Molluscum Contagiosum Virus

Abstract and Introduction

Abstract

The molluscum contagiosum (MC) virus (MCV) is a dermatotropic poxvirus, and the

causative agent of MC. Unlike smallpox and human monkeypox diseases, MC is

nonlethal, common and worldwide. Additionally, little inflammation is associated

with MC papules, and MC can persist for months to years. Such a prolonged infection

implies that MCV successfully manipulates the host environment. This review

highlights recent findings that reveal how MCV infections manipulate localized host

immune responses and which immune response are key for the eventual resolution of

MC. Also highlighted here are the MCV proteins that inhibit apoptosis, inflammation

and immune cell recruitment or that induce cellular proliferation, with discussion as

to how these proteins dampen localized antiviral immune responses. Lastly, this

review discusses how the immune evasion tactics of MCV have led to insights about

specific functions of the human innate and adaptive immune responses.

Introduction

The molluscum contagiosum (MC) virus (MCV) is a dermatotropic poxvirus that

causes umbilicated skin papules (MC) in humans. While MC is a benign disease, it is

important to human health because of its common incidence, its persistence and the

lack of a cure. As such, MC impacts patients and their families psychologically and

economically.

Here, we review recent histological studies of MC and epidemiological studies of

MCV infections, which show that inflammatory responses control MCV-induced

disease. However, MCV encodes proteins that inhibit apoptosis, inflammation and

immune cell recruitment or induce cellular proliferation, and these proteins are

presumably a means to battle the immune response. Potential cures for MCV may lie

in neutralizing these immune evasion proteins to tip the scales in favor of the host

immune system to eliminate MCV infections. Moreover, systems must be developed

to propagate MCV and to recapitulate the disease in an animal model for future

studies. This review highlights recent advances made in the field of MCV

pathogenesis, and the clinical and biological implications of these findings in

understanding how MCV manipulates the host cell and its surrounding environment

to cause persistent disease.

MCV: A Unique Poxvirus

MCV is a member of the Poxviridae family. This is an extensive group of viruses

with a broad host range that includes both vertebrates and invertebrates.[1] Poxviruses

are characterized by a large, dsDNA genome of 130–300 kbp, which encodes nearly

200 proteins. Poxviruses are unique from other viruses with DNA genomes, because

poxviruses replicate and transcribe their genome exclusively in the cytoplasm of their

host cell. To perform this task, poxviruses encode their own DNA replication and

transcription machinery. Poxviruses encode a diverse set of proteins that modulate

host–pathogen interactions.[1,2] The study of poxviruses has led to significant

advancements in the fields of virology, cellular biology and immunology.

The MCV is the sole member of the Molluscipoxvirus genus, and causes MC.[1,2] The

best-studied poxviruses belong to theOrthopoxvirus genus, and include: variola virus,

the causative agent of smallpox; and monkeypox virus, which causes monkeypox in

humans. Vaccinia virus (VACV) is highly similar to the variola and monkeypox

viruses, and it is used as a vaccine to protect against both diseases.[1,2]

There are several striking characteristics of MCV that makes it unique among other

poxviruses that cause disease in humans. First, MCV is the only poxvirus known to

cause a persistent disease (MC) in humans. By contrast, the monkeypox and variola

viruses cause acute diseases. Smallpox and human monkeypox also have much higher

morbidity and mortality rates than MC, which is a nonlethal disease.[1,2] Moreover,

while MCV infection remains limited to keratinocytes, both the variola and

monkeypox viruses have a systemic disease effect. Finally, MCV is the only poxvirus

other than the variola virus that is exclusively pathogenic to humans.[1,3] Variola virus

was eradicated owing to an extensive vaccination campaign. As such, MCV is the

only remaining poxvirus that solely infects humans worldwide.

Epidemiology of MCV Infections

MCV infections are common in healthy individuals, with incidents in humans ranging

from 2.7% (Spain) to as high as 17% (The Netherlands).[4,5] Serological studies of

Australians show that 6–26% of persons test positive for anti-MCV antibodies,[6] indicating that subclinical infections are also common.

