Immune evasion strategies of molluscum contagiosum virus.

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Immune Evasion Strategies of Molluscum Contagiosum Virus Joanna L. Shisler1 Department of Microbiology, College of Medicine, University of Illinois, Urbana, Illinois, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4.

Introduction Characteristics of the MCV Genome and Insights into MCV Replication MC Lesion Development Characterization of MC Lesions 4.1 Initial stages of infection 4.2 Host cell response to MCV infection 5. Immune Responses to MCV Infection 6. MCV Epidemiology 7. MCV Diagnosis and Treatment 8. Current Roadblocks in Propagating MCV in Tissue Culture Systems 9. MCV Immune Evasion Mechanisms 10. Limitations and Caveats When Studying MCV Immune Evasion Proteins 11. The FLIP Family of Viral and Cellular Proteins 11.1 Introduction to FLIPs 11.2 Signaling events triggered by the TNFR1 11.3 The FLIP family and control of NF-κB activation 11.4 The FLIP Family and Control of IRF3 Activation 12. Other MCV Immune Evasion Molecules 12.1 MCV MC54, an IL-18-binding protein 12.2 MCV MC148, a viral chemokine 12.3 MCV MC007, a pRb-binding protein 12.4 MC66, a glutathione peroxidase homolog 13. Conclusions References

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Abstract Molluscum contagiosum virus (MCV) is the causative agent of molluscum contagiosum (MC), the third most common viral skin infection in children, and one of the five most prevalent skin diseases worldwide. No FDA-approved treatments, vaccines, or commercially available rapid diagnostics for MCV are available. This review discusses several aspects of this medically important virus including: physical properties of MCV, MCV

Advances in Virus Research, Volume 92 ISSN 0065-3527 http://dx.doi.org/10.1016/bs.aivir.2014.11.004

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pathogenesis, MCV replication, and immune responses to MCV infection. Sequencing of the MCV genome revealed novel immune evasion molecules which are highlighted here. Special attention is given to the MCV MC159 and MC160 proteins. These proteins are FLIPs with homologs in gamma herpesviruses and in the cell. They are of great interest because each protein regulates apoptosis, NF-κB, and IRF3. However, the mechanism that each protein uses to impart its effects is different. It is important to elucidate how MCV inhibits immune responses; this knowledge contributes to our understanding of viral pathogenesis and also provides new insights into how the immune system neutralizes virus infections.

1. INTRODUCTION The Poxviridae family is an extensive group of viruses with a broad host range that includes both vertebrates and invertebrates. The best-studied poxviruses belong to the Orthopoxvirus genus and include variola virus (VAR; the causative agent of smallpox) and monkeypox virus (MPX; the causative agent of monkeypox). Vaccinia virus (VACV) is highly similar to VAR and MPX viruses and is used as a vaccine to protect against both infections (Moss, 2013). Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus (Moss, 2013). MCV is a dermatotropic poxvirus that causes benign epithelial neoplasms (molluscum contagiosum, MC) in humans. There are several striking characteristics of MCV that make it unique in comparison to the well-studied members of the Orthopoxvirus genus. First, MCV causes a persistent infection with little to no inflammation. In contrast, MPX and VAR viruses cause acute diseases that have significantly higher morbidity and mortality rates than MC (Moss, 2013). Second, MCV infection remains limited to keratinocytes, while VAR and VAC viruses infect many different cell types and tissues. Third, the immune evasion molecules encoded by MCV are distinct from those encoded by the members of the Orthopoxvirus genus. This difference in immune evasion molecules likely reflects the different tissue tropisms of these viruses and the different types of diseases they cause. MCV is the only poxvirus other than the VAR that is exclusively pathogenic to humans (Moss, 2013). Since VAR has been eradicated due to an extensive vaccination campaign, MCV remains the only circulating poxvirus that solely infects humans worldwide. To understand how MCV evades skin immune responses and why MCV infection is limited to human keratinocytes will greatly benefit the field of virology, likely identifying new mechanisms of

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immune evasion and also giving new insights into virus tropism. This information will also add to our understanding of immunology because studies of viral immune evasion proteins identified new cellular mechanisms that regulate signal transduction events critical for activation of the immune system.

2. CHARACTERISTICS OF THE MCV GENOME AND INSIGHTS INTO MCV REPLICATION This review will discuss some characteristics of the MCV genome that directly relate to the pathogenesis of MCV infections. Poxviruses possess linear, double-stranded DNA genomes ranging from 130 to 300 kb in size, and a dumbbell-shaped core protects the genetic material. A lipoprotein envelope composed of post-Golgi-derived membranes surrounds poxvirus cores. Also included in the virion is a DNA-dependent RNA polymerase which is required for poxvirus replication in the cytoplasm (Moss, 2013). There are four known subtypes of MCV (MCV-1, -2, -3, and -4), identifiable by restriction fragment length polymorphisms of their genomes (Nakamura, Muraki, Yamada, Hatano, & Nii, 1995; Porter & Archard, 1992). MCV-1 is the most prevalent in healthy humans, while MCV-2 is more common in HIV-infected patients. An interesting characteristic of the MCV genome is its high guanidine–cytosine (GC) content (64%) as compared to the well-characterized VACV (34%) (Senkevich, Koonin, Bugert, Darai, & Moss, 1997). MCV infects skin, an organ that receives ultraviolet radiation present in sunlight. Thus, it has been speculated that the high GC content may decrease the potential of UV-induced mutagenesis of the MCV genome. The complete sequence of an MCV-1 genome was published in 1996 (Senkevich et al., 1996). Sequence analysis revealed that MCV encodes approximately 182 predicted proteins. Senkevich et al. compared the MCV genome to genomes from members of the Orthopoxvirus genus, including the highly related VAR and VACV (Senkevich et al., 1997). This comparison was performed because the functions of many VACV genes are known and was expected to facilitate the identification of conserved genes between MCV and VACV (Moss, 2013). Such genes would be predicted to be involved in basic aspects of virus replication. Importantly, VACV has a broad host range, but MCV has yet to be propagated successfully in cultured cells (Damon, 2013). Thus, it was also expected that this comparison would identify genes potentially responsible for these different properties, shedding light on the narrow host tropism of MCV.

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Comparative analyses revealed that 105 of the 182 predicted MCV proteins have homologs in VACV (Senkevich et al., 1997). These homologs are known to be essential for virus structure, replication, and transcription. The MCV and VACV genes with the highest conservation control viral gene transcription (e.g., polymerase subunits and genes encoding early and late transcription factors), implying that the MCV lifecycle is similar to VACV. Like in other poxvirus genomes, these MCV genes are located toward the center (Moss, 2013). MCV lacks homologs to VACV genes that are involved in nucleotide biosynthesis and a kinase (B1) that phosphorylates the cellular protein BAF (Nichols, Wiebe, & Traktman, 2006; Senkevich et al., 1996, 1997). Each of these VACV genes is required for replication in cultured cells. Whether the presence of one or more of these VACV genes would rescue the replication deficiency of MCV in cultured cells remains to be tested. Surprisingly, the ends of the VACV and MCV genomes are strikingly different (Senkevich et al., 1996, 1997). These regions of the poxvirus genome encode immune evasion genes. MCV and VACV did not share any immune evasion genes except for a gene that encodes an IL-18-binding protein. Since MCV infection is limited to keratinocytes, it may express a unique set of immune defense molecules to counteract the immune response threats specific for the skin. MCV is the sole member of the Molluscipoxvirus genus (Damon, 2013). Phylogenetic analysis of five highly conserved poxvirus genes (DNA polymerase, DNA uracil glycosylase, early transcription factor subunit, NTPase 1, and rifampicin-sensitivity factor) show that MCV and members of the Orthopoxvirus and Leporipoxvirus genera evolved from a common ancestor (Senkevich et al., 1997). More recently, the MCV genome was compared to Orf virus, a member of the Parapoxvirus genus. Orf virus causes a skin infection in sheep and can also infect humans. Analysis of the sequence of Orf virus detected several striking similarities to MCV, despite the fact that MCV and Orf are in different genera (Delhon et al., 2004). These similarities include a high GC content of the genome, the presence of three putative immune evasion orthologs, and the lack of viral genes involved in nucleotide metabolism (Delhon et al., 2004).

3. MC LESION DEVELOPMENT MCV has yet to be successfully propagated in cultured cells or in animals (Damon, 2013). As a result, most of our understanding of MCV


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pathogenesis comes from histopathological studies of patients with past or current MCV infections. These studies indicate that the severity and persistence of an MCV infection likely are due to a combination of factors, including the status of the immune system and the robustness of innate immune responses to initial MCV infections. In this section, I summarize the known and predicted steps in MCV replication and lesion formation (Fig. 1). MCV is transmitted via direct skin-to-skin contact when there are abrasions in the skin (Damon, 2007; Tyring, 2003). Although enveloped MCV virions may quickly become inactivated when exposed to the environment, MCV transmission via fomites is not excluded (Wilson, Deweber, Berry, & Wilckens, 2013). Light and electron microscopy, histology, and immunohistochemistry have been used to assess the initial events of virus infection, evaluating the presence of MCV virions and virus-induced cytopathic effects in different layers of the epidermis. A detailed description of the MCV infection process was recently provided (Chen, Anstey, & Bugert, 2013). MCV infection is restricted to cells in the epidermis and does not cross the basement membrane, where MCV morphogenesis is likely intimately linked to the differentiation of its host keratinocyte. MCV cores are more common in the two deepest layers of the epidermis [the stratum basale (SB) and stratum spinosum (SS)] than in the upper cellular layers [the stratum granulosum (SG) and stratum lucidum (SL)] (Epstein & Fukuyama, 1973). In addition, more mature

Figure 1 Electron microscopy of molluscum contagiosum virus. Transmission electron micrograph of molluscum contagiosum virus (MCV) showing the typical morphology of a poxvirus. Image by courtesy of the Centers for Disease Control (CDC).

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MCV particles are present in the upper layer of the epidermis than in cells in the SB and SS (Epstein & Fukuyama, 1973). These data suggest a polarity in the development of the infection process, with MCV initially (and perhaps preferentially) targeting and infecting cells in SB and SS, and MCV maturation occurring as these cells differentiate. In support of this model, MCV-induced cytopathic effects increase as keratinocytes differentiate (Vreeswijk, Leene, & Kalsbeek, 1976). Specifically, there is a redistribution of mitochondria and empty cytoplasmic areas (presumably to allow for the development of a viral replication factory) and also an increase in mature viruses as cells differentiate from the SB and SS to the SG and SL (Vreeswijk et al., 1976). This process of virus morphogenesis takes about 5 days (Epstein & Fukuyama, 1973). The last stage of keratinocyte differentiation is the stratum corneum. During this differentiation process, molluscum bodies (MBs), which are inclusion bodies that contain large number of virions, increase. These MBs are in a lipid-rich sac-like structure, which may provide a mechanism for MCV to remain undetected by intracellular pattern recognition molecules (Diven, 2001). Eventually, these MBs are trapped in the horny layer of the fibrous network, which eventually dissolves in the center of the lesion, forming a central core that is composed of viruses. If MCV replication is dependent upon the differentiation state of its host keratinocyte, then perhaps a productive MCV infection in cultured cells can only occur when a distinct set of host keratinocyte proteins is expressed. This could explain why MCV cannot be propagated in continuous cell lines.

