TRENDS in Microbiology
Vol.12 No.8 August 2004
| Research Focus
The Toll of herpes simplex virus infection Lynda A. Morrison Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104, USA
Herpes simplex virus (HSV) infections provoke an inflammatory cytokine response, but the innate pathogen-sensing mechanisms that transduce the signal for this response are poorly understood. Recent findings have revealed that Toll-like receptor (TLR) 2 initiates the inflammatory process, and surprisingly that the response the TLR triggers might be overzealous in its attempt to counter the attack by the virus. Other recent findings suggest complexity in the array of TLRs that are triggered by HSV and the cell types they activate. Here we discuss the new revelations about these guardians against HSV infection and the consequences of the alarms raised in the host that they are assigned to protect. Herpes simplex virus (HSV)-1 and HSV-2 are common human pathogens, infecting approximately 90% and 22%, respectively, of individuals in the United States . HSV-1 typically initiates infection upon contact with oral mucosa or corneal epithelium, where replication causes vesicular lesions. Sensory nerve endings innervating the infection site are rapidly invaded and HSV is transported within axons to the peripheral ganglion, where the virus establishes latent infection. Sporadic perturbations of neuronal homeostasis result in virus reactivation and recurrent epithelial disease. In rare cases, HSV will continue to spread centripetally into the central nervous system (CNS) to cause devastating encephalitis. Similar to HSV-1, HSV-2 establishes latency in sensory neurons after replicating in epithelial cells, but has a predilection for the genital mucosa. Primary HSV-2 infection or reactivation in an expectant mother can result in perinatal transmission to her newborn. HSV-2 infections in the newborn are particularly severe and frequently involve the CNS. As with most virus infections, the host mounts an orchestrated series of responses against HSV that result in inflammation and Th1-dominated immunity. A longstanding essential question that remains to be answered is what mechanism is used to initiate the inflammatory response to HSV infection. A recent report by Kurt-Jones et al.  reveals that a Toll-like receptor (TLR) senses HSV and triggers the inflammatory response, and surprisingly, the role it plays in alerting the innate response has pathological rather than protective consequences.
proteins . TLRs are transmembrane proteins that detect redundant molecular patterns in broad classes of microbial pathogens. They alert the host to the presence of a pathogen by initiating intracellular signaling events through recruitment of adaptor proteins, such as MyD88 (common to all TLRs), TIRAP [Toll –interleukin (IL)-1 receptor (TIR)-associated protein] (TLRs 2 and 4) and TICAM (TIR-containing adaptor molecule-1; TLRs 3 and 4), to the intracellular TIR domain. Sequential phosphorylation and activation events result in nuclear translocation of nuclear factor (NF)-kB, which stimulates transcription of antimicrobial/antiviral and pro-inflammatory cytokine genes. Ten human TLRs have now been identified, along with natural or synthetic ligands for nine of these (Table 1). For example, detection of bacteria and fungi can occur through lipopolysaccharide-binding TLR4, and through TLR2, which recognizes a broad range of ligands including peptigoglycan. The signals transmitted by these TLRs in many cases are essential for pathogen clearance and host survival, as demonstrated in many mouse models of infection. Although originally described as receptors for molecular patterns found in bacterial and fungal pathogens, TLR recognition of viral products has more Table 1. The Toll-like receptor (TLR) family and its ligands
Ligands (organism of origin)a
Triacylated lipopeptides (bacteria) Soluble factors (bacteria) Envelope protein (virus) Lipopeptide (virus) Lipoprotein (bacteria) Peptidoglycan (bacteria) Atypical LPS (bacteria) Porin (bacteria) Lipoarabinomannan (mycobacteria) Glycolipids (spirochetes) GPI anchor (parasite) Zymosan (yeast) HSP60, HSP70, HSP90, HSPgp96 (host) Double-stranded RNA (virus) LPS (Gram-negative bacteria) Envelope protein (virus) Taxol (plant) HSP60, HSP70, HSPgp96 (host) Flagellin (bacteria) Diacylated lipopeptides (bacteria) Single-stranded RNA (virus) Single-stranded RNA (virus) (in humans) Unmethylated CpG DNA (virus, bacteria) Chromatin-IgG complexes (host) ??
