNAs. The basis for the differential expression of HSVgenes during lytic infection of epithelial cells versus the expressionof only the LAT and miRNAs during latent infection has been animportant unanswered question. It has been increasingly recognizedthat epigenetic mechanisms are central in regulation of eukaryoticgene expression (Gardner et al., 2011), and these regulatory mechan-isms also apply to HSV gene expression (Knipe and Cliffe, 2008; Knipeet al., 2013).

Herpesviral DNA in the virion is not associated with histones, butinstead the negative charges are apparently neutralized by the poly-amine spermine. Upon entry into the host cell nucleus of dividingcells, HSV DNA is rapidly associated with histones (Cliffe and Knipe,2008; Oh and Fraser, 2008), and heterochromatin modifications arerapidly put onto the chromatin by 1–2 hpi (Liang et al., 2009; Raja, Lee,and Knipe, manuscript in preparation). The HSV VP16 virion proteinassembles into a transactivator complex with the host cellular proteinshost cell factor 1 (HCF-1) and octamer binding factor 1 (Oct-1), andOct-1 and VP16 bind to sequences in the viral immediate-earlypromoters, bringing the activator complex to the promoter, whichactivates transcription of the immediate-early genes (Kristie, 2015;Roizman and Zhou, 2015). HCF-1 recruits (1) the LSD1 histonedemethylase to demethylate H3K9me2 and H3K9me, (2) the JMJD2demethylases to demethylate H3K9me3, and (3) the H3K4 methyl-transferases to put on this euchromatin modification. Thus, VP16 isrequired for euchromatin modifications as well as reduced histoneloading on viral IE promoters (Herrera and Triezenberg, 2004). IEgenes are then expressed, including infected cell protein 0 (ICP0). ICP0promotes histone removal and acetylation of histones on E and Lgenes (Cliffe and Knipe, 2008), thereby allowing their transcription.ICP0 disrupts the CoRest-HDAC1 complex (Gu et al., 2005) and recruitsthe CLOCK histone acetyltransferase to the viral genome (Kalamvokiand Roizman, 2010). The mechanisms by which ICP0 affects histonemethylation have not been defined yet.

In contrast, during latent infection of sensory neurons, the virallytic genes are associated with heterochromatin (Wang et al., 2005),primarily facultative heterochromatin (Cliffe et al., 2009; Kwiatkowskiet al., 2009), and only the LAT promoter and enhancer are associatedwith euchromatin (Kubat et al., 2004a, 2004b). In neurons, HSV DNAtakes several days to become associated with histones (Cliffe et al.,2013;Wang et al., 2005), a much longer time than in epithelial cells orfibroblasts, likely because the pool of histones is smaller in the non-dividing neurons. From days 7–14 postinfection, histones accumulateon the viral lytic genes and heterochromatin modifications are put onthe histones (Cliffe et al., 2013; Wang et al., 2005). Viral lytic gene

expression is very inefficient because HCF-1 is in the cytoplasm ofsensory neurons (Kristie et al., 1999), and VP16 may also not localizeinto the nucleus of the neurons. A neuron-specific promoter/enhancerdrives the expression of LAT (Zwaagstra et al., 1990) and the precursorof a series of miRNAs (Kramer et al., 2011), some of which inhibit ICP4and ICP0 expression (Umbach et al., 2008). LAT expression reduceslytic gene expression in the neurons (Garber et al., 1997); furthermore,LAT expression increases H3K9me2, H3K9me3, and H3K27me3modifications on viral chromatin (Cliffe et al., 2009; Wang et al.,2005). One study reported that LAT decreased H3K27me3 modifica-tion (Kwiatkowski et al., 2009), but in this study the levels ofH3K27me3 reported on cellular genes were different for the wild-type samples versus the LAT promoter mutant samples. In total, theliterature supports the concept that LAT reduces lytic gene expressionduring acute infection (Garber et al., 1997) and latent infection (Chenet al., 1997) of sensory neurons, promotes heterochromatin on virallytic genes in sensory neurons (Cliffe et al., 2009; Wang et al., 2005),and reduces acute infection death of neurons and increases neuronalsurvival (Nicoll et al., 2012; Thompson and Sawtell, 2001). Thus, ourcurrent working model is that HSV gene products regulate theepigenetic modifications on the HSV genome (Fig. 1), with VP16and ICP0 promoting euchromatin during lytic infection and LATpromoting heterochromatin during latent infection. Many importantquestions on the mechanisms of epigenetic regulation of HSV geneexpression remain as exciting areas for future study.

Attempts to cure individuals of latent viruses such as HIV havefocused on activating the virus from latency by epigenetic drugsand then treatment with antiviral drugs (Shirakawa et al., 2013).Reactivation of HSV from latent infection in the peripheral nervoussystem and potentially in the central nervous system has thepotential for harm to the individual; therefore, the concept of lock-ing in HSV latency with epigenetic drugs has been raised (Lianget al., 2009). Recent studies have shown that rabbits, guinea pigs,and mice treated with an epigenetic drug that inhibits the LSD1histone demethylase show reduced reactivation and increasedlevels of heterochromatin in vivo (Hill et al., 2014). If safe epi-genetic drugs can be discovered that block HSV at this very earlystage of reactivation, they could have great therapeutic potential.

Sensing of foreign DNA

Mammalian cells have a number of receptors at various siteswithin the cell that detect different kinds of foreign nucleic acidsand initiate innate immune responses (Iwasaki and Medzhitov,2013). These include Toll-like receptors (TLRs) in endosomes, RIG-like receptors in the cytosol, and DNA sensors in the cytosol andnucleus. Organisms have also evolved mechanisms to detect foreignDNA and degrade it or restrict its expression. Bacteria have modi-fication-restriction systems to detect foreign DNA and cleave it(Youell and Firman, 2012) as well as CRISPR-CAS systems to cleaveand delete sequences from the foreign DNA (Sander and Joung,2014; Kennedy and Cullen, 2015). Mammalian cells assemblechromatin on transfected DNA and silence its expression within afew days (Cereghini and Yaniv, 1984). Similarly, as discussed above,viral DNA genomes, such as those of the herpesviruses, are rapidlyassociated with histones upon infection of cells, but viruses encodeproteins that combat the epigenetic silencing of their ge-nome (Knipe et al., 2013).