4. CHARACTERIZATION OF MC LESIONS MC lesions are characterized histologically by a localized mass of hypertrophied and hyperplastic epidermis, projecting above the surface as a small papule. In healthy people, a typical lesion is 2–5 mm in width and appears waxy or pearl-like. The lesion is umbilicated and the central plug of the neoplasm is a lipid-rich material that contains MCV particles. There is very little inflammation associated with these lesions. As such, these benign MCV lesions are quite different from the necrotic lesions induced by VAR or other poxviruses that cause zoonotic infections in humans (Damon, 2013).

4.1. Initial stages of infection Immunohistochemical staining of MC lesions and surrounding cells identifies features that give insight into the relationship between the virus and


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the host cell. Several lines of evidence suggest that MCV infection alters the cellular environment to the benefit of the virus. For example, the epidermal growth factor receptor (EGFR) is upregulated in MCV-infected epidermal cells (Viac & Chardonnet, 1990). Epidermal growth factor (EGF)–EGFR interactions induce events that can stimulate cellular proliferation (Prenzel, Fischer, Streit, Hart, & Ullrich, 2001). Interestingly, VACV infection activates the EGFR to stimulate macropinocytosis of VACV (Mercer et al., 2010). Whether this same event would enhance the uptake of MCV in keratinocytes is unknown. VACV encodes vaccinia virus growth factor (VGF), a homolog to EGF (Twardzik, Brown, Ranchalis, Todaro, & Moss, 1985). There are several benefits that VGF confers during infection. VGF binds to the EGFR to induce proliferation in uninfected cells presumably to make a host cell more suitable for virus infection (Buller, Chakrabarti, Moss, & Fredrickson, 1988). There is no obvious VGF homolog encoded by MCV, begging the question if MCV possesses a strategy to take advantage of this increase in EGFR protein expression to enhance virus replication. Similarly, MCV infection manipulates keratinocyte differentiation (Callegaro & Sotto, 2009). Using immunohistochemistry, these authors found that MCV infection alters the expression of the K14 and K16 keratin proteins (Callegaro & Sotto, 2009). K14, which normally is expressed in SB cells only, is found also in other populations of differentiated keratinocytes during MCV infection. K16 is another keratin protein only expressed in hyperproliferating cells. During MCV infection, K16 is present in MCVinfected and neighboring cells (Callegaro & Sotto, 2009). Together, these results suggest that MCV infection may induce proliferation of host cells to provide a more suitable environment for virus replication.

4.2. Host cell response to MCV infection Virus infection typically induces an antiviral state to make the host cell recalcitrant to viral infection. Viruses then must neutralize these responses to replicate. MCV infection upregulates the immune response in host and neighboring uninfected cells. For example, inoculation of MCV onto murine embryo fibroblasts (MEFs), conditions which will not result in production of virus, induces the production of type I interferon (IFN) (Postlethwaite, 1970; Postlethwaite & Lee, 1970). Moreover, pretreatment of MEFs with MCV blocks the replication of VACV, herpesviruses, and ectromelia virus (Postlethwaite, 1964; Postlethwaite & Lee, 1970). Other cellular antiviral proteins also are upregulated by MCV infection. Human

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β-defensin-3 belongs to a family of antimicrobial peptides, which stimulate innate immunity. Its levels are increased in MCV lesions versus normal skin (Meyer-Hoffert, Schwarz, Schroder, & Glaser, 2010). Tumor necrosis factor (TNF) and IFNβ are highly expressed in MCV lesions and surrounding tissue (Ku et al., 2008). Toll-like receptors 3 and 9, which detect viral RNA and DNA, respectively, also are upregulated in MCV lesions (Ku et al., 2008), suggesting that MCV infection triggers the downstream activation of IRF-3 and NF-κB, resulting in expression of IFN-regulated genes including the production of cytokines. Despite the upregulation of these proinflammatory host cell genes, there is little inflammation associated with MC lesions (Damon, 2007; Epstein & Fukuyama, 1973; Gottlieb & Myskowski, 1994). This implies that the initial response to MCV infection is not sufficient to induce classical innate cell recruitment to the area of infection. In support of this hypothesis is the finding that immune cells involved in inflammatory responses (T cells, Langerhans cells, and NK cells) are not recruited to the site of MCV infection (Viac & Chardonnet, 1990). Thus, the prevailing theory is that MCV encodes molecules that dampen or inhibit these antiviral immune responses. Indeed, several MCV proteins with these properties are now known and will be discussed in this review.

5. IMMUNE RESPONSES TO MCV INFECTION In healthy individuals, MC lesions typically are present for weeks to months. However, they can persist for up to 5 years (Damon, 2013). It is not clear which factor(s) determine whether the MC lesions are short-lived or persistent. The severity and persistence of an MCV infection likely are due to a combination of factors, including the status of a patient’s immune system and the robustness of innate immune responses to initial MCV infections. Regardless of how long these lesions persist, in general, MCV infections are self-limiting in healthy individuals. It is still unclear what aspects of the immune response mediate resolution. A recent study reveals some of the innate immune components that are important for MC resolution. Histological evidence shows that plasmacytoid dendritic cells, IFN-induced dendritic cells, immune cell infiltration, and innate immune signaling are responsible for spontaneous regression of MCV lesions (Vermi et al., 2011). Vermi et al. propose that two types of MCV lesions exist in healthy individuals: inflammatory MC (I-MC) and noninflammatory MC (NI-MC) lesions (Vermi et al., 2011). I-MC lesions display many immunogenic


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properties including the expression of MHC class I and II proteins, and infiltration of immune cells including CTLs, NK cells, and plasmacytoid DCs. Furthermore, they found evidence of type I IFN activation, apoptosis, and NF-κB activation in cells near I-MC lesions. Conversely, NI-MC lesions did not express MHC or IFN molecules or exhibit immune cell infiltration. The concept that inflammatory processes may dictate the clearance of an MCV infection is also supported by studies showing that patients with inflamed MC lesions are less likely to develop additional lesions than those with noninflamed MC lesions (Vermi et al., 2011). These data suggest that a strong host immune response is important for the clearance of MC lesions. It remains unclear, however, what triggers events for an NI-MC lesion to become an I-MC and what is required for the resolution of MC lesions. Acquired immunity is also important for the resolution of MCV infections. For example, giant MC is common among HIV-positive patients and patients on immunosuppressive therapy, such as transplant recipients (Mansur, Goktay, Gunduz, & Serdar, 2004), suggesting that the immune system of an individual is important in controlling the size and spread of MC lesions. Furthermore, MCV infections in these patients can persist indefinitely (Buckley & Smith, 1999; Tyring, 2003). With respect to B cellmediated immunity, 58–77% of MCV-infected individuals possess antibodies against MCV (Konya & Thompson, 1999; Watanabe et al., 2000). However, anti-MCV antiserum is not sufficient for curing MCV infections because many individuals with persistent MCV infections have high antiMCV antibody titers (Konya & Thompson, 1999).

6. MCV EPIDEMIOLOGY Most childhood cases of MC occur on the face and trunk and resolve within 3 months (Berger, Orlow, Patel, & Schaffer, 2012). Of note, ocular and oral MC in immunocompetent children has also been reported (de Carvalho et al., 2012; Pandhi & Singhal, 2005). In contrast, when MCV infections are sexually transmitted in young adults, the MC lesions initially are present in the groin area. It is difficult to track the incidence of MCV cases annually in the USA and other countries where medical professionals are not required to report cases of MC to local or national health organizations. The incidence of past MCV infections is quantified by measuring the presence of anti-MCV antibodies in blood. Using this approach, anti-MCV antibodies have been

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detected in 6% and 22% of the general population in Japan and Australia, respectively (Konya & Thompson, 1999; Watanabe et al., 2000), whereas 14.8% of a surveyed German and approximately 30% of a tested UK population were seropositive for MCV (Sherwani et al., 2014). Another method to track the incidence of MC is to ask physicians to record MCV infections in their own practices. Of 100 pediatric dermatologists surveyed in the USA in 2009, approximately two-thirds of respondents reported seeing between 1 and 10 cases of MC per week. Less than 7% of clinicians reported seeing an average of less than one case of MC per week. A study of MCV incidence in American Indians and Alaskan native populations found the majority of MC cases to be in individuals under 15 years, with the peak incidence in the 1- to 4-year range (Reynolds, Holman, Yorita Christensen, Cheek, & Damon, 2009). Pannell et al. gathered information from general practitioners in England and Wales and found that 90% of new MCV cases were reported in children up to 14 years (Pannell, Fleming, & Cross, 2005). Two studies in the Netherlands found the childhood incidence of MCV to be 3% (Mohammedamin et al., 2006) and 17% (Koning, Bruijnzeels, van Suijlekom-Smit, & van der Wouden, 1994). It is well documented that MCV infection rates are higher in patients that are immunocompromised. MCV infections are present in 5–8% of the HIVpositive population (Gottlieb & Myskowski, 1994), and HIV-positive individuals have up to a 33% increased risk of contracting MCV as compared to healthy individuals (Aldabagh, Ly, Hessel, & Usmani, 2010; Cursiefen et al., 2002; Mansur et al., 2004; Sung, Lee, Choi, Seo, & Yoon, 2012). In children with HIV/AIDS, MCV infections occur in 5–18% of patients (Wananukul, Deekajorndech, Panchareon, & Thisyakorn, 2003). Patients receiving anti-TNF therapy for rheumatoid arthritis also show increased susceptibility to MCV infection (Cursiefen et al., 2002) as do patients with congenital immune suppressive conditions. For example, mutations in the DOCK8 (dedicator of cytokinesis 8) gene, a guanine nucleotide exchange factor predicted to play a role in cytoskeleton rearrangement, have been linked to recurrent cutaneous viral infections including MCV (Zhang et al., 2009). One patient with a homozygous DOCK8 gene deletion was reported to have extensive, disseminated MC on the hands and anogenital regions (Aan de Kerk et al., 2013). MC is common in student athletes who wrestle (Wilson et al., 2013). Two reports show that there is an association between swimming and MC, with swimming increasing the chances of MC by twofold (Castilla, Sanzo, & Fuentes, 1995; Niizeki, Kano, & Kondo, 1984).