MCV can be transmitted via direct skin-to-skin or mucous membrane contact, or by

fomites.[7] MC is most commonly diagnosed in children, and it appears that children

involved in close contact sports, such as wrestling, have a higher risk of MC. Other

risk factors for childhood MCV infection are age, fomite sharing, skin-to-skin

contact, tropical climate and living in close contact with others.[8] A recent

epidemiology study in Japan estimates that 20% of children contract MCV before the

age of 6 years.[9] Similar studies in The Netherlands (in 1987 and 2001) and UK

(1994–2003) estimate the childhood incidence of MCV to be approximately 17%

with the majority of cases being in children <14 years of age.[5,10,11] A study of MCV

incidence in American–Indians and Alaskan native populations found the majority of

MC cases to be in individuals <15 years old.[12] Most childhood cases of MCV occur

on the face and trunk and resolve within 3 months.[13] Of note, ocular and oral MCV

infections in immunocompetent children have also been reported.[14,15] MC is also

sexually transmitted. When this occurs, the MC lesions initially are present in the

groin area.[7]

MCV infection rates and the risk of contracting MCV are higher in

immunocompromised versus healthy patients.[7] This implies that the immune

response is important for limiting or resolving MCV infections. For example, MCV

infections are present in 5–8% of the HIV-positive population, which is higher than in

noninfected people.[16] Additionally, HIV-positive individuals have up to a 33%

increased risk of contracting MCV as compared with noninfected individuals.[17–

21] Patients receiving anti-TNF therapy for rheumatoid arthritis are also reported to

have increased susceptibility to MCV infection.[20] People with congenital immune-

suppressive conditions are also at increased risk for MCV infection. For example,

mutations in the DOCK8 gene, a guanine nucleotide exchange factor predicted to play

a role in cytoskeleton rearrangement, have been linked to recurrent cutaneous viral

infections including MCV.[21] Several reports show that atopic dermatitis increases the

risk of a MCV infection, the number of MC lesions and the risk of relapse.[9,13,22,23] The continued study of these and other populations that have increased rates

of MCV infection are expected to enable scientists to identify the important host anti-

MCV immune responses that are required for resolution of infection.

Characteristics of MC Papules & Treatments for MC

MCV only replicates in human keratinocytes.[7,24] This is in contrast to the variola

virus, which is known to infect many different tissues, including the skin, and cause

systemic infections.[1] Thus, MCV is transmitted via abrasions in the skin, followed

by a 14–50-day incubation period before MC lesions are detectable.[25] MCV causes

the formation of benign, umbilicated papules in the epidermis. Lesions may group in

clusters or appear individually and, in healthy individuals, these lesions are usually

less than 1 cm in diameter, and appear waxy or pearl-like. MC lesions can persist for

6 months to 5 years.[25] The persistent nature of MCV infections can be considered in

two manners. First, individuals may have areas of lesions that clear, with new lesions

appearing in new areas over a prolonged period of time. This is a pattern common

among children, and likely occurs owing to autoinoculation. A second mode of

persistence, often observed in immunocompromised individuals (see below), would

be a small number of lesions that persist over a long period of time. Regardless, the

MCV infection is self-limiting.

It is still unknown what portion(s) of the immune response mediates the resolution.

Immunocompromised patients usually possess much larger MC lesions (greater than

2 cm in size), referred to as giant MC, and MCV infections in these patients can

persist indefinitely.[7,26] Giant MC is common among HIV-positive patients and

patients on immunosuppressive therapies such as transplant recipients.[19] The

presence of giant MC in immunocompromised patients suggests that the immune

system of an individual is important in controlling the size and spread of MC lesions.

The histological hallmarks of MC lesions are hypertrophy and proliferation of

keratinocytes, and viral inclusions called molluscum bodies.[27] Not surprisingly,

MCV encodes proteins that stimulate proliferation (MC007) and inhibit apoptosis

(MC159), and it is likely that these proteins contribute to this phenotype.[28–30] Recent

evidence suggests that MCV infection manipulates the keratinization process in MC

lesions and adjacent epidermis.[27] Variation in lesion appearance, number and

location all affect the likelihood of proper diagnosis.[31] An MCV infection is readily

diagnosed when lesions are present with characteristic dome-shaped umbilicated

papules. Expression of these papules will reveal an umbilicated core and a white

curd-like substance. MC is also identified by the relative absence of inflammation

surrounding the papules. When MCV causes atypical lesions or when MCV

infections occur in conjunction with other dermatological conditions, diagnosis is

more difficult. In these cases, a biopsy and histopathological evaluation of the lesion

is required to determine the presence of MCV, and is determined, in part, by the

presence of hyperplastic epidermal cells. Dermoscopy, a noninvasive method for

visualizing lesions, represents the most popular tool for diagnosis.[32] Recently,

reflectance confocal microscopy, a noninvasive technique for imaging skin at the

cellular level, has emerged as a secondary method for examining potential MCV

lesions.[33] The validity of both techniques has been confirmed using histological

methods.[32,33]

