Herpes simplex virus (HSV) is a very common human pathogenic virus. Clinically, HSV infection may give rise to gingivostomatitis, cold sore, keratitis, encephalitis, and genital herpes [1]. The diseases are normally self-limiting but in immunocompromised individuals, such as newborns, transplantation patients, and AIDS-patients, the virus may cause devastating dissimilated infections and encephalitis [1].

Type I interferons (IFN)s, also known as IFN-α/β, and to a lesser extent type III IFNs (IFN-λ1-3) are important for antiviral response against HSV [2-5]. In addition to their direct antiviral effect, IFNs together with proinflammatory cytokines are important signalling molecules for activation and attraction of leukocytes to the site of infection.

Pathogens are a highly diverse group of microbes and viruses. Their detection is therefore a major challenge to the immune system. Host defense against infections is initiated by the innate immune system, operating on the basis of general pathogen features via pattern recognition receptors (PRRs). PRRs detect pathogen-associated molecular patterns (PAMPs) and signal the presence of infection to the host, activating host defense, including antiviral (IFN-α/β) and pro-inflammatory cytokine (interleukin-1β (IL-1β), IL-6, IL-18 and tumor necrosis factor α (TNF-α)) production. In addition, PRRs often initiate programmed cell death (apoptosis) of infected cells.

Toll-like receptors (TLRs) are the best characterized family of PRRs that recognize PAMPs in mammals. There are ten characterized TLRs in humans and they are a class of membrane receptors that sense microbes either in the extracellular space or in intracellular endolysosomal compartments. The second important group of PRRs that have an important role in recognition of HSV infection are retinoid acid inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated gene-5 (MDA-5). They are focused on detecting viral genomic RNA or its replication intermediates in the cytoplasm [6]. Finally a third pathway is presumed to recognize DNA in the cytoplasm, potentially also genomic DNA from HSV. One suggested DNA receptor is DNA-dependent activator of IFN Regulatory Factors (DAI) that is able to activate type I IFN production in response to cytosolic DNA [7]. However, the role of DAI in the in vivo recognition of cytoplasmic DNA has remained controversial. Recently, cytosolic DNA-dependent RNA polymerase III has been shown to recognize cytosolic DNA and linked to recognition of HSV and the production of IFN-β [8,9]. Further studies are required to reveal the specific role of DAI, RNA polymerase III and other putative other DNA receptors in activation of innate immune response during virus infection in humans.

To mount an efficient antiviral immune response the cell must recognize the virus and activate a number of signalling pathways, including Nuclear Factor kappa B (NF-κB), IFN Regulatory Factors (IRFs) and Mitogen-Activated Protein Kinase (MAPK) pathways. As summarized in Figure 1, the recognition of HSV includes (i) recognition of viral surface glycoprotein via either TLR2 or and yet unidentified receptor [10-12], (ii) recognition of HSV viral DNA by TLR9 in endosomes, or in the cytosol via either RNA polymerase III or potentially DAI [7,9,13-16], and (iii) the recognition of virally-derived double-stranded (ds)RNA recognized by RLRs [16]. Finally, TLR3 plays a role in restricting HSV infection evidenced by a recent study showing that humans that bear mutations in TLR3 are predisposed to HSV-associated encephalitis [17].

To gain time, and potentially also to allow chronic infection, HSV has evolved numerous strategies to evade IFN signalling at different stages, including directly affecting JAK/STAT signalling and interactions with the IFN-induced antiviral proteins Protein Kinase R (PKR) and the 2’-5’-Oligoadenylate Synthethase (2’-5’-OAS)/RNaseL system. Furthermore, HSV has means of escaping cytokine and IFN production via inhibition of the transcription factors NF-κB and IRF3 and IRF7 signalling pathways, reduction of cytokine mRNA stability and interfering with translation.

This review presents the current knowledge on antiviral escape mechanisms employed by HSV.

HSV-1 and HSV-2 are closely related, enveloped, nuclear replicating, double-stranded (ds)DNA viruses belonging to the subgroup of alphaherpesvirinae. The two subtypes share 83% homology in the protein coding regions of the genome [18], and exhibit many similar biological functions [1]. Because of similarities between the two subtypes, no distinction between HSV-1 and HSV-2 will be made in the following sections. The reader is encouraged to consult the primary literature for data on the individual HSV subtypes.