TLR5 TLR6 TLR7 TLR8 TLR9
TLR signaling of virus infection Pathogen recognition mediated by the host often occurs through a phalanx of ancient sentinels known as TLR Corresponding author: Lynda Morrison ([email protected]
). Available online 2 July 2004
Abbreviations: HSP, heat shock protein; LPS, lipopolysaccharide.
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recently been appreciated. Among these, TLR3 recognizes double-stranded RNA, which is produced by viruses in the course of their replication cycle. Therefore, it was originally expected that TLR3 would be the principal mediator of viral recognition. However, four other TLRs have now been associated with virus recognition. TLR7 interaction with single-stranded RNA of influenza virus or human immunodeficiency virus (HIV) delivers the signal for production of interferon (IFN)-a and inflammatory cytokines [4,5]. Once thought reserved for bacteria, TLR4 activates the immune response to respiratory syncytial virus (RSV) and retrovirus infections [6– 8]. Finally, TLR2 and TLR9 have been described as recognition molecules for members of the Herpesviridae [2,9– 11]. Viruses once again are revealing complexities in host immune responses, even in a primordial signaling system. TLRs 2 and 9 initiate innate responses to herpesviruses The picture of TLR recognition of viruses that has emerged from initial reports fits neatly into place on the existing mural depicting inflammatory cytokine and chemokine induction in response to virus infection. Viruses stimulate type I IFN responses, tissue-specific chemokines that recruit and activate inflammatory cells, and cytokines that drive lymphocyte activation and Th1 differentiation, all of which are induced by TLR signaling. Herpes virions interact with host cells in several ways that facilitate virus entry and permit the IFN and cytokine intruder alert systems to be activated by the host. A series of herpesvirus glycoproteins mediates attachment to host cells and fusion of the viral and cellular membranes. Infection of fibroblasts with human cytomegalovirus (HCMV) strongly stimulates transcription of the IFN pathway and inflammatory cytokine genes. Inactive virus particles or even glycoprotein B, the HCMV entry mediator, largely mimic the response to intact virus , suggesting that interaction of CMV envelope proteins with a cell surface sensing mechanism transduces the signal for antiviral activation. TLR2 and CD14 have been identified as the sensors of HCMV infection that stimulate IFN and inflammatory cytokine responses . HSV also induces a type I IFN response and stimulates IL-12 production. Similar to HCMV, the HSV entry mediator glycoprotein D alone provides sufficient stimulus . A role was recently discovered for TLR9 in IFN and IL-12 responses to HSV-2 infection ; the ligand for this TLR is unmethylated CpG , an abundant motif in the GC-rich HSV genome . TLR9 signals HSV-2 interaction with bone marrow-derived, CD11cþCD11b2B220þ plasmacytoid dendritic cells (DCs) in vitro (Figure 1). This MyD88-dependent signal results in IFN-a and IL-12 secretion [9,10] and upregulates CD86 costimulation molecules . These data provide a link between TLR9 response to HSV infection and induction of Th1-dominated immune responses. Both live and UV-inactivated HSV are competent to activate the inflammatory response of DCs differentiated in vitro, which are phenotypically equivalent to plasmacytoid DCs in the spleen that produce opious amounts of IFN-a in response to murine CMV and UV-inactivated HSV that have been administered intravenously [16,17]. However, whether TLR9 is the www.sciencedirect.com
Vol.12 No.8 August 2004