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7. MCV DIAGNOSIS AND TREATMENT There are several features of MC that make it easily diagnosed by visual inspection, including its small size, its characteristic umbilicated crater with a white core, and its pearl-like coloring. An atypical presentation, such as MC papules in the anogenital area or coinfection with another agent, can be a diagnostic challenge for clinicians (Cribier, Scrivener, & Grosshans, 2001). Dermoscopy is a noninvasive method for visualizing lesions, and it represents a popular tool for diagnosis (Ianhez, Cestari Sda, Enokihara, & Seize, 2011). It allows for the magnification of the cutaneous lesions and can facilitate the visualization MB as white, shiny clods (Mun, Ko, Kim, & Kim, 2013). Reflectance confocal microscopy, a noninvasive technique for imaging skin at the cellular level, has emerged as a secondary method for examining potential MCV lesions (Scope et al., 2008). The validity of both techniques has been confirmed using histological methods (Ianhez et al., 2011; Scope et al., 2008). An alternative tool for diagnosis involves PCR amplification of parts of the MCV genome directly from an individual lesion (Nunez et al., 1996). Two real-time PCR assays have been developed to detect MCV DNA, although they cannot distinguish between MCV subtypes (Trama, Adelson, & Mordechai, 2007). Recently, a FRET-based real-time PCR has been developed that now allows for differentiation between MCV-1 and MCV-2, taking advantage of single nucleotide polymorphisms that exist in the MC021L gene (Hosnjak, Kocjan, Kusar, Seme, & Poljak, 2013). Currently, there are several therapeutic treatments for MCV infection. Also, no treatment is 100% effective for curing MC lesions. Since MCV lesions will resolve without intervention, education of the patient to prevent autoinoculation to other areas of the body is important. However, patients (or the parents of children with MC) will often seek treatment because of the social stigma associated with visible lesions. The most common treatment strategies include administration of cantharidin or imiquimod, curettage, cryotherapy, retinoids, cimetidine, salicylic acid, duct tape, Candida antigen, potassium hydroxide, and cidofovir. In a poll of 100 pediatric dermatologists, cantharidin, a compound secreted by the blister beetle, is the most commonly used therapeutic (Coloe & Morrell, 2009). It has been used to treat MC since the 1950s (Coloe & Morrell, 2009). It is applied topically and causes the blistering of skin at the area of application. It is thought that cantharidin has two mechanisms of action to aid in curing MC. It kills

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virus-infected cells and it induces an inflammatory response, which may be sufficient to trigger an antiviral immune response. A 5% imiquimod cream is sometimes preferred over cantharidin because of the reduced pain with application and the ability for administration at home. Similar to cantharidin, imiquimod application upregulates cytokines that possess antiviral properties. A more recent MC treatment directly injects Candida antigen into the MC lesion weekly for 3–5 weeks until infection is cured. Curettage and cryotherapy are traditional treatments to remove MC lesions. As could be expected, both methods are quite painful and have scarring potential.

8. CURRENT ROADBLOCKS IN PROPAGATING MCV IN TISSUE CULTURE SYSTEMS MCV is distantly related to other well-known poxviruses such as VAR and VACV (Damon, 2007). Like all members of the Poxviridae family, MCV has a large (190 kb), linear double-stranded DNA genome and replicates in the cytoplasm of cells (Moss, 2013). Similarly, MCV is predicted to have staged transcription consisting of early, intermediate, and late stages (Senkevich et al., 1996, 1997). For VACV, early gene synthesis is initiated in the cytoplasm, while the genome is still encapsidated. Early gene synthesis triggers uncoating of the genome and is followed by genome replication which must occur for intermediate gene synthesis, followed by late gene synthesis and virion morphogenesis. Staged transcription occurs through a cascade effect with specific transcription factors being synthesized in the stage immediately preceding the stage where they are utilized. A block in any of these stages inhibits all subsequent steps. While the lack of a suitable cell culture model hinders MCV morphogenesis studies, it is assumed that the MCV lifecycle mirrors that of other poxviruses in permissive host cells. MCV replication has been tested in several primary and established human cell lines and tissues (Barbanti-Brodano, Mannini-Palenzona, Varoli, Portolani, & La Placa, 1974; Damon, 2007, 2013; Dourmashkin & Duperrat, 1958; Mitchell, 1953; Neva, 1962; Pirie, Bishop, Burke, & Postlethwaite, 1971; Postlethwaite, 1970; Postlethwaite & Lee, 1970; Shcherbakov, 1966). However, to date, there are no confirmed reports of MCV cultivation in vitro. One study analyzed several cell lines and found only early gene expression, leading to the theory that the MCV core does not uncoat, precluding DNA replication, and subsequent intermediate and late gene expression (McFadden, Pace, Purres, & Dales, 1979). Recently, MCV late gene mRNA was reported to be detectable by reverse transcriptase PCR starting


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at 5 days after infection of the human lung fibroblast cell line MRC-5 (Bugert, Lohmuller, & Darai, 1999; Bugert, Melquiot, & Kehm, 2001), implying that some virions uncoat and some viral DNA is synthesized. However, neither an increase in MCV genomes as measured by PCR amplification nor any virion morphogenesis stages were detected (Bugert et al., 2001). It is widely accepted that poxvirus DNA replication is required for intermediate and late gene expression and viral morphogenesis (Moss, 2013). Thus, perhaps sufficient DNA replication occurred for limited late gene expression, but it was insufficient for virion morphogenesis. MCV was reported to replicate in human foreskin fragments that were implanted into the renal capsule of mice (Fife et al., 1996). However, these viruses were not infectious (Fife et al., 1996). Although MCV cannot produce infectious progeny in cell culture, a new method that uses quantitative PCR and a luciferase reporter construct under the control of an early/late poxvirus promoter has recently been developed to assess MCV infectivity (Sherwani, Blythe, Farleigh, & Bugert, 2012). Why is there insufficient MCV genome replication in cultured cells to support virion morphogenesis? This could be due to the inability of MCV to either directly produce or co-opt the components required. The MCV genome sequence (Senkevich et al., 1996, 1997) reveals information that supports the former possibility. MCV lacks homologs to the six VACV virus genes involved in nucleotide biosynthesis (thymidine kinase, thymidylate kinase, guanylate kinase, deoxyuridine triphosphatase, and the two ribonucleotide reductase subunits), in addition to the B1 kinase that is important for VACV DNA replication (Senkevich et al., 1997; Wiebe & Traktman, 2007). In addition, MCV only possesses one of VACV genes that encode proteins associated with the formation of the extracellular enveloped form of VACV (Senkevich et al., 1997). Thus, MCV morphogenesis may differ from VACV and may only be completed in differentiated keratinocytes. An alternative model is that MCV is unable to overcome the innate immune defenses in cell lines used to date. For example, MCV encodes no poxvirus homolog for proteins that inhibit protein kinase R, a cellular protein that blocks viral protein synthesis, or other proteins that could potentially inhibit cellular intrinsic innate immune responses including soluble IFN inhibitors (Perdiguero & Esteban, 2009; Smith et al., 2013). Thus, MCV replication may not occur in cultured cells that are permissive for VACV infection because of the absence of one or more of these genes. Yet a third possibility is that MCV replication requires specific cofactors that are present only during keratinocyte differentiation. Until this technical barrier is overcome, researchers will continue to study MCV genes either by

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expressing genes independent of infection, by a surrogate virus, or by using MCV collected from lesions of patients for experimental infections (Randall, Jokela, & Shisler, 2012). The MCV host range is limited to humans. Interestingly, an MCV-like disease has recently been described in animals including donkeys, chickens, sparrows, pigeons, chimpanzees, kangaroos, dogs, and horses (Fox et al., 2012). There is tantalizing evidence that these viruses are members of the poxvirus family: hybridization between MCV DNA and DNA from an MCV-like virus isolated from a horse has been observed, suggesting that the MCV-like equine virus is very similar to MCV (Thompson, Yager, & Van Rensburg, 1998). Unfortunately, all attempts to culture these MCV-like viruses also were unsuccessful. In addition, no animal MCV infection has been successfully experimentally transmitted from one animal to another. A very exciting area of future research is to compare the sequenced genomes of these MCV-like viruses to MCV as a strategy to identify common mechanisms these viruses share for virus replication and viral immune evasion.

9. MCV IMMUNE EVASION MECHANISMS The sequencing of the MCV genome revealed the presence of several viral genes with similarity to cellular molecules that control the immune response, or with no homology to known genes (Senkevich et al., 1996, 1997). In total, 77 of the 182 MCV genes are predicted to be involved in immune evasion based on (i) their location at the ends of the linear MCV genome and (ii) the lack of homology to other poxvirus proteins involved in the virus replication cycle. Surprisingly, only 6 of these 77 proteins, including MC159 and MC160, have been studied in any detail (Randall & Shisler, 2013) (Table 1). Thus, the remaining MCV genes represent a potential gold mine to discover new strategies that viruses use to counteract innate antiviral immunity to ensure MCV replication and spread.

10. LIMITATIONS AND CAVEATS WHEN STUDYING MCV IMMUNE EVASION PROTEINS Despite its high impact on human health MCV is understudied. There is neither a system to propagate stocks of MCV nor an animal model that recapitulates MCV replication, which contributes to the slow pace of MCV research. Current studies require a cumbersome process of purifying

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Table 1 Immune defense molecules of MCV discussed in this review MCV protein Function Mechanism Homologs

MC159 Inhibits apoptosis Inhibits NF-κB

Binds FADD

MC160, HHV-8 K13, EHV E8, cFLIPS, cFLIPL

Binds IKKγ

Inhibits IRF3 Binds TBK1-IKKε MC160 Inhibits NF-κB MC54

Binds caspase-8, Hsp90

Inhibits IL-18 Binds IL-18

MC148 Inhibits chemotaxis

Binds CCR8, CXCL12-α

MC159, HHV-8 K13, EHV E8, cFLIPS, cFLIPL VAR D7, ECT p13, huIL-18bp MC148R1 and MC148R2

MC007 Inhibits pRb Binds pRb

LxCxE motif containing proteins

MC66

Glutathione peroxidase

Inhibits apoptosis

Inhibits hydrogen peroxide

HHV-8, human herpes virus 8; EHV, equine herpes virus; VAR, variola virus; ECT ectromelia virus.

virus from primary lesions donated by patients with no method for determination of infectious virus yields as convenient as those used for VACV (Randall et al., 2012; Sherwani et al., 2012). In addition, the use of primary lesions has the potential for strain variation, and large differences in viral yields between lesions (up to 10-fold) (Sherwani et al., 2012) make the planning and execution of experiments difficult. In lieu of isolating MCV from primary lesions, individual MCV genes are studied either by expression in a surrogate virus, typically (VACV), or using transient transfection (reviewed in Chen et al., 2013; Randall & Shisler, 2013). These strategies have provided important information regarding the biological function of individual MCV genes (see below). However, one should be aware of the caveats when studying MCV genes independent of MCV infection. For example, it is not known if the expression of MCV genes by transfection or surrogate infection is equivalent to that of a natural MCV infection. The use of VACV to express MCV proteins can be advantageous. Both VACV and MCV immune evasion genes are usually early genes that are transcribed prior to viral genome replication (Senkevich et al., 1997). Thus, an MCV gene that is controlled by a natural early VACV promoter is most likely to be closest to MCV gene expression in natural

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infections. A great weakness to using surrogate poxviruses is that they may express known or as-yet-unidentified functional homologs of MCV genes. For example, we discuss below that MC159 inhibits TNF-induced NF-κB activation. VACV expresses at least 10 proteins that also inhibit NF-κB activation (Smith et al., 2013). Thus, the molecular mechanism of MC159 may be masked if it is expressed within the context of a VACV construct that expresses these 10 molecules. Moreover, infection of a human cell line with a surrogate poxvirus may not accurately reflect the properties of an MCV infection of differentiating keratinocytes. Nevertheless, the rigorous study of these viral proteins is essential to the fields of virology and immunology. They can be used as tools to identify novel cellular signaling mechanisms that up- or downregulate innate immune responses, leading to a greater understanding of the signal transduction events that control the immune system. We will now describe the MCV immune evasion proteins that have been characterized to date.