Sensing of HSV DNA, innate responses, and epigeneticregulation of foreign DNA

What are the sensors that detect the foreign DNA and initiate theloading of heterochromatin on that DNA to silence it? The first evi-

D.M. Knipe / Virology 479-480 (2015) 153–159154

dence for DNA sensors came from studies looking at the induction ofinnate responses by detection of foreign DNA. There are a number ofDNA sensors that have been reported to detect viral DNA, and thesehave been reviewed in a number of excellent reviews (Orzalli andKnipe, 2014; Paludan and Bowie, 2013; Dempsey and Bowie, 2015),to which the reader is referred for more details about these sensors.The goal of this mini-review is to discuss the relationship betweenDNA sensing, innate immune responses, and epigenetic regulation.

DNA sensing in general leads to activation of cytokine expressionthrough the STING-IRF-3/NF-κB pathway. Induction of the antiviraltype I IFN response is regulated by a cellular signaling cascadethat results in the activation of IFN regulatory factor-3 (IRF-3) andnuclear factor–kappa B (NF-κB) transcription factors. A critical com-ponent of this signaling cascade is stimulator of IFN genes (STING)(Ishikawa et al., 2009), an endoplasmic reticulum–localized, trans-membrane protein that bridges DNA-sensing mechanisms to down-stream signaling events. Upon microbial DNA stimulation, STINGre-localizes to distinct intracellular foci (Saitoh et al., 2009) andpromotes TANK-binding kinase 1 (TBK1)-dependent phosphoryla-tion of IRF-3 by a direct interaction with both proteins (Tanaka andChen, 2012). Phosphorylated IRF-3 dimerizes, translocates to thenucleus, and binds to IFN-stimulated regulatory elements (ISREs) inthe promoters of IRF-3-responsive genes, including type I IFNs. IFNis subsequently secreted from cells and can act in an autocrine orparacrine fashion to upregulate IFN-stimulated genes (ISGs) via thetype I IFN receptor and a JAK-STAT signaling cascade (reviewed inStark and Darnell, 2012). A subset of ISGs can also be inducedindependently of IFN, but IFN significantly amplifies this induction(Wathelet et al., 1992).

A number of foreign DNA sensors have been reported that arelocated within the cell at various sites within different cells. (1) TLR9,located in endosomes of plasmacytoid dendritic cells, is activated bynonmethylated CpG DNA, and TLR9� /� mice are more susceptible toHSV infection. (2) The DNA-dependent activator of interferon (DAI),located in the cytosol, is required for IFN response to HSV-1 asshown by siRNA depletion (Takaoka et al., 2007) but not in knockoutcells (Ishii and Akira, 2006). (3) RNA polymerase III (Ablasser et al.,2009; Chiu et al., 2009) was reported to be required for sensing ofHSV-1 DNA (Chiu et al., 2009), but others have not seen a require-ment (Melchjorsen et al., 2010; Monroe et al., 2014; Unterholzneret al., 2010). (4) Absent in Melanoma 2 or AIM2 (Fernandes-Alnemriet al., 2009; Hornung et al., 2009) is the prototypic member ofthe PYHIN family of proteins, named because of their pyrin protein–

protein interaction/signaling domain and HIN (hematopoietic IFNinducible nuclear antigen) DNA binding domain. AIM2 is involvedin the sensing of vaccinia virus and murine cytomegalovirus DNAbut not HSV DNA (Hornung et al., 2009; Rathinam et al., 2010) toactivate inflammasomes. (5) Interferon-inducible protein 16 or IFI16(Unterholzner et al., 2010) is, like AIM2, a member of the PYHINfamily of proteins because it has a pyrin domain and two HIN do-mains. IFI16 was identified originally as a cytoplasmic DNA-bindingprotein and innate sensor (Unterholzner et al., 2010), but IFI16 canbe nuclear or cytoplasmic depending on the cell type (Choubey et al.,2008). Nuclear localization of IFI16 is now known to be regulatedby acetylation (Li et al., 2012). IFI16 is capable of binding DNAdirectly through sequence-independent contacts with the sugarphosphate backbone (Jin et al., 2012), providing a biochemical basisfor DNA sensing. IFI16 is required for both activation of inflamma-somes (Johnson et al., 2013; Kerur et al., 2011) and induction of IFN-β in HSV-infected cells (Li et al., 2012; Orzalli et al., 2012). (6) Cyclicguanosine monophosphate-adenosine monophosphate synthase orcGAS is required for IRF-3 activation in response to transfected DNAand HSV-1 infection (Sun et al., 2013; Wu et al., 2013). Because IFI16and cGAS are the most extensively studied and best documentedsensors of HSV DNA in infected cells, most of the rest of mydiscussion will focus on these two DNA sensors.

HSV infection induces innate responses by a number of mechan-isms (Roizman et al., 2013). Among these, DNA-sensing plays a rolein type I interferon responses to HSV infection. UV-inactivated HSVand HSV recombinants that do not express viral proteins induceinterferon-β in human fibroblasts, but expression of viral proteins orjust ICP0 prevented induction of IFN-β (Eidson et al., 2002; Mossmanet al., 2001; Preston et al., 2001). Consistent with this, infection ofhuman fibroblasts with the HSV-1 d109 recombinant virus that exp-resses no viral proteins in normal cells leads to IFI16-dependent IFN-βinduction, and IFN-β induction is dependent on viral DNA entry intothe nucleus (Li et al., 2012; Orzalli et al., 2012). IFI16 must have afunctional nuclear localization signal and be localized into the nucleusto sense HSV-1 DNA and induce IFN-β expression (Li et al., 2012).These results are consistent with a model in which HSV DNA is rele-ased from the capsid into the nucleus, and nuclear IFI16 binds to theviral DNA (Fig. 2). Interestingly, as an exception to this, in humanmacrophages HSV capsid proteins are ubiquitinated in the cytoplasmand the capsid is degraded by the proteasome, but IFI16 is cytoplas-mic in these cells and required for IFN-β induction (Horan et al.,2013).