Currently there is no cure for MCV infection. Since MCV lesions will resolve

without intervention, treatment goals are often limited to preventing autoinoculation

and minimizing the duration of infection. For children, treatment of MC usually

occurs only after the lesions persist for more than a few months. While no treatment

is 100% effective for clearing MC lesions, the most common treatment strategies

include topical administration of cantharidin or imiquimod, or curettage.[7,34] Curettage has been found to have fewer adverse effects than the other methods.[35] Interestingly, both cantharidin and imiquimod activate immune responses when

applied topically, implying that the normal anti-MCV immune response is not

vigorous enough or is sufficiently inhibited by MCV immune evasion molecules to

eliminate virus-infected cells.[36] It should be noted that cidofovir, a nucleoside analog

that inhibits the replication of other poxviruses, is often used for treating MC in

immunocompromised patients.[37]

The MCV Genome

There are four subtypes of MCV: MCV-1; MCV-2; MCV-3; and MCV-4.[38] MCV-1

is the most commonly reported subtype found.[39] The MCV-1 genome was sequenced

in 1996.[40]Sequence analysis revealed that MCV encodes approximately 182

predicted proteins, 105 of which have homologs in VACV, a poxvirus that is in the

same genus as the variola and monkeypox viruses.[40,41] The genes that are most

conserved between MCV and VACV are localized at the center of the genome, and

encode proteins that are essential for structure, replication and transcription. Genes

unique to MCV are located at the ends of its DNA genome, and these genes likely

encode proteins that function as host-range and immunomodulatory factors.[40–42] As

such, the MCV genome has the same organizational pattern as other poxviruses.[1]

The MCV host range is limited to humans. Interestingly, recent reports have

described a MCV-like disease in animals including donkeys, chickens, sparrows,

pigeons, chimpanzees, kangaroos, dogs and horses.[43] In situ hybridization analysis of

viral DNA from human MCV and a MCV-like virus isolated from a horse suggests

that considerable homology exists between human and equine MCV.[44] Similar to

human MCV, all attempts to culture these MCV-like viruses have been unsuccessful.

In addition, no animal MCV infection has been successfully experimentally

transmitted from one animal to another. Further investigation is needed to understand

how these MCV-like viruses are related to MCV in terms of genomic sequences,

immune evasion molecules and host range. Such information may shed light on the

virus–host interactions that are important for productive MCV infections.

Orf is a poxvirus that causes a dermatological infection in sheep.[45] Delhon et

al. published the sequence of the Orf virus and found several striking similarities to

MCV, despite the fact that MCV and Orf are in different genera. These similarities

include a high CG content of the genome, the presence of three putative immune

evasion orthologs and the lack of viral genes involved in nucleotide metabolism.

Based on these findings, the authors suggest that Orf and MCV are distinct from

other poxvirus genera.[45] These similarities may make Orf an attractive candidate for

a surrogate virus to study the role of MCV immunomodulatory proteins in

pathogenesis.

MCV Propagation

Attempts to propagate infectious MCV in tissue-cultured cells have been

unsuccessful.[46] One reason for this difficulty may lie in the fact that MCV has a

narrow tissue tropism (keratinocytes). Interestingly, two reports identify initiation of

MCV replication, but not MCV propogation. First, Bugert et al.show that MCV

undergoes an abortive infection in a human primary fibroblast cell line (MRC-5).[47] In these cells, MCV expresses early and late proteins, but no viable progeny is

produced.[47] MCV also replicates in human foreskin fragments that were implanted

into the renal capsule of mice.[48] While there was virus replication in the implanted

tissue, as measured by genome replication, no infectious virions were produced.[48]It

is possible that MCV replication requires factors only present in skin cells;[3] yet,

another possibility is that an as-yet-unidentified intracellular antiviral host factor is

triggered in cell lines during an MCV infection, and that MCV does not encode an

immune evasion protein that inhibits that particular innate immune defense. A similar

scenario occurs for myxoma virus, a poxvirus for which replication is only supported

in rabbit cells. It was observed that myxoma virus replication occurs in human cells

only when type I interferons (IFNs)are inhibited.[49]Another hypothesis is that MCV

cannot be propagated because it lacks some genes that are otherwise present in other

poxviruses that allow for replication in multiple cell types.[3,41] For example, MCV

lacks the viral kinase B1 encoded by VACV.[40,41] B1 plays a critical role in

replication by phosphorylating the cellular protein BAF.[50] Indeed, a mutant VACV

lacking the B1R gene is replication incompetent.[50]Until this technical barrier is

overcome, researchers will continue to use MCV collected from lesions of patients as

the source of virus for infections.[51] It should be noted that, 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.[52]

Immune Response to MCV Infection

The study of MCV pathogenesis remains limited owing to the lack of a propagation

system and a suitable animal model that recapitulates human disease. Despite these

limitations, studies of healthy and immunocompromised human patients with current

or past MCV infections have given us greater understanding of the roles of the innate

and acquired immune responses in clearing MCV infections.