11. THE FLIP FAMILY OF VIRAL AND CELLULAR PROTEINS 11.1. Introduction to FLIPs The MCV MC159 and MC160 proteins belong to a family of proteins known as FLIPs (FLICE (FADD-like interleukin-1 beta-converting enzyme)-inhibitory proteins) (Yu & Shi, 2008). This family of proteins was discovered during early studies of signal transduction pathways that are responsible for apoptosis of cells, prompting the identification of other viral FLIP (vFLIP) and cellular FLIP (cFLIP), that also regulate apoptosis. More recently, this family of proteins was found to regulate NF-κB and IRF3 activation (Shisler, 2014). The next section of this review highlights the similarities and differences in functions and mechanisms for these vFLIP and cFLIP followed by discussion of the current knowledge about signal transduction events that result in apoptosis and NF-κB activation as triggered through the death receptor (DR) family, focusing on signaling triggered through the tumor necrosis factor receptor 1 (TNFR1) and CD95 (Fas). With this framework in place, I will then discuss how each FLIP modulates these signal transduction events.

11.2. Signaling events triggered by the TNFR1 The TNFR1 is well studied, and there is much interest in this receptor because of its ability to stimulate NF-κB activation and apoptosis

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(Ofengeim & Yuan, 2013). TNFR1-mediated NF-κB activation begins when a trimeric TNF complex binds to the extracellular portion of the TNFR1 (Fig. 2). Next, TRADD, RIP1, and TRAF2 or TRAF5 migrate to the cytoplasmic portion of TNFR1 (Hsu, Huang, Shu, Baichwal, & Goeddel, 1996; Hsu, Shu, Pan, & Goeddel, 1996; Hsu, Xiong, & Goeddel, 1995). cIAP molecules, which are inhibitors of apoptosis, also migrate to this complex (Rothe, Pan, Henzel, Ayres, & Goeddel, 1995; Shu, Takeuchi, & Goeddel, 1996). Importantly, the LUBAC (linear ubiquitin chain assembly complex) is recruited to ubiquitinate RIP1. This signalosome is often referred to as complex I. RIP1 then receives Lys64 polyubiquitination and linear Met1 ubiquitination. This posttranslational

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Figure 2 TNFR1-induced signalosome complex formation. A trimeric TNF binds to the TNFR1, which induces the migration of the TRADD, RIP1, and TRAF2 molecules. In addition, cIAP and the LUBAC complex (not shown here) migrate to the TFNR1 to form complex I. In this complex, RIP1 is ubiquitinated. Next, the IKK complex docks to this signalosome platform. IKK is activated and phosphorylates the IκBα molecule to stimulate IκBα degradation. Freed NF-κB migrates to the nucleus to act as a transcription factor. When the A20 and CYLD deubiquitinases are present, RIP1 is deubiquitinated. RIP1 then interacts with FADD and procaspase-8 to form complex IIa. When a second procapase-8 interacts with the complex IIa, procaspase-8 autocleaves, such that the non-DED-containing region of caspase-8 is separated from the complex. This form of caspase-8 is further processed, resulting in the formation of a heterodimer that possesses full caspase activity.

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modification of RIP1 triggers the recruitment of the IKK complex to the signalosome (Tokunaga et al., 2009). NF-κB resides in the cytoplasm bound by its inhibitor, IκB (Hayden & Ghosh, 2004, 2008) which inhibits NF-κB activity by preventing NF-κB translocation to the nucleus. Upon stimulation of the NF-κB pathway, the IKK complex phosphorylates IκB resulting in IκB ubiquitination and degradation (Hayden & Ghosh, 2004, 2008). Freed NF-κB migrates to the nucleus where it binds to specific promoter sequences to trigger transcription. The TNFR1 can also signal apoptosis. In this case, the two cellular proteins A20 and CYLD deubiquitinate RIP1 and IKK in complex I (Ofengeim & Yuan, 2013). This releases RIP1 to the cytoplasm, to become part of a complex IIa which triggers caspase-8-induced apoptosis, or complex IIb which triggers necroptosis. For this review, complex IIa is most interesting (Fig. 2). For apoptosis to occur, the released RIP1 molecule forms a complex with FADD and procaspase-8 (O’Donnell, LegardaAddison, Skountzos, Yeh, & Ting, 2007). When a second procaspase-8 molecule interacts with procaspase-8 in the complex, both zymogens undergo autoproteolysis. The 55-kDa procaspase-8 is cleaved in such a manner that its C-terminus, which lacks the death effector domain (DED) motif, is cleaved. The freed caspase-8 is processed further, resulting in an active caspase-8 composed of two 18 kDa subunits and two 10 kDa subunits (Taylor, Cullen, & Martin, 2008). The mature, highly active caspase-8 then initiates procaspase-3 activation, eventually resulting in poly (ADP-ribose) polymerase (PARP) and DNA cleavage (Taylor et al., 2008). CD95 is very similar to the TNFR1 in activation of apoptosis. However, there are slight differences in the signal transduction pathways triggered by CD95 versus TNFR1 (Li, Yin, & Wu, 2013; Wallach, 2013). When CD95-induced apoptosis occurs, FADD and procaspase-8 form a signalosome (DISC, death-induced signaling complex) with the cytoplasmic tail of CD95. As above, homodimeric interactions of procaspase-8 at the DISC result in caspase-8 activation and apoptosis. 11.2.1 Discovery of viral and cellular homologs of procaspase-8 FADD and procaspase-8 possess one and two DED motifs, respectively, which are responsible for FADD–procaspase-8 interactions (Fig. 3). A DED does not possess enzymatic activity, but its structure of 6 α-helices promotes interactions with other proteins (Valmiki & Ramos, 2009). As shown in Fig. 3, the sequencing of several viral genomes revealed the presence of proteins that, like procaspase-8, contained two tandem DEDs

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Figure 3 FLIPs and other cellular proteins that possess DED motifs. Proteins that possess the death effector domain (DED) motives. All proteins contain two DEDs except for FADD, which has one DED and an additional death domain (DD) motif. Procaspase-8 has an additional caspase domain that has catalytic activity. The cFLIPL protein has a caspase-like domain that lacks enzymatic activity. The length of each protein in amino acids is listed.

including the MCV MC159 and MC160 products, as well as the Kaposi’s sarcoma herpesvirus (KSHV) K13 and the equine herpesvirus E8 proteins (Bertin et al., 1997; Hu, Vincenz, Buller, & Dixit, 1997; Irmler et al., 1997). Because of the presence of these domains, it was thought that these viral homologs would either act similar to procaspase-8 to induce apoptosis or act as a procaspase-8 antagonist by competitively binding to procaspase-8. Several studies show that the second hypothesis is correct: MC159, K13, and E8 each inhibit procaspase-8-induced apoptosis triggered either through TNFR1 or CD95 (Bertin et al., 1997; Hu et al., 1997; Irmler et al., 1997). Procaspase-8 was originally named FLICE, and these viral proteins were named “FLIPs” (FLICE-inhibitory proteins). Although an antiapoptotic property of MC160 had been reported (Hu et al., 1997), other studies show that MC160 does not inhibit apoptosis (Shisler & Moss, 2001; Shisler, Senkevich, Berry, & Moss, 1998). Soon after the discovery of vFLIPs, cFLIP was discovered. The role of cFLIP in apoptosis originally was unclear because of conflicting reports showing cFLIP with pro- or antiapoptotic activities. It is now understood that these differences are likely due to the intracellular ratios of cFLIP to procaspase-8. In the next section, the antiapoptosis mechanism of the MC159 will be discussed first. Then, the molecular functions of other FLIPs will be discussed and compared to highlight the similarities and differences between these proteins.

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11.2.2 MC159 and inhibition of apoptosis Apoptosis is a host defense to eliminate virus-infected cells, and it is proposed that vFLIPs present a strategy to evade this immune response. While there is no current model for MCV pathogenesis, one prediction is that MCV that lacks MC159 or MC160 would be attenuated. The ca. 25-kDa MC159 protein encompasses 241 amino acids (Fig. 3). It is characterized by the presence of two tandem DEDs (DED1 and DED2) separated by a 14-amino acid linker sequence and a 64-amino acid carboxy terminal tail. Like the FADD DED, the MC159 DEDs are comprised of six α-helices (Eberstadt et al., 1998). In addition, these DED-containing proteins possess an RXDL motif at the carboxy terminal end of each DED. Despite these similarities, it should be noted that these DEDs, while similar, are not structurally or functionally equivalent, as discussed below. The crystal structure of a truncated MC159 protein has been identified (Li, Jeffrey, Yu, & Shi, 2006; Yang et al., 2005) as a rigid dumbbell in which its two DEDs tightly associate (Li et al., 2006; Yang et al., 2005). Both MC159 DEDs have a hydrogen-bonded charge triad on one face of their surfaces, which is part of the RXDL motif. Thus, it is thought that this triad plays a role in the ability for protein–protein interactions (Li et al., 2006; Yang et al., 2005). In addition, the DEDs interact with each other through hydrophobic interactions. The DED1 of MC159 is highly divergent from DED2 and from other DEDs (Yang et al., 2005). There are several physical properties that are responsible for this divergence including a missing helix loop 3 in MC159; two additional α-helices (H0 and H7) in MC159 that are not present in other DEDs; and a helix 2 that is shorter than in other DEDs (Li et al., 2006; Yang et al., 2005). The MC159 protein inhibits TNFR1- and CD95-induced apoptosis (Bertin et al., 1997; Hu et al., 1997; Irmler et al., 1997; Murao & Shisler, 2005; Shisler & Moss, 2001). The molecular mechanism for this MC159inhibitory function has been studied intensely (Fig. 4). Both DEDs of MC159 must be present, and in their proper orientation, to provide antiapoptotic function (Garvey, Bertin, Siegel, Lenardo, & Cohen, 2002). Within each DED, the RXDL motif is critical for antiapoptosis function (Garvey, Bertin, Siegel, Lenardo, et al., 2002). The wild-type MC159 protein coimmunoprecipitates with both FADD and procaspase-8 (Bertin et al., 1997; Garvey, Bertin, Siegel, Wang, et al., 2002; Shisler & Moss, 2001), and these data suggested that MC159 prevents FADD–procaspase-8 interactions. Curiously, point mutational analysis revealed the presence of mutant MC159 proteins that still interact with FADD and procaspase-8 but lose the

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Figure 4 The effect of individual FLIPs on apoptosis. Complex IIa comprises FADD, RIP1, and procaspase-8. Under proapoptotic conditions, a second procaspase-8 binds to complex IIa. This results in autocleavage of the 55-kDa procaspase-8 to an active heterotetrameric caspase-8. The cFLIPS, cFLIPL (when expressed at high levels), and E8 proteins bind to procaspase-8 to prevent the formation of this heterotetramer. MC159 and E8 are also known to bind to FADD. The MC160 protein does not inhibit apoptosis even though it binds to procaspase-8, suggesting that MC160 does not prevent the formation of the caspase-8 heterodimer. When cFLIPL is expressed at low levels in the cells, procaspase-8 is cleaved to active p43 and p41 forms, resulting in apoptosis.