Fig. 1. Model for epigenetic regulation of the lytic versus latent infection decision by HSV. (A). Following infection of epithelial cells, the capsid is transported to the nuclearpore where the viral DNA is released into the nucleus where it rapidly circularizes and becomes associated with histones bearing heterochromatin marks. VP16 from thevirion tegument forms a complex with HCF-1 and Oct1 that binds to viral IE promoters and HCF-1 recruits histone modification enzymes and chromatin remodelingcomplexes that decrease histone association with viral IE genes and increase euchromatin marks on the remaining associated histones. ICP0 is expressed as an IE protein andit promotes similar processes on the rest of the genome. (B). Following infection of neuronal cells, the capsid is also transported to the nuclear pore where the viral DNA isreleased into the nucleus where it rapidly circularizes and becomes associated with histones. VP16 cannot be transported into the neuronal nucleus, and HCF-1 is notlocalized in the nucleus so viral IE genes are not transcribed efficiently. Instead, the latency-associated transcript is expressed and it promotes the association of facultativeheterochromatin marks on the viral chromatin (Copyright, Lynne Chang, Priya Raja, and David Knipe).

D.M. Knipe / Virology 479-480 (2015) 153–159 155

Following binding to HSV DNA in the nucleus, IFI16 is proposedto undergo a conformational change that allows oligomerizationas shown by intranuclear filaments or aggregates (Li et al., 2013;Orzalli et al., 2013). The formation of higher-order complexes ofthese signaling molecules is consistent with the “supramolecularorganizing centers” or SMOCs that are assembled with other innatesignaling molecules (Kagan et al., 2014). This initiates a signal thatactivates STING in the cytoplasm, which activates TBK-1 phosphor-ylation, phosphorylation and dimerization of IRF-3, and IRF-3localization into the nucleus to bind to the IFN-β gene promoterand activate its transcription (Orzalli et al., 2012) (Fig. 2). The natureof the signal that moves from the nucleus to the cytoplasm toconnect IFI16 and STING is not known. In some studies, IFI16 doesnot move detectably from the nucleus (Li et al., 2013, 2012; Orzalliet al., 2012) while in others IFI16 is reported to move to thecytoplasm and co-localize with inflammasome structures (Johnsonet al., 2013; Kerur et al., 2011). Therefore, the mechanism by whichIFI16 signals to STING remains a mystery. IFI16 may also play adirect transcriptional role in innate immune induction in that itpromotes IFN-α reporter gene expression, increases RNA polymer-ase II loading on the IFN-α gene promoter, and can bind to the IFN-α promoter sequences (Thompson et al., 2014), although thespecificity of the in vitro binding remains to be demonstrated.

Sensing of HSV DNA and epigenetic silencing

Three of the components of the ND10 bodies, PML, hDaxx, andSp1, which are localized near incoming HSV genomes early duringinfection, have been shown to restrict viral IE gene expression (Boutell

and Everett, 2013). Although the PML-containing ND10 structures orbodies have been described variously as localizing “near” the viralgenomes (Maul et al., 1996) or “at the site of viral DNA” (Maul, 1998),there no evidence that PML or other ND10 proteins associate directlywith viral DNA. Furthermore, the mechanisms of this viral restrictionhave not been defined. IFI16 acts as a restriction factor for HSV-1 andHCMV replication in human embryonic lung fibroblasts (Garianoet al., 2012). The mouse homolog of IFI16 has not been definedbecause mice contain ten PYHIN genes while humans encode four,but the closest functional homolog appears to be p204, and depletionof p204 in the mouse cornea increased HSV-1 replication (Conradyet al., 2012).

We found that depletion of IFI16 in HFF cells resulted in increasedreplication and IE gene expression by an ICP0- mutant virus and thatectopic expression of IFI16 in U2OS cells resulted in decreased IE geneexpression (Orzalli et al., 2013). Depletion of IFI16 resulted in dec-reased H3K9me3 modification and increased H3K4me3 modificationon the ICP4 gene promoter, consistent with the increased ICP4 exp-ression. The restriction effect of IFI16 was also observed on transfectedplasmids; therefore, the results were consistent with IFI16 recruitinghistone modification enzymes that place H3K9 heterochromatinmarks on foreign DNA introduced into the cell in an unchromatinizedform. These results were recently confirmed by studies in which theIFI16 gene was deleted in U2OS cells, and the IFI16 knockout cellsshowed enhanced expression of HSV IE genes, reduced heterochro-matin, and increased euchromatin and RNA polymerase II loading onIE genes in wildtype virus-infected cells (Johnson et al., 2014).

How does IFI16 distinguish between viral and host DNA in theinfected cell nucleus? We initially hypothesized that the IFI16 speci-ficity for viral DNA in the nucleus could involve IFI16 recognizing the

Fig. 2. Model of nuclear HSV-1 DNA sensing, innate signaling, and epigenetic regulation by IFI16 and inhibition by ICP0. HSV capsids in the cytoplasm traffic to nuclear poreswhere viral DNA is released into the nucleus. The viral DNA rapidly circularizes, and nuclear IFI16 binds to the viral DNA and multimerizes, initiating a nuclear-to-cytoplasmicsignaling cascade that activates IRF-3, which dimerizes and translocates to the nucleus. Immediate-early expression of ICP0 promotes degradation of IFI16 to inhibitsubsequent IRF-3 signaling and IFN-β expression. The multimerized IFI16 on the viral DNA also recruits histone modification complexes that add H3K9me3 modifications tothe viral chromatin, resulting in epigenetic silencing of the viral genes. (Copyright, Megan Orzalli and David Knipe).

D.M. Knipe / Virology 479-480 (2015) 153–159156

underchromatinized viral DNA to initiate innate responses (Orzalliet al., 2012). Consistent with this, we observed that IFI16 restric-ted expression of the SV40 large T antigen when expressed froma transfected plasmid but did not restrict T antigen expression inSV40 virus-infected cells (Orzalli et al., 2013). Because SV40 DNA isassembled in a mini-chromosome in the virion, this may make itresistant to IFI16-mediated silencing. A biochemical explanation forthis specificity of IFI16 was provided by a recent paper which showedthat IFI16 binds cooperatively to double-stranded DNA that is 33base pairs or longer and does not bind efficiently to DNA the sizeof the linker DNA between nucleosomes or at a transcription bubble(Morrone et al., 2014). Those authors also speculated that IFI16would not bind efficiently to cellular DNA that is assembled innucleosomes.