predominant inflammatory signal in vivo in response to HSV infection and what the consequences of infection would be in the absence of the TLR signal remain unresolved. TLR2 can mediate a detrimental inflammatory response to HSV Depending on the route of infection in vivo, HSV interacts with a variety of cell types in addition to plasmacytoid DCs. Kurt-Jones et al.  demonstrated that peritoneal macrophages require TLR2 for IL-6 and monocyte chemotactic protein (MCP)-1 secretion in response to HSV-1, and that association of TLR2 with TLR6 is not essential for signal transduction . In addition, exposure of human embryonic kidney cells that express TLR2, 3, 4 or 9 to infection with HSV-1 indicated that TLR2 is sufficient for NF-kB translocation to the nucleus and that the signal strength is proportional to the dose of virus added. Myeloid DCs differentiated in vitro (which express TLR2) can be non-productively infected with HSV-1 and receive signals from the virus that stimulate expression of maturation marker CD83, costimulation molecules CD80 and CD86, and major histocompatiblity complex (MHC) class II molecules [19,20]. Therefore, TLR2 expressed on immune and non-immune cell types recognizes and responds in vitro to a molecular pattern present in the HSV virion (Figure 1). But would TLR2 provide the necessary signals for an inflammatory response to HSV in vivo? Wild-type mice infected intraperitoneally with a high dose of HSV-1 developed a strong inflammatory response, which was characterized by IL-6 in the blood and MCP-1 in the brain and was absent in TLR22/2 mice . However, HSV-1 infection caused significantly less mortality in TLR22/2 mice than in wild-type mice. Reduced mortality was also observed in 4-day-old TLR22/2 pups infected with a lower dose of HSV-1. Despite perivascular cuffing and mononuclear cell infiltrates in the brains of wild-type mice, virus titers were not statistically different than those in TLR22/2 mice. These data suggested that HSV infection detected by TLR2 resulted in an overzealous cytokine response that had lethal consequences for the host. Perhaps the ancient sentinels of the body have not received the benefits of evolutionary refinement. Paradoxical lack of effect on virus replication The observation that lack of TLR signaling had no effect on virus replication was particularly striking. In the paradigmatic view of innate host responses, an acute inflammatory cell influx in infected tissue aids in virus clearance; therefore, TLR signaling-deficient mice should have higher viral titers because they cannot mount an inflammatory response. Inoculation of HSV-1 onto the corneal epithelium of wild-type, TLR92/2 or MyD882/2 mice yielded similar virus titers in eye swabs and trigeminal ganglia , suggesting that a possible TLR9- and MyD88independent inflammatory response held virus replication in check. The iconoclastic observation by Kurt-Jones et al. was that TLR22/2 mice lacked a cytokine response or inflammatory infiltrates in the HSV-infected brain, but they did not have higher virus titers than wild-type mice. The TLR-initiated response of systemically infected
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Vol.12 No.8 August 2004
(b) TLR1 or TLR6
TLR2 TLR9 MyD88
MyD88 IRAK4 P
TRAF6 IRAK4 P
TAK1 TRAF6 TAK1
IL-1 IL-6 IL-8
IL-1 IL-6 TNFα
IFNα/β IL-12 TRENDS in Microbiology
Figure 1. Mechanism of signal transduction upon activation of TLRs by HSV. TLR2 or TLR9 recognition of HSV by different cell types initiates signaling pathways. (a) TLR2 is expressed on the plasma membrane of cells of myeloid lineage. (b) HSV-mediated activation of TLR9 occurs within acidified endosomes of plasmacytoid DCs. MyD88 is an essential adaptor molecule linking TLR engagement to IRAK activation. IRAK4 phosphorylation of IRAK1 liberates TRAF6 to join the complex of TAB1, TAB2 and TAK1. TAK1 activates IKK and MKK complexes. NF-kB translocates to the nucleus upon phosphorylation of IkBa, stimulating inflammatory cytokine production. Selective expression of TLR9 on plasmacytoid DCs might correlate with type I IFN-producing capacity through an unidentified mechanism. Abbreviations: DCs, dendritic cells; HSV, herpes simplex virus; IFN, interferon; IKK, inhibitor of NF-kB; IL, interleukin; IRAK, IL-1 receptor-associated kinase; MAP, mitogen-activated protein; MCP-1, monocyte chemotactic protein 1; MIP-2, macrophage inflammatory protein 2; MKK, MAP kinase kinase; NF-kB, nuclear factor-kB; TAK1, TGFb-activated kinase 1; TGFb, transforming growth factor b; TLRs, Toll-like receptors; TNF, tumor necrosis factor; TRAF, TNF-receptor-associated factor.