There is growing evidence that innate immune signaling molecules are key in the

clearance of MCV infections. It appears that TNF-α is a crucial host defense against

MCV. First, TNF-α is highly expressed in MCV lesions and the surrounding tissues.[53] Second, patients who are being treated with anti-TNF therapy for rheumatoid

arthritis have an increased susceptibility to MCV infection.[20] Expression of TLR3

and 9, which detect viral RNA and DNA, respectively, is also upregulated in MCV

lesions,[53] suggesting that cellular molecules controlled by the IRF-3 and NF-κB

transcription factors (i.e., cytokines and IFN-regulated genes) are synthesized in

response to MCV infections. As such, we predict that the MC159 and MC160

proteins, which each inhibit NF-κB (see below section), are important in

downregulating this portion of the innate immune response.

A recent study by Vermi et al. presents histological evidence showing that infiltration

of plasmacytoid dendritic cells (DCs) and IFN-induced DCs along with innate

immune signaling are responsible for spontaneous regression of MCV lesions.[54] They propose that two types of MCV lesions exist in healthy individuals:

inflammatory MC (I-MC) lesions and noninflammatory MC (NI-MC) lesions.[54] I-

MC lesions display many immunogenic properties including the expression of MHC

class I and II proteins, and infiltration of immune cells such as cytotoxic T cells, NK

cells and plasmacytoid DCs. Furthermore, they found evidence of type I IFN

activation, TNF-α-induced apoptosis and NF-κB activation in cells near I-MC

lesions. Conversely, NI-MC lesions did not express MHC or IFN molecules, nor was

there immune cell infiltration. It is likely that the many immune evasion genes

encoded by MCV (e.g., MC54, MC148,MC159 and MC160) contribute to the low

levels of inflammation and immune cell infiltration observed in NI-MC lesions. 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.[13,54] When taken together, these data imply that a strong host immune response is

important for the clearance of MC lesions. What remains a mystery, however, is what

immune system events tip the scales for an NI-MC lesion to become an I-MC lesion

during MC infections, and what events are required for the resolution of MC lesions.

To further confound the issue is the fact that MCV encodes and expresses immune

evasion genes,[47] raising the question of how the increases of inflammatory signals

are detected in some MCV lesions despite the presumed presence of viral immune

evasion proteins (see below).

MCV infection also induces the acquired immune response. With respect to B-cell-

mediated immunity, two publications show that 58–77% of MCV-infected

individuals possess antibodies against MCV.[6,39] It should also be noted that anti-

MCV antibodies, which are indicative of a past MCV infection, are detected in 6–

23% of the general population.[6,39] However, it is unclear whether anti-MCV

antiserum is sufficient for clearing MCV infections, because many individuals with

persistent MCV infections have high anti-MCV antibody titers.[39] Less is known

about cytotoxic T-cell responses to MCV. Perhaps the strongest evidence that T-cell-

mediated immunity is important in resolving MC lesions are data showing that

patients who lack appropriate T-cell responses (i.e., AIDS patients) have larger MC

lesions that persist.

Like most poxviruses, nearly 30% of the MCV genome encodes putative immune

evasion molecules.[40,41] However, most immunomodulatory proteins encoded by

MCV are not present in variola virus or other well-studied poxviruses. One

hypothesis is that the unique immune defense molecules of MCV are responsible for

its narrow host range and limited tropism, as well as the persistent nature of MC

infections. Alternatively, MCV may possess a unique set of immune evasion

molecules to counteract the immune response threats specific to the skin

environment. Either way, neutralizing such viral strategies is a potential means of

ameliorating the viral pathology. Indeed, the rigorous study of these viral proteins

could identify ways to reconstitute the immune response and prevent MC. As an

equally important correlate, the identification of these immune evasion molecules

could identify novel cellular signaling mechanisms that up- or down-regulate innate

immune responses, leading to a greater understanding of the immune system and

allowing for prevention of many immune-mediated diseases. Here we will describe

five MCV immune evasion proteins that are characterized (Table 1)

MCV MC159: Modulator of Cell Death & Inflammation

The MC159 protein is 241 amino acids in length and is approximately 25 kDa.[55,56] MC159 is characterized by the presence of two tandem death effector domains