Viral evasion of IFI16

The herpesviruses have been shown to evade the effects ofIFI16 by two mechanisms. First, ICP0 promotes the degradation ofIFI16 in HFF cells in a RING domain- and proteasome-dependentmanner (Johnson et al., 2013; Orzalli et al., 2012). Although onestudy reported that ICP0 was not necessary or sufficient for IFI16degradation (Cuchet-Lourenco et al., 2013), most of the experi-ments in this study used tumor cells where IFI16 is often notfunctional and not activated by binding to HSV DNA (Li et al., 2012;Orzalli et al., 2013). Further studies in our lab have shown thatIFI16 has a short half-life, and HSV infection can lead to both ICP0-dependent and ICP0-independent loss of IFI16, the latter likelyinvolving vhs-dependent degradation of IFI16 mRNA (Orzalli,Broekema, and Knipe, manuscript in preparation). Second, HCMVpUL83 binds to the pyrin domain of IFI16 and prevents its multi-merization and initiation of the IRF-3 signaling pathway (Li et al.,2013). Therefore, both viruses target IFI16 to remove it or block itssignaling, supporting the importance of IFI16 in initiating innateand epigenetic responses to herpesviral infection.

cGAS

In 2013, the Chen laboratory reported the discovery of the cGASenzyme, whose catalytic activity is activated upon binding to DNAto synthesize cyclic guanosine monophosphate-adenosine mono-phosphate (cGAMP) (Sun et al., 2013; Wu et al., 2013). cGAMP bindsto STING and activates the IRF-3 signaling pathway. This novelsensor and signaling mechanism is required for HSV-1 activation ofIRF-3 dimerization (Wu et al., 2013). Cells from cGAS� /� mice showreduced antiviral responses, and the cGAS� /� mice are moresusceptible to DNA virus infection (Li et al., 2013). These resultsshowed that cGAS is required for IRF-3 signaling and IFN-β induc-tion in HSV-1 infected cells and mice. Surprisingly, Schoggins et al.(2014) showed that cGAS� /� mice are also more susceptible toRNA virus infection, indicating that cGAS activity may be pan-

antiviral (Schoggins et al., 2014). Schoggins et al. (2014) also obser-ved a reduction in the basal levels of IFN-β and several ISGs incGAS-deficient bone marrow-derived macrophages, implicatingcGAS in the basal homeostasis of innate signaling (Schogginset al., 2014). cGAMP can also spread to neighboring cells via gapjunctions to activate STING (Ablasser et al., 2013). Many proposedDNA sensors, including IFI16, are IFN inducible; therefore, cGASdepletion could indirectly affect the ability of additional DNAsensors to induce antiviral cytokine production. No microbial geneproducts that specifically inhibit the activity of cGAS have beendocumented in publications, but we have recently identified anHSV gene product that appears to be required for wildtype HSVinhibition of cGAS activity (Broekema and Knipe, manuscript inpreparation).

cGAS and IFI16

In combination, these results have raised the question of howthe cytosolic cGAS and the nuclear IFI16 DNA sensors are bothrequired for IRF-3 signaling and interferon-β induction in HSV-infected cells. Our own results have shown that both IFI16 and cGASare required for IFN-β induction by HSV-1 infection in HFFs (Orzalliet al., 2015). However, cGAS plays a unique role in human HFF cellsin that it shows low levels of cGAMP production in HSV-infectedcells. Instead, it localizes partially in the nucleus, interacts withIFI16, and stabilizes IFI16 against proteasomal degradation. There-fore, cGAS may have different activities in different cell types andunder different conditions.

Role of IFI16 and cGAS in sensing DNA of other viruses

Similar to the situation with HSV, both cGAS and IFI16 have beenreported to be required for IRF-3 signaling in HIV-infected cells.Detection of HIV reverse transcriptase products required cGAS forIRF-3 dimerization (Gao et al., 2013). In another study, IFI16 wasfound to sense HIV DNA and to restrict HIV-1 gene expression andreplication (Jakobsen et al., 2013). Induction of pyroptosis in quies-cent CD4þ T cells requires IFI16 (Monroe et al., 2014). Therefore,there is evidence that cGAS and IFI16 are both involved in innateresponses to and epigenetic silencing of HIV infection, and furtherstudies are needed to define the precise role of each in the variouscell types that HIV infects.

Normal roles of IFI16 in human cells

IFI16 has been implicated in innate responses to and restric-tion of microbial DNA, but what physiological roles does IFI16 playin cells not exposed to microbes? IFI16 has also been implicated incell senescence and cell growth control. IFI16 levels increase inaging cells (Xin et al., 2004; Angelova and Knipe, unpublished),and this has been implicated in the aging process. Furthermore,IFI16 is often not expressed or defective in cancer cells (Li et al.,2012; Orzalli et al., 2013, 2012; Xin et al., 2003). When introducedinto tumor cells, IFI16 inhibits cell growth (Xin et al., 2003; Tarnita,Orzalli and Knipe, unpublished results), consistent with a role incontrolling normal cell growth. It will be interesting to see howthe functional mechanisms of these IFI16 activities compare withthe IFI16 mechanisms that function during innate sensing andepigenetic silencing of foreign DNA and to see whether they arerelated or distinct functions of IFI16.