wild-type mice contributes to inflammation that leads, in the CNS, to pathological consequences. The experiments of Kurt-Jones et al.  suggest that innate responses of infants to disseminated HSV infection and of adults to encephalitis might be the principal source of their sepsislike symptoms. But is the ‘double-edged sword’ of TLR2 activation in response to virus infection and the inflammatory response provoked necessarily pathologic in its consequences, or could it be protective under other circumstances? Chlamydia-infected TLR22/2 mice also show reduced tumor necrosis factor (TNF)-a and macrophage inflammatory protein (MIP)-2 levels in vaginal secretions and reduced tissue pathology but no alteration in microbial count . However, the TLR2-mediated response to Staphylococcus aureus and the TLR4-mediated response to RSV infection are associated with reduced pathogen loads [6,22]. Future painstaking correlations of virus replication in various tissues with signs of the inflammatory response might reveal whether the paradigm of protective inflammatory responses to virus infection needs to be shifted to accommodate distinct tissue types. www.sciencedirect.com
Concluding remarks The particular TLR transmitting the pathogen invasion signal might be dependent on the means of virus entry into the cell and the properties of the cell engaged by virus. HSV is now known to trigger innate, and ultimately adaptive, cellular immune responses through interaction of virions and/or virion components with TLR2 and TLR9. Although TLR9 binds HSV DNA CpG motifs, and CpG alone can be immunostimulatory , encapsidated DNA much more efficiently stimulates TLR recognition than naked DNA . TLR9 is localized to the endosomal membrane rather than the cell surface, and inhibitors of endosome acidification prevent TLR9 recognition of HSV DNA by plasmacytoid DCs . These observations suggest that the mechanism of TLR9 recognition involves uptake of hematogenously spreading virus by plasmacytoid DCs, which are specialized for antigen uptake, are ideally located for surveillance of the blood, and preferentially express TLR9 but not TLR2 . TLR2 could be distributed on other cell types in solid tissues as a cellsurface viral sensor, suggesting that recognition receptors
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for molecular patterns of pathogens might also be arrayed in patterns throughout the body, with certain TLRs responsible for surveillance of distinct body compartments rather than solely classes of microbes . Future studies on the role of TLR2 in response to HSV infections by other routes and lower doses of virus will provide important clarification of the role of cell- and tissue-type on the outcome of infection, as will a more detailed assessment of nuances in the intracellular signals transmitted by TLR2 – TLR1 and TLR2 – TLR6 heterodimers , and TLR2 complexes containing integrins, which might also facilitate virus infection . Acknowledgements
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I gratefully acknowledge the contributions of Becky Duerst in revision of the manuscript, and the thoughtful comments of Pat Stuart. I also extend an apology to many colleagues whose contributions to the research described here could not be cited owing to space constraints.
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0966-842X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.06.001
EBV-associated nasopharyngeal carcinomas: from epidemiology to virus-targeting strategies Pierre Busson1, Ce´cile Keryer1, Tadamassa Ooka2 and Marilys Corbex3 1
UMR 8126, CNRS and Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France UMR 5537, CNRS and Faculte´ de Me´decine Laennec, rue Guillaume Paradin, 69372 Lyon, France 3 Genetic Epidemiology Unit, International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon, France 2
Nasopharyngeal carcinoma is a human malignancy consistently associated with the Epstein– Barr virus. Corresponding author: Pierre Busson ([email protected]
). Available online 2 July 2004 www.sciencedirect.com
Exposure to non-viral carcinogens and genetic predisposition are other crucial etiologic factors. Tumor development appears to require the expression of a small subset of transforming viral RNAs and proteins with concomitant silencing of most other viral genes.