(DEDs). This protein exhibits a diffuse staining pattern via fluorescence microscopy

suggesting it primarily localizes to the cytoplasm.[29]The MCV MC159 and MC160

proteins belong to a family of proteins known as FLICE;FADD-like interleukin-1 β-

converting enzyme-inhibitory proteins (FLIPs).[57] This family of proteins was

discovered during early studies of the signal transduction pathways that are

responsible for the apoptosis of cells, and their discovery resulted in the identification

of novel cellular FLIPs (cFLIPs) that regulate apoptosis. Here, we will review the

common signaling events involved in apoptosis and then the mechanism that MC159

and its homologs use to inhibit apoptosis (Figure 1).

(Enlarge Image)

Figure 1.

Modulation of either TNFR-1-induced apoptosis or NF-κB activation by the

molluscum contagiosum virus MC159 and MC160 proteins. TNF binds to the

TNFR-1 receptor to initiate either apoptosis or NF-κB activation. (A) For apoptosis,

TNF–TNFR-1 interactions initiate recruitment and activation of DISC, composed of

TRADD, FADD and procaspase-8. The MC159 protein binds to FADD and prevents

DISC assembly, inhibiting TNF-induced apoptosis during MCV infection. (B) NF-κB

activation is initiated by TNF binding to TNFR-1, which recruits TRADD, TRAF2,

RIP and MEKK, resulting in complex activation and the degradation of IκB proteins,

thus enabling NF-κB activation. Evidence suggests that MC159 inhibits IKK

activation by binding to IKKγ. Other studies report MC159 activates NF-κB because

it enhances RIP–TRADD interactions to stimulate IKK. The MC160 protein inhibits

IKK activation by binding to HSP90, which triggers IKKα degradation.

DISC: Death-inducing signaling complex; MCV: Molluscum contagiosum virus.

Adapted with permission from [51]. © 2012 The American Association of

Immunologists, Inc.

Apoptosis is an effective mechanism to destroy virus-infected cells. Central to this

process is the interaction of FADD and procaspase-8.[58] During TNFR-1-induced

apoptosis, FADD interacts with procaspase-8 via DED motifs that are present in both

proteins. Next, procaspase-8 undergoes autoproteolysis, in which the N-terminal

tandem DEDs are cleaved, resulting in a mature, highly active enzyme that activates

the downstream procaspase-3, which in turn cleaves other proteins to initiate

apoptosis.

The sequencing of several viral genomes revealed the presence of proteins that, like

procaspase-8, contained two tandem DEDs: the MC (MCV) MC159 and MC160

products, and the Kaposi's sarcoma herpes virus K13 protein.[59–61] Interestingly,

MC159 and K13 inhibit TNF- and procaspase-8-induced apoptosis,[59–61] while

MC160 does not appear to have this function.[62] As such, these viral proteins are

named FLIPs.

The study of viral FLIPs (vFLIPs) identified a novel mechanism of viral inhibition of

apoptosis. Further inspection of the human genome revealed the presence of a cFLIP.[57,63] There are three isoforms of cFLIP due to splice variation: cFLIPL, cFLIPS and

cFLIPR. Like their viral counterparts, cFLIPL and cFLIPS inhibit apoptosis.[57,63–65] One

current idea in the field is that viruses may have 'stolen' genes encoding cFLIPs as a

means to inhibit apoptosis and aid in viral spread.

MC159: Inhibition of Apoptosis

When the amino acid sequence of MC159 was aligned to other DED-containing

proteins, it was determined that each DED in MC159 comprises six α-helices, when

the amino acid sequence of MC159 was aligned with other DED-containing proteins.

Two groups published the crystal structure of a truncated MC159 protein, in which

the first six residues of MC159 were absent from the crystalized protein.[55,56] Both

publications describe the MC159 protein as having a rigid dumbbell structure in

which its two DEDs tightly associate.[55,56] The structure of the highly similar Kaposi's

sarcoma herpes virus K13 vFLIP has also been solved.[66] In comparing the 3D crystal

structures of MC159 and K13, Bagneris et al. argue that the first DED of MC159 has

a truncated α-helix 1, which is predicted to affect the 3D structure and potential

functions of the MC159 N-terminus.[66] However, the solved MC159 crystal structure

lacks these six N-terminal residues, making it difficult to assess the true structure of

the MC159 α-helix 1.[55,56]