Conclusion

Host cell mechanisms attempt to silence HSV DNA throughepigenetic mechanisms, but HSV and other viruses fight back. Inepithelial cells the HSV VP16 virion protein promotes euchromatinand reduced histone loading on viral immediate-early genes toallow their transcription, and the immediate-early ICP0 proteinthen promotes euchromatin and reduced histone loading on viralearly and late genes to along their expression. In neurons, LATpromotes epigenetic silencing of viral lytic genes. As a result, viralgene products drive the epigenetic landscape on the viral genomeduring both lytic and latent infection. However, little has beenknown about the host receptor that “senses” the foreign viral DNAand promotes heterochromatin assembly on the viral genome. Thenuclear IFI16 protein is required for the major part of the IRF-3signaling in response to HSV infection in the absence of viralprotein expression, identifying it as a potential sensor of HSV DNA.A major remaining question is how innate signaling pathways

D.M. Knipe / Virology 479-480 (2015) 153–159 157

connect the nuclear IFI16 with the cytoplasmic STING. IFI16 alsopromotes the attachment of heterochromatic histone H3 K9 tri-methylation marks to viral chromatin and the resulting epigeneticsilencing of the viral lytic genes. A second major question is themechanism(s) by which IFI16 recruits the histone modificationenzymes to the viral chromatin for this process. It is interesting tonote that while IFI16 is hypothesized to bind to unchromatinizedviral DNA to initiate innate signaling, it promotes the chromatini-zation of the viral DNA, so the innate signaling would be down-regulated. Perhaps this provides a mechanism for controlling thelevels of innate responses, and an extrapolation of this is thatgenetic defects in the epigenetic silencing mechanisms could leadto uncontrolled inflammation. In addition to IFI16, cGAS has alsobeen shown to play a role in sensing of HSV DNA, and it has beenpuzzling how the cytoplasmic cGAS and the nuclear IFI16 couldboth be required for sensing of HSV DNA. Recent results haveshown that in human fibroblasts, cGAS is partially localized to thenucleus, interacts with IFI16, and stabilizes the IFI16 protein. Athird major question is why cGAS shows low levels of enzymaticactivity in HSV-infected cells and how viral gene products mightinhibit its activity. In conclusion, sensing of nuclear HSV DNA playsan important role in the host innate response to viral infection andin epigenetic regulation of viral gene expression, and there aremany exciting avenues of research that remain to be explored tofully understand this critical area of interaction of herpesviruseswith the host cell.

Acknowledgments

I thank Priya Raja and Megan Orzalli for preparation of figures,Megan Orzalli and Nicole Broekema for comments on the manuscript,and Patrick T. Waters for assistance in preparation of the manuscript.Research in the laboratory of the author cited in this review wassupported by NIH grants AI106934 and AI098681 from NIAID, andDE023909 from the National Institute of Dental and Cranial Research.

References

Ablasser, A., Bauernfeind, F., Hartmann, G., Latz, E., Fitzgerald, K.A., Hornung, V.,2009. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNApolymerase III-transcribed RNA intermediate. Nat. Immunol. 10, 1065–1072.

Ablasser, A., Schmid-Burgk, J.L., Hemmerling, I., Horvath, G.L., Schmidt, T., Latz, E.,Hornung, V., 2013. Cell intrinsic immunity spreads to bystander cells via theintercellular transfer of cGAMP. Nature 503, 530–534.

Boutell, C., Everett, R.D., 2013. Regulation of alphaherpesvirus infections by the ICP0family of proteins. J. Gen. Virol. 94, 465–481.

Cereghini, S., Yaniv, M., 1984. Assembly of transfected DNA into chromatin:structural changes in the origin-promoter-enhancer region upon replication.EMBO J. 3, 1243–1253.

Chen, S.H., Kramer, M.F., Schaffer, P.A., Coen, D.M., 1997. A viral function repressesaccumulation of transcripts from productive-cycle genes in mouse ganglialatently infected with herpes simplex virus. J. Virol. 71, 5878–5884.

Chiu, Y.H., Macmillan, J.B., Chen, Z.J., 2009. RNA polymerase III detects cytosolicDNA and induces type I interferons through the RIG-I pathway. Cell Biochem.Biophys. 138, 576–591.

Choubey, D., Deka, R., Ho, S.M., 2008. Interferon-inducible IFI16 protein in humancancers and autoimmune diseases. Front. Biosci. 13, 598–608.

Cliffe, A.R., Coen, D.M., Knipe, D.M., 2013. Kinetics of facultative heterochromatinand polycomb group protein association with the herpes simplex viral genomeduring establishment of latent infection. MBio, 4.

Cliffe, A.R., Garber, D.A., Knipe, D.M., 2009. Transcription of the herpes simplexvirus latency-associated transcript promotes the formation of facultativeheterochromatin on lytic promoters. J. Virol. 83, 8182–8190.

Cliffe, A.R., Knipe, D.M., 2008. Herpes simplex virus ICP0 promotes both histoneremoval and acetylation on viral DNA during lytic infection. J. Virol. 82,12030–12038.

Conrady, C.D., Zheng, M., Fitzgerald, K.A., Liu, C., Carr, D.J., 2012. Resistance to HSV-1infection in the epithelium resides with the novel innate sensor, IFI-16. MucosalImmunol. 5, 173–183.

Cuchet-Lourenco, D., Anderson, G., Sloan, E., Orr, A., Everett, R.D., 2013. The viralubiquitin ligase ICP0 is neither sufficient nor necessary for degradation of thecellular DNA sensor IFI16 during herpes simplex virus 1 infection. J. Virol. 87,13422–13432.

Dempsey, A., Bowie, A.G., 2015. Innate immune recognition of DNA: a recenthistory. Virology, in press.

Eidson, K.M., Hobbs, W.E., Manning, B.J., Carlson, P., DeLuca, N.A., 2002. Expressionof herpes simplex virus ICP0 inhibits the induction of interferon-stimulatedgenes by viral Infection. J. Virol. 76, 2180–2191.

Fernandes-Alnemri, T., Yu, J.W., Datta, P., Wu, J., Alnemri, E.S., 2009. AIM2 activatesthe inflammasome and cell death in response to cytoplasmic DNA. Nature 458,509–513.

Gao, D., Wu, J., Wu, Y.T., Du, F., Aroh, C., Yan, N., Sun, L., Chen, Z.J., 2013. Cyclic GMP–AMP synthase is an innate immune sensor of HIV and other retroviruses.Science 341, 903–906.