The transcription of viral genes is tightly regulated and has two key features. First, herpes virus proteins fall into groups, whose synthesis is coordinately regulated, i.e., all genes within a given group are expressed sequentially starting with the immediate early (IE) gene group, followed by the early (E) gene group, and finally the late (L) group (Figure 2). The sequential expression occurs because IE proteins are required for expression of the E proteins, and E proteins generally are essential for viral genome replication, which is a prerequisite for the expression of L proteins [1]. The tegument protein VP16 associates with cellular transcription factors to promote the transcription of the five IE mRNAs encoding infected cell protein 0 (ICP0), ICP4, ICP22, ICP27, and ICP47. The IE genes are expressed in the presence of inhibitors of protein synthesis. The four IE proteins, ICP0, 4, 22, and ICP27, are transcriptional and translational regulators of the E and L genes, as well as host genes, whereas ICP47 interferes with the transporter associated with antigen presentation (TAP)1/TAP2 complex and hence antigen presentation through major histocompatibility complex class I (MHC I) [19]. Later sections will further address the evasion strategies employed by HSV. Besides being a positive regulator of IE and L genes, ICP4 also inhibits expression of the IE genes. ICP4´s negative regulation of IE genes provides a halt of IE gene expression during later stages of viral infection. Accumulation of ICP4 and ICP27 initiates the expression of E genes, which primarily encode proteins involved in DNA replication. E gene products include scavenger enzymes, such as the viral thymidine kinase and ribonucleotide reductase, as well as enzymes directly involved in DNA replication, such as the viral DNA polymerase and DNA helicase. Finally, the L genes are made after DNA replication has occurred. These include structural proteins required for the progeny viral particles, as well as VP16 and vhs, both of which are incorporated into the viral tegument. The capsid is assembled in the nucleus and takes up individual DNA genomes once the capsid is fully assembled [20]. Subsequently, the capsids associate with the tegument proteins and bud through the viral glycoprotein-containing nuclear membrane. The virions are hereafter incorporated into vesicles and released by exocytosis. Alternatively, the virus is released by cell lysis or spread by syncytia formation [1].

The first response in a HSV-infected cell is an inflammatory reaction that includes secretion of antiviral substances, such as defensins and nitric oxid and most importantly the production of cytokines, including IFNs and chemokines. The aim of the initial innate immune response is to limit spread of the infection and, if possible, to eliminate the pathogen. The secreted substances activate and attract immune cells and thus help to organize an effective antiviral response.

The type I IFNs IFN-α/β are key cytokines produced within the very first hours after HSV-infection [15,21]. IFN-α/β are important in direct control and inhibition of HSV replication [2,22,23], and this occurs in synergy with IFN-γ [24]. In vivo studies show that IFN-α/β alone can control early HSV infection independently of natural killer (NK) cell and lymphocyte activity [25]. Experiments in mice also demonstrate the importance of the IFN system in the antiviral defense against HSV infection because uncontrolled virus replication is seen in mice genetically lacking functional IFN-α/β-receptors or when mice are administered neutralizing antibodies against IFN-α/β [3,26]. The studies in mice have been supported by the recording that patients lacking functional STAT1, an essential components of the IFN pathways, are more prone to HSV encephalitis [4]. Furthermore, IFN-α/β is part of a positive feedback loop that amplifies the cytokine response during HSV infection [27]. Though studies have established an important role for IFN-α/β in antiviral defense, it is difficult to determine whether IFN-α/β´s contribution to viral clearance is primarily mediated by direct antiviral mechanisms or by a indirect immunoregulatory mechanisms.

IFN exerts its activities through induction of hundreds of IFN-stimulated genes (ISGs) [28]. ISGs especially important in antiviral defense are the PKR and the 2’-5’-OAS/RNAse L system (alternatively referred to as the 2’-5’A system). PKR is an important mediator of resistance against HSV because the absence of PKR in murine cells enhances HSV replication [29]. In addition, the HSV-induced expression of IFN-α/β and the proinflammatory chemokine RANTES is dependent on functional PKR [15,30]. The 2’-5’A system seems to play an important role after eye infections, since mice lacking RNAseL and thus the 2’-5’A system have higher mortality rates and display more severe disease progression [31]. In addition, studies in epithelial cells have revealed that IFN-α and especially IFN-β suppress HSV-1 replication through an RNAseL-dependent pathway [32]. It has also been shown that 2’-5’-OAS is a potent inhibitor of HSV replication in BHK cells [33]. As will be discussed later, the activity of PKR and the 2’-5’ A system is counteracted by HSV emphasizing the importance of PKR and the 2’-5’ A system in the innate response against HSV. Additional important ISGs produced during virus infections are chemokines, including IFN-γ-inducible protein of 10 kDA (IP-10, CXCL10), the transcription factor IRF7, and several cellular pattern recognition receptors (PRRs), such as the TLRs and the RLRs [34-39].