The MC159 protein inhibits TNFR-1-induced apoptosis (Figure 1).[51,59,62,67] The

molecular mechanism of the MC159 protein in this apoptosis pathway has been

intensely studied. Garvey et al. show that prevention of apoptosis by the MC159

protein is correlated with MC159 binding to either FADD or procaspase-8 through an

RXDL motif present in each of the MC159 protein DEDs.[29,30] Moreover, both DEDs

of MC159 must be present for MC159 to provide its antiapoptotic function.[29] Structural studies suggest that the MC159 protein binds FADD, thus preventing its

self-oligomerization and, in turn, death-inducing signaling complex assembly.[55,56] Additionally, the antiapoptotic properties of MC159 are bolstered by its

interaction with the adaptor protein TRAF3.[68] Although there has been debate

concerning which binding partners are correlated with apoptosis inhibition, the

general mechanism of blocking death-inducing signaling complex assembly is

accepted.

Woelfel et al. capitalized on this antiapoptosis function of MC159 and created a

transgenic mouse in which the MC159 protein is expressed under the control of the

promoter of an MHC class I gene as a means to study how inhibition of apoptosis

would affect the function of the acquired immune response and development of the

immune cell repertoire.[69] As would be expected, MC159-expressing immune cells

were resistant to apoptosis triggered by several stimuli, and the immune response to

virus infection was normal. Wu et al. created a transgenic mouse in which only T

cells express MC159.[70] This latter transgenic mouse is not meant to mimic MCV

infection, as MCV is not thought to infect T cells. Rather, the goal of developing

these transgenic mice was to develop a unique system in MC159 that would inhibit

apoptosis in a specific set of immune cells as a means to study how abnormal

regulation of apoptosis affects the immune response. Wu et al. [70] reported similar

findings to Woelfel et al..[69]However, these two studies reported differences when

studying T-cell activation and T-cell development. 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.[70] By 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 is most

likely due to the fact that apoptosis is blocked in immune cells that otherwise would

have been eliminated during thymic maturation.[69]

MC159: Modulation of NF-κB Activation

Several publications identified a second function of MC159, that of inhibiting NF-κB.[51,67,71] More recently, one publication reports that MC159 activates NF-κB.[72] In this

section, the signaling pathway resulting in NF-κB activation will be discussed. Then,

the molecular mechanisms used by MC159 to inhibit or induce NF-κB will be

discussed (Figure 1).

NF-κB is a cellular transcription factor activated by a diverse set of stimuli, such as

cytokines, dsRNA and viruses.[73] As such, myriad viruses encode molecules that

inhibit NF-κB. For other animal models of infection with other poxviruses, it is

known that viruses that lack these NF-κB-inhibitory proteins are attenuated.[74,75] While no animal model for MCV infection is available, it is assumed that a

mutant MCV lacking NF-κB-inhibitory proteins would be attenuated as well.

Recent reports have shown that MC159 inhibits NF-κB activation,[51,67,71] in which

TNF is used to stimulate NF-κB. When TNFR-1 stimulates NF-κB activation instead

of apoptosis, there is a different protein complex that forms at the cytoplasmic tail of

TNFR-1.[76] In this scenario, TRADD, RIP and TRAF2 migrate to the TNFR-1. This,

in turn, activates RIP, which results in stimulation of the IKK complex. NF-κB

resides in the cytoplasm bound by its inhibitor, IκB.[73,77] IκB proteins inhibit NF-κB

activity by preventing NF-κB translocation to the nucleus.[77] Upon stimulation of the

NF-κB pathway, the IKK complex phosphorylates IκB, resulting in IκB

ubiquitination and degradation.[73,77] Released NF-κB migrates to the nucleus where it

binds to specific promoter sequences to trigger transcription.

Previous studies from our laboratory show that there is a correlation between NF-κB

inhibition and TRAF2 binding when using mutant MC159 proteins with deletions in

large segments of amino acids.[67]These data suggest that MC159–TRAF2 interactions

prevent RIP activation and subsequent IKK activation. More recently, two pieces of

evidence suggest that MC159–TRAF2 interactions are not the mechanism that

inhibits NF-κB.[51] First, the use of smaller point-mutant MC159 proteins identified

some mutant MC159 proteins that still coimmunoprecipitate with TRAF2 but no

longer inhibit NF-κB.[51]Second, MC159 inhibits NF-κB activation stimulated through

several RIP- or TRAF2-independent pathways.[51]