Garber, D.A., Schaffer, P.A., Knipe, D.M., 1997. A LAT-associated function reducesproductive-cycle gene expression during acute infection of murine sensoryneurons with herpes simplex virus type 1. J. Virol. 71, 5885–5893.

Gardner, K.E., Allis, C.D., Strahl, B.D., 2011. Operating on chromatin, a colorfullanguage where context matters. J. Mol. Biol. 409, 36–46.

Gariano, G.R., Dell'Oste, V., Bronzini, M., Gatti, D., Luganini, A., De Andrea, M.,Gribaudo, G., Gariglio, M., Landolfo, S., 2012. The intracellular DNA sensor IFI16gene acts as restriction factor for human cytomegalovirus replication. PLoSPathog. 8, e1002498.

Gu, H., Liang, Y., Mandel, G., Roizman, B., 2005. Components of the REST/CoREST/histone deacetylase repressor complex are disrupted, modified, and translo-cated in HSV-1-infected cells. Proc. Natl. Acad. Sci. USA 102, 7571–7576.

Herrera, F.J., Triezenberg, S.J., 2004. VP16-dependent association of chromatin-modifying coactivators and underrepresentation of histones at immediate-earlygene promoters during herpes simplex virus infection. J. Virol. 78, 9689–9696.

Hill, J.M., Quenelle, D.C., Cardin, R.D., Vogel, J.L., Clement, C., Bravo, F.J., Foster, T.P.,Bosch-Marce, M., Raja, P., Lee, J.S., Bernstein, D.I., Krause, P.R., Knipe, D.M.,Kristie, T.M., 2014. Inhibition of LSD1 reduces herpesvirus infection, shedding,and recurrence by promoting epigenetic suppression of viral genomes. Sci.Transl. Med. 6, 265ra169.

Horan, K.A., Hansen, K., Jakobsen, M.R., Holm, C.K., Soby, S., Unterholzner, L., Thompson,M., West, J.A., Iversen, M.B., Rasmussen, S.B., Ellermann-Eriksen, S., Kurt-Jones, E.,Landolfo, S., Damania, B., Melchjorsen, J., Bowie, A.G., Fitzgerald, K.A., Paludan, S.R.,2013. Proteasomal degradation of herpes simplex virus capsids in macrophagesreleases DNA to the cytosol for recognition by DNA sensors. J. Immunol. 190,2311–2319.

Hornung, V., Ablasser, A., Charrel-Dennis, M., Bauernfeind, F., Horvath, G., Caffrey, D.R.,Latz, E., Fitzgerald, K.A., 2009. AIM2 recognizes cytosolic dsDNA and forms acaspase-1-activating inflammasome with ASC. Nature 458, 514–518.

Ishii, K.J., Akira, S., 2006. Innate immune recognition of, and regulation by, DNA.Trends Immunol. 27, 525–532.

Iwasaki, A., Medzhitov, R., 2013. Innate responses to viral infections. In: Knipe, D.M.,Howley, P.M. (Eds.), “Fields Virology”, 6th ed. Lippincott Williams & Wilkins,Philadelphia, pp. 189–213.

Ishikawa, H., Ma, Z., Barber, G.N., 2009. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792.

Jakobsen, M.R., Bak, R.O., Andersen, A., Berg, R.K., Jensen, S.B., Tengchuan, J., Laustsen, A.,Hansen, K., Ostergaard, L., Fitzgerald, K.A., Xiao, T.S., Mikkelsen, J.G., Mogensen, T.H.,Paludan, S.R., 2013. IFI16 senses DNA forms of the lentiviral replication cycle andcontrols HIV-1 replication. Proc. Natl. Acad. Sci. USA 110, E4571–E4580.

Jin, T., Perry, A., Jiang, J., Smith, P., Curry, J.A., Unterholzner, L., Jiang, Z., Horvath, G.,Rathinam, V.A., Johnstone, R.W., Hornung, V., Latz, E., Bowie, A.G., Fitzgerald, K.A.,Xiao, T.S., 2012. Structures of the HIN domain:DNA complexes reveal ligand bindingand activation mechanisms of the AIM2 inflammasome and IFI16 receptor.Immunity 36, 561–571.

Johnson, K.E., Bottero, V., Flaherty, S., Dutta, S., Singh, V.V., Chandran, B., 2014. IFI16restricts HSV-1 replication by accumulating on the HSV-1 genome, repressingHSV-1 gene expression, and directly or indirectly modulating histone mod-ifications. PLoS Pathog. 10, e1004503.

Johnson, K.E., Chikoti, L., Chandran, B., 2013. Herpes simplex virus 1 infectioninduces activation and subsequent inhibition of the IFI16 and NLRP3 inflam-masomes. J. Virol. 87, 5005–5018.

Kagan, J.C., Magupalli, V.G., Wu, H., 2014. SMOCs: supramolecular organizingcentres that control innate immunity. Nat. Rev. Immunol. 14, 821–826.

Kalamvoki, M., Roizman, B., 2010. Circadian CLOCK histone acetyl transferaselocalizes at ND10 nuclear bodies and enables herpes simplex virus geneexpression. Proc. Natl. Acad. Sci. USA 107, 17721–17726.

Kennedy, E.M., Cullen, B.R., 2015. Bacterial CRISPR/Cas DNA endonucleases: Arevolutionary technology that could dramatically impact viral research andtreatment. Virology, in press.

Kerur, N., Veettil, M.V., Sharma-Walia, N., Bottero, V., Sadagopan, S., Otageri, P.,Chandran, B., 2011. IFI16 acts as a nuclear pathogen sensor to induce theinflammasome in response to Kaposi Sarcoma-associated herpesvirus infection.Cell Host Microbe 9, 363–375.

Knipe, D.M., Cliffe, A., 2008. Chromatin control of herpes simplex virus lytic andlatent infection. Nat. Rev. Microbiol. 6, 211–221.

Knipe, D.M., Lieberman, P.M., Jung, J.U., McBride, A.A., Morris, K.V., Ott, M., Margolis, D.,Nieto, A., Nevels, M., Parks, R.J., Kristie, T.M., 2013. Snapshots: chromatin control ofviral infection. Virology 435, 141–156.