ability to inhibit apoptosis (Garvey, Bertin, Siegel, Wang, et al., 2002). These data suggest that the initial model of MC159 disrupting procaspase-8–FADD interactions to inhibit apoptosis is an oversimplification. Interestingly, MC159 possesses a TRAF-binding motif in its C-terminus, a region that is not part of either DED, suggesting that MC159 may have more than one antiapoptosis mechanism. This motif is important because the antiapoptotic properties of MC159 are bolstered by its interaction with the adaptor protein TRAF3 (Thurau, Everett, Tapernoux, Tschopp, & Thome, 2006). It is still not clear how the MC159–TRAF3 interactions would inhibit apoptosis because TRAF3 is not part of classical complex I. TRAF3 can interact with both TRAF2 and cIAP molecules at the cytoplasmic portion of CD40, a receptor that is similar to TNFR1. Thus, one possibility is that MC159–TRAF3 interactions disturb the formation of complex I. Moreover, a correlation between mutant MC159 proteins that do not inhibit apoptosis and a loss of ability to form a ternary complex with Fas and FADD has been demonstrated (Yang

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et al., 2005). This implies that MC159–FADD interactions prevent FADD– CD95 interactions to inhibit formation of a DISC (Yang et al., 2005). A question that remains is what is the importance of MC159–procaspase-8 interactions? It is now appreciated that procaspase-8 has other physiological functions, including mediating CD95-induced NF-κB activation (Imamura et al., 2004; Kawadler, Gantz, Riley, & Yang, 2008; Kreuz et al., 2004; Su et al., 2005) and inhibiting IRF3 activation (Rajput et al., 2011; Sears, Sen, Stark, & Chattopadhyay, 2011), as discussed below. Perhaps these functions would be altered in the presence of MC159. Two groups capitalized on the antiapoptosis function of MC159 and created transgenic mice expressing MC159 as a means to evaluate the role of apoptosis on T and B cell populations in vivo. Woelfel et al. established a transgenic mouse in which the MC159 is expressed under the control of the promoter for a MHC class I gene (Woelfel, Bixby, Brehm, & Chan, 2006), indicating that MC159 is expressed in almost all cells. In contrast, Wu et al. developed a transgenic mouse that expresses MC159 in T cells only (Wu et al., 2004). For both systems, MC159-expressing cells were resistant to apoptosis triggered by several stimuli. For mice in which MC159 was expressed only in T cells, CD8+ T cells were activated in response to a heterologous antigen, but no memory T cells specific for that antigen persisted (Wu et al., 2004). In contrast, Woefel et al. found that B and T cell proliferation occurred in their transgenic mice, with populations of the expanded immune cells similar to those found in mice with autoimmune diseases. It is thought that this proliferation of immune cells most likely is due to the fact that apoptosis is blocked in immune cells that otherwise would have been eliminated during thymic maturation (Woelfel et al., 2006). 11.2.3 MC160 and apoptosis MCV MC160 is a 52-kDa protein (Shisler & Moss, 2001). Like MC159, it has two tandem DEDs (residues 5–79 and 97–175), and each DED contains an RXDL motif (Fig. 3). In addition, MC160 has a unique C-terminal region (residues 175–371) that has no homology to other known proteins. The MC160 N- and C-terminal DEDs are 45% and 33% similar to the N- and C-terminal DEDs of MC159, respectively (Shisler & Moss, 2001). An original prediction was that MC160 would inhibit apoptosis like other vFLIPs (Senkevich et al., 1996, 1997). While one report indeed showed antiapoptosis function (Hu et al., 1997), others demonstrated that MC160 does not inhibit apoptosis, whether expressed transiently or by a


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surrogate poxvirus (Shisler & Moss, 2001; Shisler et al., 1998). MC160 expression also does not trigger apoptosis (Shisler & Moss, 2001; Shisler et al., 1998). These data initially were surprising because MC160 interacts with FADD and procaspase-8 (Shisler & Moss, 2001). However, with the finding that MC159 function does not correlate with FADD or procaspase-8 interactions (Garvey, Bertin, Siegel, Lenardo, et al., 2002), we now know that MC160–FADD or MC160–procaspase-8 interactions do not solely dictate apoptosis inhibition. These data would suggest that MC160 can interact with complex IIa, but this interaction is not sufficient to prevent procaspase-8 activation (Fig. 4). There are several putative caspase cleavage sites within the MC160 protein (Shisler & Moss, 2001), and MC160 is cleaved when cells are triggered to undergo apoptosis (Shisler & Moss, 2001). Whether this cleavage occurs with an MC160 that is associated with complex IIa or in a free form is unknown. This cleavage is blocked when MC159 is coexpressed with MC160 (Shisler & Moss, 2001), an event that would occur during a natural MCV infection. It is interesting to note that cFLIPL is also cleaved by caspase-8 and that the p42 and p22 cleavage products of cFLIPL activate NF-κB. Whether the MC160 cleavage products also have biological functions remains unknown. 11.2.4 Gammaherpesvirus FLIPs and inhibition of apoptosis Of the gamma herpesvirus vFLIPs, K13 (KSHV/HHV-8), E8 (equine herpesvirus-2, EHV-2), and ORF71 (herpesvirus saimiri) each have antiapoptosis properties. The ectopic expression of E8 inhibits apoptosis triggered by either TNFR1 or CD95 (Bertin et al., 1997; Hu et al., 1997; Thome et al., 1997). While MC159 targets FADD to inhibit apoptosis (Yang et al., 2005), E8 is thought to interact with procaspase-8 instead (Hu et al., 1997) as shown by yeast two-hybrid analysis. Contradictory to this model are the findings that E8 inhibits apoptosis triggered by overexpression of FADD, but not by overexpression of procaspase-8 (Hu et al., 1997), and that E8 coimmunoprecipitates with FADD in overexpression systems (Thome et al., 1997). Thus, whether E8 binds preferentially to FADD or procaspase-8 to inhibit apoptosis remains unclear (Fig. 4). KSHV is associated with Kaposi’s sarcoma (KS) and lymphoproliferative disorders including primary effusion lymphoma and multicentric Castleman’s disease (Gaidano, Castanos-Velez, & Biberfeld, 1999; Ganem, 2006; Said et al., 1996). K13 is expressed by KSHV during latency, where it inhibits lytic replication of KSHV (Guasparri, Keller, & Cesarman,

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2004; Keller, Schattner, & Cesarman, 2000). It is well established that K13 is important for the pathogenesis of KSHV infections, and K13 is essential for the survival of KSHV-infected primary effusion lymphomas (Guasparri et al., 2004) and other B cell lymphomas (Graham et al., 2013). K13 inhibits apoptosis triggered via several insults, including growth factor withdrawalinduced apoptosis and apoptosis triggered through CD95 and TNFR1 (Efklidou, Bailey, Field, Noursadeghi, & Collins, 2008; Sun, Matta, & Chaudhary, 2003). This antiapoptosis function of K13 has physiological relevance since there is a correlation between increase in the expression of K13 and reduction in apoptosis in KS lesions (Sturzl et al., 1999). The similarity of K13 to MC159 and other FLIPs predicted that K13 also prevented FADD–procaspase-8 interactions to inhibit apoptosis. However, it is now known that K13 uses other mechanisms, namely activation of NF-κB, for this purpose. K13-induced activation of NF-κB initiates the expression of several antiapoptotic genes, including manganese superoxide dismutase, Bcl-XL, and cFLIPL and cIAP (Guasparri et al., 2004; Sun, Matta, et al., 2003; Thurau et al., 2009; Tolani, Matta, Gopalakrishnan, Punj, & Chaudhary, 2014). In addition, the inhibition of the NF-κB-activating effect of K13 also abrogates the K13 ability to inhibit apoptosis (Guasparri et al., 2004; Sun, Matta, et al., 2003; Tolani et al., 2014). This antiapoptosis mechanism appears to be unique to K13 because E8 does not possess NF-κB-activating properties but still inhibits apoptosis (Bertin et al., 1997; Chaudhary, Eby, Jasmin, & Hood, 1999; Hu et al., 1997; Thome et al., 1999, 1997). Interestingly, while the ORF71 vFLIP inhibits CD95-induced apoptosis of virus-infected cells, ORF71 is not essential for virus replication, transformation, T cell lymphoma induction (Glykofrydes et al., 2000). Together, these data reveal that K13, E8, and ORF71, despite sharing homologous DEDs, use distinct mechanisms to inhibit apoptosis. 11.2.5 cFLIP and apoptosis cFLIP was identified soon after the discovery of vFLIPs (Golks, Brenner, Fritsch, Krammer, & Lavrik, 2005; Krueger, Baumann, Krammer, & Kirchhoff, 2001; Krueger, Schmitz, Baumann, Krammer, & Kirchhoff, 2001; Scaffidi, Schmitz, Krammer, & Peter, 1999). While there are many splice variants detected at the mRNA level, only three cFLIP proteins have been reported: cFLIPL, cFLIPS, and cFLIPR (Golks et al., 2005; Irmler et al., 1997; Scaffidi et al., 1999). cFLIPL is a 55-kDa protein, while the cFLIPS and cFLIPR proteins are 26 and 24 kDa, respectively (Fig. 4). cFLIPL most


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closely resembles procaspase-8. Both cFLIPL and procaspase-8 contain tandem DEDs but cFLIPL lacks the catalytic function of procaspase-8 due to the absence of a catalytic cysteine residue that is otherwise present in procaspase-8 (Irmler et al., 1997). In contrast, the two short isoforms (cFLIPS and cFLIPR) consist mainly of the two tandem DEDs and short C-terminal tails that are unique to each form. cFLIPL and cFLIPS are present in human cells, while cFLIPL and cFLIPR are expressed in mouse cells (Budd, Yeh, & Tschopp, 2006). One current idea is that viruses may have hijacked genes encoding cFLIP as a means to inhibit apoptosis and aid in viral spread. The short forms of cFLIP are reported to inhibit apoptosis when members of the DR family are triggered (Golks et al., 2005; Krueger, Baumann, et al., 2001; Krueger, Schmitz, et al., 2001; Scaffidi et al., 1999). Under these circumstances, cFLIPS or cFLIPR binds to procaspase-8, which prevents procaspase-8 homodimerization and subsequent caspase-8 activation (Golks et al., 2005; Krueger, Baumann, et al., 2001; Ueffing et al., 2008) (Fig. 4). Regulation of apoptosis by cFLIPL is more complicated because it can show pro- or anti-apoptotic functions (Fig. 4). cFLIPL is anti-apoptotic when it is expressed at high concentrations, and under these conditions, cFLIPL uses a mechanism similar to cFLIPS/R to inhibit apoptosis (Chang et al., 2002; Krueger, Schmitz, et al., 2001). The selective knockdown of cFLIPL sensitizes cells to apoptosis, also suggesting that cFLIPL is an antiapoptotic molecule (Sharp, Lawrence, & Ashkenazi, 2005). However, when cFLIPL is expressed at lower concentrations, it triggers procaspase-8 activation to stimulate apoptosis (Chang et al., 2002) because, in this case, cFLIPL– procaspase-8 interactions stabilize procaspase-8, triggering procaspase-8 cleavage to a proapoptotic dimer of 43 and 41 kDa (Micheau et al., 2002; Pop et al., 2011; Yu, Jeffrey, & Shi, 2009). A more recent paper shows that the function of cFLIPL is also dependent upon other events such as strength of receptor stimulation and relative amounts of cFLIPR/S (Fricker et al., 2010). The study of cFLIP is of great importance to cancer biology (Safa, 2012; Shirley & Micheau, 2013). cFLIP is overexpressed in many types of malignancies (de Hooge et al., 2007; Griffith, Chin, Jackson, Lynch, & Kubin, 1998; MacFarlane et al., 2002; Mathas et al., 2004; Okano et al., 2003; Olsson et al., 2001; Zhang et al., 2004; Zong et al., 2009). Furthermore, high levels of cFLIPL correlate with more aggressive tumors (Djerbi et al., 1999) and with a poor prognosis (Korkolopoulou et al., 2004;

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Ullenhag et al., 2007; Valente et al., 2006; Valnet-Rabier et al., 2005; Wang et al., 2007). In addition, cFLIPS upregulation is observed in gastric carcinoma and pancreatic cell lines (Mori et al., 2005; Nam et al., 2003). Preclinical data show that selective inhibition of cFLIP in combination with a reagent to trigger DR-induced apoptosis relieves the antiapoptosis function of cFLIP and induces apoptosis of cancer cells (Safa, 2012; Shirley & Micheau, 2013). Thus, it is important to understand how cFLIP regulation of apoptosis occurs.