Further study of the MC159-inhibitory function yielded findings consistent with the

mechanism that MC159 inhibits activation of the IKK complex. The IKK complex is

a large multiprotein complex that is composed of three main subunits: two

catalytically active kinases, IKKα and IKKβ; and one regulatory subunit, IKKγ. An

active IKK complex will phosphorylate the IκB protein, resulting in IκB

ubiquitination and subsequent degradation. The degradation of IκB frees NF-κB and

allows it to translocate to the nucleus.[73,77] The MC159 protein coimmunoprecipitates

with the IKK complex via its interactions with IKKγ.[51]As such, it is expected that

MC159 prevents IKK activation as its mechanism to inhibit NF-κB. Initial mutational

analysis of MC159 shows that the N-terminal DED of MC159 is required for

inhibition of NF-κB and associating with IKKγ.[51]

It should also be noted that a recent report by Challa et al. showed that MC159

stimulates NF-κB activation when using MC159-expressing Jurkat T cells, and that

MC159 increases RIP1–TRADD interactions,[72] which would activate IKK and NF-

κB (Figure 1). Similarly, the ectopic expression of MC159 in HEK293T cells also

activates NF-κB, albeit very low levels.[51,67] It is not yet fully understood how the

MC159 protein possesses both NF-κB-activating and NF-κB-inhibiting functions.

Different cell lines and means of expressing MC159 were used in these studies. Thus,

one possibility is that the concentration of MC159 protein in the cell could have

opposing effects on its IKK complex, with lower amounts of MC159 binding to IKKγ

to stimulate the IKK complex, while higher concentrations of MC159 inhibits IKK

activation.

While it is well known that TNFR-1 triggers either apoptosis or NF-κB activation,[76] what is less clear is the molecular mechanism by which TNF-α dictates either cell

death or survival. Identification of this cellular regulation may be extremely helpful

for designing future TNF-α therapies as it may give scientists the ability to control

whether a cell will undergo apoptosis or NF-κB activation. When mapping the

regions of MC159 sufficient for each function, both MC159 DEDs must be present to

inhibit apoptosis,[29] while only the N-terminal DED of MC159 is sufficient to inhibit

NF-κB activation.[51,67] How these distinct surfaces of MC159 compete for different

cellular targets in virus-infected cells, especially when host cells are receiving

multiple extracellular stimulatory signals, is still unclear.

MCV MC160: An NF-κB-inhibitory Protein

The MCV MC160 protein is an intracellular 52-kDa protein, and is a vFLIP.[62] When

aligning the MC159 and MC160 protein sequences, the MC160 N- and C-terminal

DEDs are 45 and 33% similar to the N- and C- terminal DEDs of MC159,

respectively.[62] In addition, MC160 has a longer, unique C-terminal tail that is absent

in MC159. Based on the presence of tandem DEDs, and the finding that MC160

coimmunoprecipitates with FADD or procaspase-8,[62] it was predicted that MC160

also would inhibit apoptosis.[40,41] While one report indeed showed antiapoptosis

function,[60] other reports show that MC160 does not inhibit apoptosis, whether

MC160 is expressed transiently or by a surrogate poxvirus infection.[62,78] These data

were initially surprising because the model for apoptosis was that DED-mediated

protein–protein interactions are sufficient to trigger apoptosis. However, more recent

studies identified that TNF-α–TNFR-1 interactions result in distinct intracellular

complexes, with complex I triggering NF-κB activation and complex II triggering

apoptosis.[79] Thus, one possibility is that MC160 still allows the formation of a

mature complex II. There are several putative caspase cleavage sites within the

MC160 protein,[62] and MC160 is cleaved when cells are triggered to undergo

apoptosis.[63] This cleavage is blocked when MC159 is coexpressed with MC160.[62] Thus, a second possibility is that MC160 may act as a competitive sink for

caspases during an MCV infection.

Similar to MC159, the MC160 protein inhibits NF-κB (Figure 1).[80,81] Interestingly,

while the MC159 protein appears to target the IKKγ subunit of the IKK complex,[82] MC160 targets IKKα to prevent IKK activation.[81] 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, interacts with

HSP90, resulting in degradation of IKKα subunits.[81] Interestingly, the second DED

(DED2) of MC160 also inhibits NF-κB activation. In this case, DED2 binds to

procaspase-8 and inhibits NF-κB activation.[81]

MCV MC54: An IL-18-binding Protein

The MC54 protein is an IL-18-binding protein (IL-18bp) of approximately 70 kDa

that is secreted from virally infected cells.[83] It is known to bind to human and murine

IL-18.[84] IL-18 responses are critical for defense against viral infection.[85,86] IL-18

potently activates macrophages, inducing production of several cytokines and

chemokines, including IFN-γ. It also potentiates NK cell and Th1 responses.[87]The

cellular human IL-18bp, huIL-18bp, is a means of regulating the potent effects of IL-