Kramer, M.F., Jurak, I., Pesola, J.M., Boissel, S., Knipe, D.M., Coen, D.M., 2011. Herpessimplex virus 1 microRNAs expressed abundantly during latent infection arenot essential for latency in mouse trigeminal ganglia. Virology 417, 239–247.

Kristie, T.M., 2015. Dynamic modulation of HSV chromatin drives initiation ofinfection and provides targets for epigenetic therapies. Virology, in press,available online 18 February 2015.

D.M. Knipe / Virology 479-480 (2015) 153–159158

Kristie, T.M., Vogel, J.L., Sears, A.E., 1999. Nuclear localization of the C1 factor (hostcell factor) in sensory neurons correlates with reactivation of herpes simplexvirus from latency. Proc. Natl. Acad. Sci. USA 96, 1229–1233.

Kubat, N.J., Amelio, A.L., Giordani, N.V., Bloom, D.C., 2004a. The herpes simplexvirus type 1 latency-associated transcript (LAT) enhancer/rcr is hyperacetylatedduring latency independently of LAT transcription. J. Virol. 78, 12508–12518.

Kubat, N.J., Tran, R.K., McAnany, P., Bloom, D.C., 2004b. Specific histone tailmodification and not DNA methylation is a determinant of herpes simplexvirus type 1 latent gene expression. J Virol 78, 1139–1149.

Kwiatkowski, D.L., Thompson, H.W., Bloom, D.C., 2009. The polycomb group proteinBmi1 binds to the herpes simplex virus 1 latent genome and maintainsrepressive histone marks during latency. J. Virol. 83, 8173–8181.

Li, T., Chen, J., Cristea, I.M., 2013. Human cytomegalovirus tegument protein pUL83inhibits IFI16-mediated DNA sensing for immune evasion. Cell Host Microbe 14,591–599.

Li, T., Diner, B.A., Chen, J., Cristea, I.M., 2012. Acetylation modulates cellulardistribution and DNA sensing ability of interferon-inducible protein IFI16. Proc.Natl. Acad. Sci. USA 109, 10558–10563.

Li, X.D., Wu, J., Gao, D., Wang, H., Sun, L., Chen, Z.J., 2013. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341,1390–1394.

Liang, Y., Vogel, J.L., Narayanan, A., Peng, H., Kristie, T.M., 2009. Inhibition of thehistone demethylase LSD1 blocks alpha-herpesvirus lytic replication andreactivation from latency. Nat. Med. 15, 1312–1317.

Maul, G.G., 1998. Nuclear domain 10, the site of DNA virus transcription andreplication. BioEssays 20, 660–667.

Maul, G.G., Ishov, A.M., Everett, R.D., 1996. Nuclear Domain 10 as preexistingpotential replication start sites of herpes simplex virus type 1. Virology 217,67–75.

Melchjorsen, J., Rintahaka, J., Soby, S., Horan, K.A., Poltajainen, A., Ostergaard, L.,Paludan, S.R., Matikainen, S., 2010. Early innate recognition of herpes simplexvirus in human primary macrophages is mediated via the MDA5/MAVS-dependent and MDA5/MAVS/RNA polymerase III-independent pathways. J.Virol. 84, 11350–11358.

Monroe, K.M., Yang, Z., Johnson, J.R., Geng, X., Doitsh, G., Krogan, N.J., Greene, W.C.,2014. IFI16 DNA sensor is required for death of lymphoid CD4 T cells abortivelyinfected with HIV. Science 343, 428–432.

Morrone, S.R., Wang, T., Constantoulakis, L.M., Hooy, R.M., Delannoy, M.J., Sohn, J.,2014. Cooperative assembly of IFI16 filaments on dsDNA provides insights intohost defense strategy. Proc. Natl. Acad. Sci. USA 111, E62–E71.

Mossman, K.L., Macgregor, P.F., Rozmus, J.J., Goryachev, A.B., Edwards, A.M., Smiley, J.R.,2001. Herpes simplex virus triggers and then disarms a host antiviral response. J.Virol. 75, 750–758.

Nicoll, M.P., Proenca, J.T., Connor, V., Efstathiou, S., 2012. Influence of herpessimplex virus 1 latency-associated transcripts on the establishment andmaintenance of latency in the ROSA26R reporter mouse model. J. Virol. 86,8848–8858.

Oh, J., Fraser, N.W., 2008. Temporal association of the herpes simplex virus genomewith histone proteins during a lytic infection. J. Virol. 82, 3530–3537.

Orzalli, M.H., Broekema, N.M., Diner, B.A., Hancks, D.C., Elde, N.C., Cristea, I.M.,Knipe, D.M., 2015. cGAS-mediated stabilization of IFI16 promotes innatesignaling during herpes simplex virus infection. Proc. Natl. Acad. Sci. USA, inpress.

Orzalli, M.H., Conwell, S.E., Berrios, C., Decaprio, J.A., Knipe, D.M., 2013. Nuclearinterferon-inducible protein 16 promotes silencing of herpesviral and trans-fected DNA. Proc. Natl. Acad. Sci. USA.

Orzalli, M.H., DeLuca, N.A., Knipe, D.M., 2012. Nuclear IFI16 induction of IRF-3signaling during herpesviral infection and degradation of IFI16 by the viral ICP0protein. Proc. Natl. Acad. Sci. USA 109, E3008–E3017.

Orzalli, M.H., Knipe, D.M., 2014. Cellular sensing of viral DNA and viral evasionmechanisms. Annu. Rev. Microbiol. 68, 477–492.

Paludan, S.R., Bowie, A.G., 2013. Immune sensing of DNA. Immunity 38, 870–880.Preston, C.M., Harman, A.N., Nicholl, M.J., 2001. Activation of interferon response

factor-3 in human cells infected with herpes simplex virus type 1 or humancytomegalovirus. J. Virol. 75, 8909–8916.

Rathinam, V.A., Jiang, Z., Waggoner, S.N., Sharma, S., Cole, L.E., Waggoner, L., Vanaja, S.K.,Monks, B.G., Ganesan, S., Latz, E., Hornung, V., Vogel, S.N., Szomolanyi-Tsuda, E.,

Fitzgerald, K.A., 2010. The AIM2 inflammasome is essential for host defense againstcytosolic bacteria and DNA viruses. Nat. Immunol. 11, 395–402.