11.3. The FLIP family and control of NF-κB activation NF-κB is a cellular transcription factor that is activated by diverse stimuli, such as cytokines, dsRNA, and viruses. It is a key initiator of the proinflammatory response, triggering the expression of over 100 cellular genes (Hayden & Ghosh, 2004, 2008; Mohamed & McFadden, 2009). Thus, many viruses encode molecules that inhibit NF-κB as a means for evading antiviral immune responses. At least for VACV, viruses that lack these NF-κB-inhibitory proteins are attenuated (Chen, Ryzhakov, Cooray, Randow, & Smith, 2008; Stack et al., 2005) in animal models of infection, indicating that NF-κB inhibition does indeed aid in viral pathogenesis and replication. NF-κB also controls the expression of genes involved in cell cycle and cellular proliferation. Some viruses such as KSHV and HIV take advantage of this characteristic and stimulate NF-κB to alter the host cell environment to make it suitable for virus replication (Guasparri et al., 2004). Two lines of evidence suggest that FLIPs may alter TNFR1-induced NF-κB activation. First, FLIPs interact with components of the TNFR1 complex I (discussed above). Second, overexpression of procaspase-8, which contains tandem DEDs, activates NF-κB (Chaudhary et al., 2000). This section compares the effects of the MC159 and MC160 FLIPs (inhibitors of NF-κB activation) and K13 and cFLIP (activators of NF-κB). 11.3.1 MC159: A protein that inhibits NF-κB activation Several reports show that MC159 protein inhibits TNF-induced NF-κB activation (Gil, Rullas, Alcami, & Esteban, 2001; Murao & Shisler, 2005; Randall et al., 2012). An initial study from our lab used large MC159 deletion mutants to identify a correlation between NF-κB inhibition and MC159–TRAF2 interactions (Murao & Shisler, 2005). These data suggest that MC159 prevents both the formation of complex I and subsequent IKK activation. More recently, evidence suggests that MC159–TRAF2

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interactions are not the sole mechanism to inhibit NF-κB (Randall et al., 2012). First, the use of smaller point mutant MC159 proteins identified mutated MC159 proteins that still coimmunoprecipitate with TRAF2 but no longer inhibit NF-κB (Randall et al., 2012). Second, MC159 inhibits NF-κB activation stimulated through several RIP- or TRAF2-independent pathways (Randall et al., 2012). A clue to the inhibitory function of NF-κB came from the finding that MC159 coimmunoprecipitates with members of the IKK complex (Randall et al., 2012) (Fig. 5). IKK consists of IKKα, IKKβ, and IKKγ in a 2:2:4 ratio, and IKKγ regulates the IKK complex (Israel, 2010). Using MEF devoid of IKKα, IKKβ, or IKKγ, it was determined that MC159 interacts with the IKKγ subunit of this complex (Randall et al., 2012). Furthermore, the MC159–IKKγ was observed when MC159 was expressed either independent of infection or within the context of a surrogate VACV infection or a wild-type MCV infection (Randall et al., 2012). Initial mutational analysis of

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Figure 5 The effect of individual FLIPs on NF-κB activation. IKK is a multisubunit complex. For this review, only the IKKα, IKKβ, IKKγ, and Hsp90 subunits are shown. IKK activation is a multistep process in which the IKKγ subunit becomes mono- and polyubiquitinated by the LUBAC complex (not shown here). These posttranslational modifications correlate with a conformational change of IKKγ from a bundled form to a linear form. This allows for IKKβ phosphorylation to occur, resulting in an active IKK complex. IKK phosphorylates IκBα, resulting in IκBα degradation and subsequent NF-κB activation. The K13 protein binds to IKK via interactions with IKKγ and this interaction is sufficient to alter the confirmation of IKKγ, resulting in NF-κB activation. It is thought that the p22 version of cFLIP functions in a similar manner to stimulate NF-κB. The MC159 protein inhibits IKK activation and this correlate with its interaction with IKKγ. The MC160 molecule also inhibits IKK activation, and does so directly by binding to Hsp90, which results in IKKα degradation.

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MC159 showed that the DED1 of MC159 is required for inhibition of NF-κB and associating with IKKγ (Randall et al., 2012), correlating the MC159-IKKγ-binding mechanism with an inhibitory phenotype. The MC159 regions required for inhibition of apoptosis are different from those required to inhibit NF-κB. Both MC159 DEDs must be present to inhibit apoptosis (Garvey, Bertin, Siegel, Wang, et al., 2002), while only the DED1 of MC159 inhibits NF-κB activation (Randall et al., 2012). How the distinct surfaces of MC159 compete for different cellular targets in virusinfected cells is still unclear. While it is well known that the TNFR1 triggers either apoptosis or NF-κB activation (Ofengeim & Yuan, 2013), the molecular switches that dictate whether a cell will die or survive when exposed to TNF are less clear. Identification of this cellular regulation may be extremely helpful for designing future TNF therapies as it may give scientists the ability to control whether a cell will undergo apoptosis or NF-κB activation. A recent report shows that MC159 stimulates NF-κB activation when using MC159-expressing Jurkat T cells (Challa, Woelfel, Guildford, Moquin, & Chan, 2010). In this case, MC159 increases RIP1–TRADD interactions suggesting that MC159 promotes the formation of complex I. Similarly, the ectopic expression of MC159 in HEK293T cells also activates NF-κB, albeit to very low levels as compared to the ability of TNF to activate NF-κB (Murao & Shisler, 2005; Randall et al., 2012). How the MC159 protein executes both NF-κB-activating and NF-κB-inhibiting functions and their impact of each phenotype on MCV pathogenesis is not yet fully understood. Given that differentiating keratinocytes are the host cells for MCV infection, perhaps NF-κB activation is beneficial to the virus by stimulating the expression of proteins that aid in cellular differentiation. In contrast, inhibition of NF-κB would be beneficial to the virus if this property diminished proinflammatory immune responses. 11.3.2 MC160, an NF-κB-inhibitory protein Similar to MC159, the MC160 protein inhibits TNF-induced NF-κB activation (Nichols & Shisler, 2006, 2009). Interestingly, while the MC159 protein targets the IKKγ subunit of the IKK complex (Randall et al., 2012), MC160 expression results in IKKα degradation, preventing the formation of an IKK complex (Nichols & Shisler, 2009) (Fig. 5). Mutational analysis reveals that two MC160 regions are important for NF-κB-inhibitory function, DED2, and the C-terminus of MC160. Hsp90 is part of the mature IKK complex, binding to and stabilizing IKKα. Further molecular characterization showed that the C-terminus of MC160, which is devoid of DEDs,


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interacts with Hsp90 and likely competes with Hsp90 for IKKα, resulting in degradation of IKKα subunits (Nichols & Shisler, 2009). Interestingly, the second DED (DED2) of MC160 also inhibits NF-κB activation. In this case, the DED2 binds to procaspase-8 and this correlates with inhibition of NF-κB activation (Nichols & Shisler, 2009). 11.3.3 The K13 vFLIP is an NF-κB-activating protein As mentioned above, some viruses activate NF-κB to stimulate production of cellular proteins involved in cell cycle regulation, ultimately aiding virus infection. For example, K13 activates NF-κB and this property is critical for KSVH to transform cells and abrogation of this K13 function results in a nonproductive KSHV infection (Chaudhary et al., 2000; Chaudhary, Jasmin, Eby, & Hood, 1999; Golks, Brenner, Krammer, & Lavrik, 2006; Kataoka et al., 2000; Liu et al., 2002; Matta, Sun, Moses, & Chaudhary, 2003; Sun, Matta, et al., 2003). A recent study shows that the IKKγ subunit of the IKK complex is critical for K13-induced NF-κB activation (Tolani et al., 2014). Here, we discuss the molecular mechanism used by K13 to activate NF-κB. K13 uses its DED1 to associate with the IKKγ subunit of IKK (Liu et al., 2002; Randall et al., 2012; Sun, Zachariah, & Chaudhary, 2003), similar to mutational analyses with MC159. When one considers that MC159 inhibits NF-κB, whereas K13 activates NF-κB, these differences offer a system to identify the molecular basis for FLIP regulation of IKKγ and cellular processes that regulate IKKγ. Both MC159 and K13 target the IKKγ protein of the IKK complex. For this reason, it is best to review the role of IKKγ to promote IKK activation (Fig. 5). For NF-κB activation to occur, IKKγ becomes ubiquitinated by LUBAC (M1-linked ubiquitin dimers) and E3 ligases (TRAF6; K63-linked polyubiquitination) (Clark, Nanda, & Cohen, 2013). It is thought that M1-linked ubiquitination converts IKKγ from the inactive (folded) to the active (linear) form. IKKγ unfolds to make extended interactions with IKKβ (Lo et al., 2009; Rahighi et al., 2009). Then, IKKγ K63-linked polyubiquitination creates a docking site for TAK1 (Clark et al., 2013; Deng et al., 2000) which then migrates to IKK and triggers the initial IKKβ phosphorylation at S177 (Clark et al., 2011). Next, ubiquitinated IKKγ continues its association with IKKα/IKKβ and alters the IKK conformation to stimulate IKKβ S181 phosphorylation. More recently, it was shown that IKKγ is posttranslationally modified by M1- and K63-linked polyubiquitination chains presumably such that linearization, TAK1 docking, and IKKβ activation occur rapidly (Emmerich et al., 2013). Regardless, these IKKγ–IKKβ