18. In Crohn's disease, there is an elevated level of huIL-18bp in the intestines of

patients,[88] suggesting that regulation of IL-18 is important for dictating appropriate

versus autoimmune responses. In other studies, high IL-18 levels are observed in

autoimmune and inflammatory disorders.[85,89]

Identification of small molecules that inhibit IL-18 could be used to develop new

therapies to treat aberrant IL-18 activation, which would presumably rebalance

inappropriate immune responses. To this end, the study of the poxviral proteins that

bind to IL-18 may be the basis for creating small molecules to inhibit IL-18–huIL-

18bp interactions. On the heels of the discovery of huIL-18bp was the identification

of the MCV MC53 and MC54 proteins as huIL-18bp homologs, binding to IL-18 and

neutralizing its biological activity.[84] Further characterization of MC54 reveals that

two forms of the protein are active: the full-length protein that binds to

glycosaminoglycans through its C-terminal tail and to huIL-18 with its N-terminus;

and a furin cleavage product that binds to huIL-18 only.[83,84]

Interestingly, MC54 is the only MCV immune defense molecule to have functionally

similar homologs in other members of the Orthopoxvirus [90–

92] and Yatapoxvirus genera.[93] Examination of the binding strategies of cellular and

viral IL-18bps to IL-18 has resulted in a deeper understanding of the surfaces

necessary for their interaction. 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.[92] By contrast, the

yatapoxvirus IL-18bp forms a dimer that has a higher binding affinity for IL-18 as

compared with a monomeric form.[93] 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.[92,93]

MCV MC148: A Viral Chemokine

MC148 is a secreted protein that is 11 kDa.[94] Immune cell recruitment is critical for

the clearance of poxvirus infection. MCVs encode the viral chemokine, MC148,

which acts to inhibit chemotaxis.[95,96] To date, the MC148 proteins from MCV-1

(MC148R1) and MCV-2 (MC148R2) have been studied. Initial characterization of

MC148R1 showed it to inhibit 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.[97,98] In agreement with these

studies, Jin et al. found that MC148R1 was capable of blocking both CXCL12α-

mediated and CCL3 (MIP-1α)-mediated chemotaxis; however, MC148R2 only

inhibited CCL3.[94] Immunoprecipitations revealed that MC148R1 interacted with

CXCL12α, preventing CXCL12α from binding its CXCR4 receptor.[94] 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,[95]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 are 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. As

such, one speculation is that MC148 contributes to the persistent nature of MCV

infections.

MCV MC007: A pRb-binding Protein

The MC007 protein is 34-kDa protein that is expressed intracellularly and localizes to

the mitochondria via an N-terminal mitochondrial targeting sequence.[28] MCV

infection produces benign lesions 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.[28] The pRb protein is a critical regulator of cell

proliferation through inhibition of the E2F family of transcription factors.[99,100] Deregulation of pRb has been linked to tumorigenesis.[99] MC007 binds to pRb

through the LXCXE pRb-binding motif, resulting in MC007-mediated sequestering

of pRb to the mitochondria.[28] Moreover, MC007's inhibitory effects were potent

enough to transform rat kidney cells, suggesting that MC007 may contribute to

persistence of MCV lesions. Further studies are needed to fully understand MC007's

role in MCV lesion production and pathogenesis.

Conclusion

MC is an understudied disease because of its low morbidity. However, this disease is

underappreciated given its common nature and the impact it has on children and their

caretakers. MCV is unique among poxviruses because of its narrow host range of

human keratinocytes, specialized immune evasion strategies and ability to cause

persistent infections. As such, 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 for the cellular mechanisms that regulate the

immune response to disease. Such information may aid in the development of new

topical therapies to resolve MCV lesions quickly and painlessly, and to counteract

other skin diseases that have similar deregulation of immune responses to those seen

during MCV infections. The impact of MC infections on human health, in terms of

the sociological impact on people infected with MC, and costs associated with

decreased productivity and increased healthcare, remains high for our own and other

societies. As such, there is a clear need for cost-effective treatments that can clear

MCV infections in a timely manner. By reviewing these publications together, we

now have a clarified picture of the immune and viral mechanisms that dictate the

prolonged nature of MC that leads to a better understanding of the viral and cellular

pathways to modulate for effective treatments in the future.

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Papers of special note have been highlighted as:

* of interest

** of considerable interest

http://www.medscape.com/viewarticle/805709_10 diakses tanggal 22 juni 2014

04:15WIT