Roizman, B., Knipe, D.M., Whitley, R.J., 2013. Herpes simplex viruses. In: Knipe, D.M.,Howley, P.M. (Eds.), “Fields Virology”, 6th ed. Lippincott Williams & Wilkins,Philadelphia, pp. 1823–1897.

Roizman, B., Zhou, G., 2015. The 3 facets of regulation of herpes simplex virus geneexpression: a critical inquiry. Virology, in press.

Saitoh, T., Fujita, N., Hayashi, T., Takahara, K., Satoh, T., Lee, H., Matsunaga, K.,Kageyama, S., Omori, H., Noda, T., Yamamoto, N., Kawai, T., Ishii, K., Takeuchi, O.,Yoshimori, T., Akira, S., 2009. Atg9a controls dsDNA-driven dynamic transloca-tion of STING and the innate immune response. Proc. Natl. Acad. Sci. USA 106,20842–20846.

Sander, J.D., Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating andtargeting genomes. Nat. Biotechnol. 32, 347–355.

Schoggins, J.W., MacDuff, D.A., Imanaka, N., Gainey, M.D., Shrestha, B., Eitson, J.L., Mar, K.B.,Richardson, R.B., Ratushny, A.V., Litvak, V., Dabelic, R., Manicassamy, B., Aitchison, J.D.,Aderem, A., Elliott, R.M., Garcia-Sastre, A., Racaniello, V., Snijder, E.J., Yokoyama, W.M.,Diamond, M.S., Virgin, H.W., Rice, C.M., 2014. Pan-viral specificity of IFN-inducedgenes reveals new roles for cGAS in innate immunity. Nature 505, 691–695.

Shirakawa, K., Chavez, L., Hakre, S., Calvanese, V., Verdin, E., 2013. Reactivation oflatent HIV by histone deacetylase inhibitors. Trends Microbiol. 21, 277–285.

Stark, G.R., Darnell Jr., J.E., 2012. The JAK-STAT pathway at twenty. Immunity 36,503–514.

Sun, L., Wu, J., Du, F., Chen, X., Chen, Z.J., 2013. Cyclic GMP-AMP synthase is acytosolic DNA sensor that activates the type I interferon pathway. Science 339,786–791.

Takaoka, A., Wang, Z., Choi, M.K., Yanai, H., Negishi, H., Ban, T., Lu, Y., Miyagishi, M.,Kodama, T., Honda, K., Ohba, Y., Taniguchi, T., 2007. DAI (DLM-1/ZBP1) is acytosolic DNA sensor and an activator of innate immune response. Nature 448,501–505.

Tanaka, Y., Chen, Z.J., 2012. STING specifies IRF3 phosphorylation by TBK1 in thecytosolic DNA signaling pathway. Sci. Signal. 5, ra20.

Thompson, M.R., Sharma, S., Atianand, M., Jensen, S.B., Carpenter, S., Knipe, D.M.,Fitzgerald, K.A., Kurt-Jones, E.A., 2014. Interferon gamma-inducible protein (IFI)16 transcriptionally regulates type i interferons and other interferon-stimulatedgenes and controls the interferon response to both DNA and RNA viruses. J. Biol.Chem. 289, 23568–23581.

Thompson, R.L., Sawtell, N.M., 2001. Herpes simplex virus type 1 latency-associatedtranscript gene promotes neuronal survival. J. Virol. 75, 6660–6675.

Umbach, J.L., Kramer, M.F., Jurak, I., Karnowski, H.W., Coen, D.M., Cullen, B.R., 2008.MicroRNAs expressed by herpes simplex virus 1 during latent infectionregulate viral mRNAs. Nature 454, 780–783.

Unterholzner, L., Keating, S.E., Baran, M., Horan, K.A., Jensen, S.B., Sharma, S., Sirois, C.M.,Jin, T., Latz, E., Xiao, T.S., Fitzgerald, K.A., Paludan, S.R., Bowie, A.G., 2010. IFI16 is aninnate immune sensor for intracellular DNA. Nat. Immunol. 11, 997–1004.

Wang, Q.-Y., Zhou, C., Johnson, K.E., Colgrove, R.C., Coen, D.M., Knipe, D.M., 2005.Herpesviral latency-associated transcript gene promotes assembly of hetero-chromatin on viral lytic-gene promoters in latent infection. Proc. Natl. Acad. Sci.USA 102, 16055–16059.

Wathelet, M.G., Berr, P.M., Huez, G.A., 1992. Regulation of gene expression bycytokines and virus in human cells lacking the type-I interferon locus. Eur.J. Biochem. 206, 901–910.

Wu, J., Sun, L., Chen, X., Du, F., Shi, H., Chen, C., Chen, Z.J., 2013. Cyclic GMP-AMP isan endogenous second messenger in innate immune signaling by cytosolicDNA. Science 339, 826–830.

Xin, H., Curry, J., Johnstone, R.W., Nickoloff, B.J., Choubey, D., 2003. Role of IFI 16, amember of the interferon-inducible p200-protein family, in prostate epithelialcellular senescence. Oncogene 22, 4831–4840.

Xin, H., Pereira-Smith, O.M., Choubey, D., 2004. Role of IFI 16 in cellular senescenceof human fibroblasts. Oncogene 23, 6209–6217.

Youell, J., Firman, K., 2012. Mechanistic insight into Type I restriction endonu-cleases. Front. Biosci. 17, 2122–2139.

Zwaagstra, J.C., Ghiasi, H., Slanina, S.M., Nesburn, A.B., Wheatley, S.C., Lillycrop, K.,Wood, J., Latchman, D.S., Patel, K., Wechsler, S.L., 1990. Activity of herpessimplex virus type 1 latency-associated transcript (LAT) promoter in neuron-derived cells: evidence for neuron specificity and for a large LAT transcript.J. Virol. 64, 5019–5028.

D.M. Knipe / Virology 479-480 (2015) 153–159 159