Type III IFNs IL-29 (IFN-λ1) and IL-28 (IFN-λ2/3) have been described to possess antiviral effects against HSV [27,40]. IFN-λs activate the same JAK/STAT signalling pathways as type I IFNs, induce resistance to viral infection in several human cell lines, and upregulate expression of ISGs, such as MHC class I antigen, MxA, and 2’-5’OAS [41,42]. IFN-λs are expressed after infection with several RNA viruses and treatment with dsRNA [37,41-43]. Recent data have shown that HSV induces expression of IFN-λs in both human monocyte-derived macrophages and DCs and in murine pDCs and cDCs [5,27]. In addition, both IFN-λ and IFN-α/β are part of a positive feedback loop that greatly enhances the expression of proinflammatory cytokines, chemokines, and IFNs after HSV infection [27]. Type III IFN inhibits HSV IE gene expression with comparable effect to IFN-α/β implying that IFN-λ has direct antiviral effect against HSV [27,40]. Recent studies examining the role of IFN-λ in genital and generalized HSV-2 infection in mice, revealed that IFN-λR knockout mice did not differ from wild-type mice in their ability to clear HSV infection [5]. Nevertheless, it would be interesting to gain insight into the role of IFN-λs during HSV infection in humans, and to establish whether the type III IFNs will have a role in the treatment of HSV infections.

In addition to type I and type III IFN, IFN-γ is a prominent mediator of antiviral immunity against HSV infection [24,44,45]. Studies show that IFN-γ-mediated control of HSV in murine macrophages correlates with production of nitric oxide [44] and that IFN-γ together with IFN-α/β and IL-12 is very important in control of HSV infection [24,25,46]. Furthermore, IFN-γ reduces reactivation of HSV from the neurons [45]. IFN-γ is mainly produced by T and NK cells [47], but also other cells, including macrophages, γδT cells, and DCs, produce IFN-γ after stimulation with IL-12 and IL-18 or after HSV infection [48-52]. Besides its direct antiviral activity, IFN-γ also plays a key role in initiating and amplifying the immune response. IFN-γ, for instance, synergizes with HSV to induce the expression of proinflammatory cytokines, such as TNF-α, IL-6, and IL-12 p40 [53,54]. Moreover, IFN-γ indirectly enhances cellular recognition of the virus and subsequent cytokine production through induced expression of potential virus PRRs, such as TLRs and RLRs [36,39,55].

The outcome of the infection is often determined by the signal transduction pathways activated. The first line of defense against virus infections requires activation of multiple signal transduction pathways, including NF-κB, MAPK pathways, and IRFs [56]. However, viruses are extremely well adapted to their host and part of this adaption is caused by virus manipulation of host signaling. HSV modulates the signaling pathways to facilitate viral gene expression and enhance viral replication, but may also down-regulate specific pathways to evade immune responses and improve survival in the host cell. During early HSV infection, NF-κB, MAPK and IRF pathways are activated [27,57]. The activated signaling pathways may have dual purposes; both activating cytokine expression and viral replication [58]. It is worth noting that the mechanism through which HSV is recognized and stimulates cellular gene expression is cell type dependent [16]. As will be discussed later the cell-dependency also seems to apply for HSV evasion strategies.

HSV does not directly activate JAK/STAT signalling pathways. JAK/STAT pathways are, nevertheless, very important for development of the proinflammatory response after HSV infection. Many HSV-induced cytokines, including IFN-α/β, IFN-γ, IL-6, and IL-12, mediate phosphorylation of JAK proteins and subsequent phosphorylation and activation of STAT proteins [59]. The activated STAT proteins dimerize, translocate to the nucleus, and activate transcription through binding to the gamma activation site or after association with IRF9 (p48) binding to IFN-stimulated response elements [59]. STAT1 is a central player for IFN-α/β and IFN-γ-activated signal transduction and activity evidenced by the fact that mice and humans deficient in STAT1 are very susceptible to HSV infections, as discussed above [60].

Like most viruses, HSV has numerous countermeasures to overcome the host innate defences including several anti-IFN and anti-cytokine weapons and the inhibition of apoptosis. The discussion below will focus on the current knowledge on interaction between HSV and the host response. Figure 3 and Table 1 summarize the strategies employed by HSV to evade the innate immune system.

Although some therapeutic and diagnostic improvements for control of HSV have been developed during the last years, the virus is by no means under control. Therefore research on the pathogenesis of HSV and knowledge of overall virus-cell interactions leading to an efficient immune response are still most needed.

The outcome of HSV infections is dependent on the equilibration between virus propagation and an effective immune response. Appropriate expression of IFNs, cytokines, and chemokines is essential for efficient host defense against infection. IFN and cytokines expression is mediated by interaction between the virus PAMPS with cellular PRRs. For HSV, TLR2, TLR3, TLR9, the MAVS pathways and the RNA polymerase III pathway have been identified as sensing the infection and inducing the production of IFN-α/β and cytokines.

HSV has evolved together with its host and developed mechanisms to overcome the effects of the immune response. The countermeasures are directed at several antiviral host defenses, in particular the cytokine and IFN system but also the complement system and antigen-dependent responses (Table 1, Figure 3). These countermeasures are essential for effective virus propagation and to secure an environment suited for virus replication and establishment of latency. Therefore investigations into the viral immune evasion strategies will hopefully provide additional understanding of the innate and adaptive immune mechanisms and reveal viral components essential for HSV. The new understanding of HSV infection and immunopathology might help in development of improved treatments and in design of vaccines.