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interactions are critical to attract IκBα to IKKβ (Schrofelbauer, Polley, Behar, Ghosh, & Hoffmann, 2012). Activated IKKβ phosphorylates IκBα, which leads to K48-linked polyubiquitination of IκBα, and its delivery to the proteasome. Subsequently, freed NF-κB migrates to the nucleus to initiate the transcription of cellular genes. The mechanism of K13-induced NF-κB activation has been reported repeatedly. K13 coimmunoprecipitates with several cellular proteins involved in the NF-κB activation pathway, including RIP, NIK, IKK1, IKK2, and IKKγ (Field et al., 2003; Liu et al., 2002; Matta et al., 2003). The importance of IKK itself in facilitating K13-induced NF-κB activation was established by the demonstration that K13 has a reduced or lost ability to activate NF-κB in cells lacking IKK1, IKK2, or IKKγ, but not in cells deficient for RIP or NIK (Liu et al., 2002; Matta et al., 2003). These K13–IKK interactions are relevant physiologically because K13 associates with the activated IKK complex (Field et al., 2003), and K13-induced IKK activation is critical for the survival of PEL cells (Field et al., 2003). Several lines of evidence now establish that K13 binds to IKKγ to associate with the IKK complex: K13 binds to IKKγ in a yeast two-hybrid assay, and K13–IKKγ interactions are detected in vitro and in vivo using mass spectrometry (Bagneris et al., 2008; Field et al., 2003). Field et al. used a panel of IKKγ mutant proteins to show that K13 binds to IKKγ residues 150–272 (Field et al., 2003). The crystal structure of K13IKKγ was solved, revealing it to be a dimer of two K13–IKKγ complexes (Bagneris et al., 2008). This results in what the authors refer to as an “insect head,” with K13 representing the eyes and IKKγ representing the antennae (Bagneris et al., 2008). The DED1 regions of K13 form two deep clefts that are critical for binding an IKKγ molecule. Most important, when IKKγ is bound to K13, the conformation of IKKγ changes from “bundled” to extended (Bagneris et al., 2008). This presumably would allow for the docking of TAK1 to trigger IKKβ phosphorylation independent of IKKγ ubiquitination. Additionally, this IKKγ conformation is presumed to prevent IKKγ from engaging with proteins that are known to downregulate IKK activation (Bagneris et al., 2008). In support of this hypothesis, K13induced IKK activation is independent of cellular molecules that trigger IKKG ubiquitination (TAK1 and LUBAC) (Matta et al., 2012). Some insights into the K13 and MC159 mechanisms, and how they can result in opposing phenotypes, can be derived by comparison of their three dimensional crystal structures which show a high level of structural conservation, including the physical positioning of both DEDs (Bagneris et al.,


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2008; Li et al., 2006). However, there are also differences. For example, K13 lacks the H0 helix predicted to be present in the MC159 DED1 (Bagneris et al., 2008). Bagneris et al. argue that the first DED of MC159 has a truncated alpha helix 1, and this is predicted to alter the conformation of MC159 such that there are no grooves for IKKγ binding (Bagneris et al., 2008). However, the available MC159 crystal structure lacks these 6N-terminal residues, making it difficult to assess the true structure of the MC159 alpha helix 1 (Li et al., 2006; Yang et al., 2005). Interestingly, MC159–IKKγ interactions are detected by coimmunoprecipitation in cultured cells (Randall et al., 2012). This would suggest that MC159 may use a different portion of its DED1 to bind to IKKγ. However, there are some differences. K13 is a protein that is important for maintaining latency of KSHV. The KSHV RTA protein is the major transactivator of lytic gene expression, and it targets proteins for degradation via its E3 ubiquitin ligase activity (Gould, Harrison, Hewitt, & Whitehouse, 2009; Sun et al., 1998; Yang, Yan, & Wood, 2008; Yu, Wang, & Hayward, 2005). A recent publication shows that RTA downregulates K13-induced NF-κB activation by targeting also K13 for ubiquitination and degradation (Ehrlich, Chmura, Smith, Kalu, & Hayward, 2014). When this occurs, there is a decrease in NF-κB activation, concomitant with a switch from latency to a lytic infection. 11.3.4 cFLIPs and their functions in regulating NF-κB activation There was initial confusion about the role of cFLIP regulation of NF-κB. Several reports show that cFLIP inhibits NF-κB activation (Imamura et al., 2004; Kavuri et al., 2011; Kreuz, Siegmund, Scheurich, & Wajant, 2001), while others demonstrated NF-κB activation (Chaudhary et al., 2000; Golks et al., 2006; Hu, Johnson, & Shu, 2000; Kataoka et al., 2000; Kataoka & Tschopp, 2004). There is now greater clarity with respect to the role of cFLIP, due to studies revealing that cFLIPS/L–procaspase-8 interactions result in different biochemical events in apoptosis versus NF-κB activation. For example, it is thought that cFLIPS–procaspase-8 interactions block recruitment of procaspase-8 to complex IIa or CD95 to inhibit NF-κB activation (Matsuda et al., 2014). As discussed previously, high cFLIPL level prevents procaspase-8 from converting to an active heterotetramer, and low cFLIPL levels will trigger the production of a p43 form of procaspase-8, which can eventually result in apoptosis (Fig. 5). During NF-κB activation, cFLIPL–procaspase-8 interactions instead result in cFLIPL cleavage either at residue 375 (resulting in a 43-kDa product; p43) or at residue 196 (resulting in a 22-kDa product;

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p22). The p43 version now allows RIP and TRAF2 to bind to the procaspase-8–p43 complex, inducing IKK activation and NF-κB activation (Kataoka & Tschopp, 2004; Matsuda et al., 2014). In contrast, p22 binds to the IKKγ subunit of the IKK complex to activate NF-κB (Golks et al., 2006). The DED1 of cFLIPL is sufficient to activate NF-κB (Golks et al., 2006) as is the K13 DED1. Interestingly, the MC159 DED1, which is most divergent from all DEDs, also is sufficient to inhibit NF-κB activation (Randall et al., 2012).

11.4. The FLIP Family and Control of IRF3 Activation IFNβ is a type I IFN with potent antiviral effects. The IRF3 transcription factor controls the production of IFNβ. One of the best-characterized IRF3 activation pathways starts with the apical RIG-I and MDA5 molecules, which are part of the DExD/H-box RNA helicase family (Chiang, Davis, & Gack, 2014). These molecules sense and bind to distinct sets of viral RNAs. RIG-I detects single-stranded RNA with a 5-triphosphate moiety and short dsRNAs, and these forms of dsRNA are produced during virus replication. Recent evidence suggests that RIG-I also detects RNA species generated in DNA virus infections (Ablasser et al., 2009; Chiu, Macmillan, & Chen, 2009; Minamitani, Iwakiri, & Takada, 2011). In contrast, MDA5 does not require a 50 -triphosphate for binding to RNA. Instead, it binds to longer dsRNA molecules, including those forming web-like aggregates (Kato et al., 2008; Pichlmair et al., 2006). Major steps in the MDA5–IRF3 activation pathway are as follows (Chiang et al., 2014). Viral RNA molecules interact with MDA5, resulting in MDA5 binding to and activating the mitochondrial antiviral-signaling protein (MAVS) (Loo & Gale, 2011) which then clusters on the mitochondrial surface (Hou et al., 2011; Moresco, Vine, & Beutler, 2011; Seth, Sun, Ea, & Chen, 2005; Xu et al., 2005) and recruits a MAVS signalosome, which includes TRADD, TANK, and TRAF3 (West, Shadel, & Ghosh, 2011). MAVS aggregation results in activation of the TBK1–IKKε complex (Fitzgerald et al., 2003; Oganesyan et al., 2006; Sharma et al., 2003). Activated TBK1–IKKε phosphorylates IRF3, which dimerizes either with itself or with IRF7 (Fitzgerald et al., 2003). An IRF3-containing complex migrates to the nucleus and binds to ISRE3 in the IFNβ gene promoter to stimulate IFNβ gene transcription. IFNβ is secreted from cells, and this cytokine binds to the IFNAR-1–IFNAR2 receptor on neighboring cells. This stimulates a second signal transduction pathway that activates JAK/STAT to activate the transcription of IFN-stimulated genes that are known to have antiviral effects.


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Identification of these cellular members of the IFNβ signal transduction pathway also resulted in the discovery of viral molecules that inhibit these events (Chiang et al., 2014; Randall & Goodbourn, 2008). Given that viral immunoevasion proteins often interact with host proteins to provide maximal efficiency of replication, the study of these viral proteins has greatly increased our understanding of IFNβ production and regulation. In this section, I discuss the recent publications that study the effect of FLIPs on this IRF3 activation pathway. 11.4.1 MC159 and inhibition of IRF3 The best-studied poxvirus is VACV, and it expresses several proteins that inhibit the MDA5–-IRF3 activation pathway, including C6 and N2 (Smith et al., 2013). C6 indirectly inhibits TBK1–IKKε activation by interfering with the activation of the MAVS signalosome (Unterholzner et al., 2011), while N2 inhibits an event post-IRF3 activation/phosphorylation (Ferguson et al., 2013). MCV has no genes that share sequence homology to the C6L or N2L genes. One possibility is that MCV infection does not trigger IRF3 activation, which would negate the need for such genes. This seems unlikely since type I IFN has been detected near MC lesions (Vermi et al., 2011). An intriguing report gave the first indication that MC159 may possess IRF3-inhibitory function: overexpression of MC159 prevented the activation of the IFNβ enhancer, a promoter that contains binding sites for IRF3 as well as other host cell transcription factors (Balachandran & Barber, 2007). Furthermore, this inhibitory event occurred upstream of IRF7 activation (Balachandran & Barber, 2007). A caveat of this study is that the IFNβ enhancer contains NF-κB-binding sites (Maniatis et al., 1998). Thus, one could not exclude the possibility that the observed MC159-inhibitory effect is indirectly due to the MC159 ability to inhibit NF-κB. Randall et al. used three approaches to clarify whether MC159 directly inhibits IRF3 (Randall, Biswas, Selen, & Shisler, 2014). First, luciferase reporter assays were performed using MEFs that lack the p65 subunit of NF-κB. Thus, there is virtually no functional NF-κB to interact with the IFNβ enhancer. Under these conditions, MC159 inhibits IFNβ-controlled luciferase activity to a level similar to that observed in wild-type MEFs, suggesting that MC159 inhibits IRF3 activation independent of its other inhibitory functions. In a second assay, a luciferase reported gene under the control of a synthetic IRF3 promoter was used. Here, MC159 protein expression prevented IRF3 activation triggered by overexpression of the upstream members of the MAVS–IRF3 activation pathway, including MAVS, TBK1, and IKKε.

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Third, the DED2 of MC159 possesses IRF3-inhibitory function. Since DED2 is not sufficient to inhibit apoptosis or NF-κB activation (Murao & Shisler, 2005; Randall et al., 2012), MC159 inhibition of IRF3 is a novel-inhibitory function. MC159 inhibits IRF3 activation when induced by the overexpression of MAVS, TBK1, or IKKε, but not by a constitutively active IRF3. These data suggest that MC159 inhibits an event occurring upstream of IRF3 activation. Because MC159 inhibits TBK1 activation and also coimmunoprecipitates with TBK1:IKKε (Randall et al., 2014), it is thought that MC159–TBK1 interactions prevent TBK1 activation. How MC159 prevents TBK1 activation remains unknown. Perhaps, the most logical model is that MC159 allows TBK1–IKKε interactions but prevents the MAVS signalosome from interacting with the TBK1–IKKε complex. 11.4.2 MC160 and inhibition of IRF3 Ectopic expression of MC160 also inhibits IRF3 and TBK1 activation (Randall et al., 2014). Surprisingly, MC160 does not coimmunoprecipitate with TBK1 or IKKε, suggesting that MC160 uses a different mechanism than MC159 to inhibit IRF3 activation. There are reports that now indicate that other DED-containing molecules (e.g., FADD, TRADD, procaspase-8) control IRF-3 activation (Balachandran, Thomas, & Barber, 2004; Kovalenko et al., 2009; Rajput et al., 2011; Sears et al., 2011). Since MC160 is reported to interact with each of these molecules, MC160 may use one or more of these interactions to inhibit IRF3 (Nichols & Shisler, 2006, 2009; Shisler & Moss, 2001). MC160 also binds to RIP1 (Nichols & Shisler, 2006), a cellular molecule that is part of the TNFR1 complex I and stimulates NF-κB activation. When mapping the DEDs of MC159 and MC160 responsible to inhibit IRF3 activation, it was found that the DED2 of MC160 has this inhibitory function, while either DED1 or DED2 of MC159 possesses this function (Rajput et al., 2011). 11.4.3 cFLIP and inhibition of IRF3 Mouse cells lacking the caspase-8 gene have higher levels of TBK1 and IRF3 activation (Kovalenko et al., 2009). Two recent papers now show the molecular mechanism of caspase-8 inhibition of IRF3. Sendai virus infection stimulates RIP1 migration to the RIG-I signaling complex (Rajput et al., 2011). This results in RIP1 polyubiquitination, a modification that enhances RIG-I–MAVS complex assembly (Rajput et al., 2011). This same


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RIP1 modification also leaves RIP1 susceptible to caspase-8 cleavage and, once RIP1 is cleaved by caspase-8, IRF3 activation is halted (Rajput et al., 2011). In a second mechanism for caspase-8 inhibition, caspase-8 cleaves IRF3, and this cleaved form of IRF3 is now targeted for polyubiquitination and degradation by the 26S proteasome (Sears et al., 2011). Since cFLIP binds to procaspase-8, a reasonable hypothesis is that cFLIP also regulates IRF3 activation. This concept is supported by results demonstrating that (i) overexpression of cFLIPL prevents MAVS-induced IRF3 activation (Randall et al., 2014), and (ii) cFLIP/ MEFs have increased levels of IRF3 activation as compared to wild-type MEFs (Handa, Tupper, Jordan, & Harlan, 2011). A logical prediction is that cFLIPS– procaspase-8 interactions would block the activation of caspase-8 to ultimately inhibit IRF3 cleavage. A recent publication from Buskiewicz supports this idea (Buskiewicz et al., 2014); cFLIPS expression reduces MAVS–caspase-8 association, and this correlates with a decrease in IFNβ production. More challenging is predicting how cFLIPL may inhibit IRF3 activation on a molecular level. While our lab shows that overexpression of cFLIPL inhibits IRF3 activation (Randall et al., 2014), Buskiewicz et al. find that cFLIPL expression induces IFNβ secretion in response to coxsackievirus B3 infection, implying that cFLIPL activates IRF3 (Buskiewicz et al., 2014). One potential reason for these discrepancies may be due to the fact that poly I:C, overexpression of TBK1, or coxsackievirus infection result in different levels of IRF3 activation.

12. OTHER MCV IMMUNE EVASION MOLECULES 12.1. MCV MC54, an IL-18-binding protein IL-18 responses are critical for defense against viral infection (Dinarello, 1999b; Okamura et al., 1995). IL-18 potently activates macrophages, inducing production of several cytokines including IFNγ. It also potentiates NK cell and Th-1 responses (Dinarello, 1999b; Okamura et al., 1995). The aberrant regulation of IL-18 can have deleterious effects in humans. Notably, IL-18 is high in several autoimmune and inflammatory disorders (Dinarello, 1999a, 1999b; Dinarello et al., 1998). As such, cells themselves have a strategy to downregulate IL-18 by expressing a cellular human IL-18-binding protein (IL-18bp). Soon after the discovery of the huIL-18bp, Xiang et al. found that the MCV MC53 and MC54 proteins act as huIL-18bp homologs binding to

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and neutralizing IL-18 (Xiang & Moss, 1999, 2001, 2003). Further characterization of MC54 reveals that two forms of the protein are active: the fulllength protein binds to glycosaminoglycans through its C-terminal tail and to IL-18 with its N-terminus, while a furin cleavage product binds to IL-18 only (Xiang & Moss, 1999, 2001, 2003). MCV and VACV do not specify homologous immune evasion proteins with the exception of a viral IL-18-binding protein (Senkevich et al., 1996, 1997). Interestingly, there are MC54 homologs in members of the Yatapoxvirus genus as well as the Orthopoxvirus genus (Esteban, Nuara, & Buller, 2004; Krumm, Meng, Li, Xiang, & Deng, 2008; Krumm, Meng, Wang, Xiang, & Deng, 2012; Smith, Bryant, & Alcami, 2000). One goal is to identify small molecules that inhibit IL-18, which would presumably rebalance inappropriate immune responses. As such, the comparative study of poxviral and human IL-18bps provides an excellent means for identifying the IL18bp–IL-18 contact points that are most important for neutralizing function. The comparison of these IL-18bps revealed that the human and orthopoxvirus IL-18bps are monomeric when binding to IL-18 and use a highly conserved phenylalanine residue to bind to IL-18 (Esteban et al., 2004; Krumm et al., 2008, 2012; Smith et al., 2000). In contrast, the yatapoxvirus IL-18bp forms a dimer that has a higher binding affinity for IL-18 as compared to a monomeric form (Krumm et al., 2008). Despite these differences, all viral IL-18bps bind to the same region of IL-18, a region that is required for IL-18 interaction with its cognate receptor (Krumm et al., 2008, 2012).

12.2. MCV MC148, a viral chemokine Immune cell recruitment is critical for the clearance of poxvirus infection. MCV encodes the viral chemokine, MC148, which acts to inhibit chemotaxis of immune cells (Damon, Murphy, & Moss, 1998; Luttichau, Gerstoft, & Schwartz, 2001; Luttichau et al., 2000). To date, the MC148 proteins from MCV-1 (MC148R1) and MCV-2 (MC148R2) have been studied. Initial characterization of MC148R1 shows that it inhibits the migration of monocytes, lymphocytes, and neutrophils in response to several types of CC and CXC chemokines, suggesting that it could function as a broad agonist of chemokines (Damon et al., 1998; Krathwohl, Hromas, Brown, Broxmeyer, & Fife, 1997). In agreement with these studies, Jin et al. found that MC148R1 was capable of blocking both CXCL12αmediated and CCL3 (MIP-1α)-mediated chemotaxis, whereas MC148R2


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only inhibited CCL3 ( Jin, Altenburg, Hossain, & Alkhatib, 2011). Immunoprecipitations revealed that MC148R1 interacted with CXCL12-α, preventing CXCL12-α from binding its CXCR4 receptor ( Jin et al., 2011). As such, one model is that MC148R1 binds to chemokines, preventing association with a chemokine receptor as a means to inhibit chemokine-signaling events. Other data show that MC148R1 binds to the CCR8 chemokine receptor (Luttichau et al., 2000), indicating that MC148 instead binds to a receptor to prevent chemokine–chemokine receptor interactions. Since different cell lines and different sources of purified MC148R1 were used in these studies, the basis for these differences is still unclear. Regardless of the mechanism, the presence of MC148R1 is expected to prevent recruitment of immune cells such as monocytes, lymphocytes, and neutrophils to the site of infection. By this, MC148 may contribute to the persistent nature of MCV infections.

12.3. MCV MC007, a pRb-binding protein MCV infection produces benign neoplasms that persist for many months. In addition, cellular proliferation is present at the sites of MCV infections. Therefore, it is not surprising that MCV encodes a viral protein (MC007) that manipulates cellular proliferation. In 2008, Mohr et al. characterized the MCV MC007 protein as an inhibitor of the retinoblastoma pRb/E2F complex (Mohr et al., 2008). The pRb protein is a critical regulator of cell proliferation through inhibition of the E2F family of transcription factors, and deregulation of pRb has been linked to tumorigenesis (Nevins, 2001). MC007 binds to pRb through the LxCxE pRB-binding motif, resulting in MC007-mediated sequestering of pRb to the mitochondria (Mohr et al., 2008). Moreover, MC007’s inhibitory effects were potent enough to transform rat kidney cells, suggesting MC007 may contribute to MCV tumor-like lesions. Further studies are needed to full understand MC007’s role in MCV lesion production and pathogenesis.

12.4. MC66, a glutathione peroxidase homolog The MC66 protein initially was identified as a homolog to the eukaryotic glutathione peroxidase (Shisler et al., 1998). Like the cellular glutathione peroxidase, MC66 is a selenoprotein, incorporating the trace element selenium (Shisler et al., 1998), which, like its cellular homolog, prevents apoptosis triggered by ultraviolet irradiation and hydrogen peroxide (Shisler et al., 1998).

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13. CONCLUSIONS MCV is unique when compared to other poxviruses because of its narrow host range of human keratinocytes and its unique immune evasion molecule. The study of MCV has its drawbacks, which have slowed the progress of understanding how this virus causes disease. Indeed, the lack of a system to propagate wild type or create mutant MCV limits the study of MCV immune evasion proteins to conditions where MCV proteins are expressed independent of infection or within the context of a surrogate virus infection. Nevertheless, the study of this virus gives us opportunities to identify novel immune evasion mechanisms of viruses, which will in turn advance the understanding of viral pathogenesis and potentially identify new cellular mechanisms that regulate the immune response to disease. No FDA-approved treatment of MCV exists; thus, the continued study of MCV immune evasion molecules may aid in the development of new topical therapies to resolve MCV lesions.

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Molluscum contagiosum virus.

Molluscum contagiosum.

Molluscum contagiosum.

Warts and molluscum contagiosum.

Two Epidemics of Molluscum Contagiosum.

Seroprevalence of Molluscum contagiosum virus in German and UK populations.

Molluscum contagiosum -- a defective poxvirus?

Giant molluscum contagiosum following splenectomy.

Lymphoma: immune evasion strategies.

Atypical infantile genital Molluscum contagiosum.

[Multiple molluscum contagiosum of the child].

Treatment of molluscum contagiosum with ingenol mebutate.

Histopathological features of molluscum contagiosum other than molluscum bodies.

Langerhans Cell Hyperplasia From Molluscum Contagiosum.

Ocular molluscum contagiosum atypical clinical presentation.

Isolated giant molluscum contagiosum mimicking epidermoid cyst.

Immune Evasion Strategies of Trypanosoma cruzi.

Characterization of a molluscum contagiosum virus homolog of the vaccinia virus p37K major envelope antigen.

Interdigital molluscum contagiosum on the foot.

Eruptive xanthomas masquerading as molluscum contagiosum.

The utility of cantharidin for the treatment of molluscum contagiosum.

Inhibition of interferon gene activation by death-effector domain-containing proteins from the molluscum contagiosum virus.

Epidemiology of molluscum contagiosum in children: a systematic review.

Treatment of molluscum contagiosum in adult, pediatric, and immunodeficient populations.