Molluscum Contagiosum Virus Protein MC008 Targets NF-κB Activation by Inhibiting Ubiquitination of NEMO.

Molluscum contagiosum virus (MCV) is a human-adapted poxvirus that causes a common and persistent yet mild infection characterized by distinct, contagious, papular skin lesions. These lesions are notable for having little or no inflammation associated with them and can persist for long periods without an effective clearance response from the host. Like all poxviruses, MCV encodes potent immunosuppressive proteins that perturb innate immune pathways involved in virus sensing, the interferon response, and inflammation, which collectively orchestrate antiviral immunity and clearance, with several of these pathways converging at common signaling nodes. One such node is the regulator of canonical nuclear factor kappa B (NF-κB) activation, NF-κB essential modulator (NEMO). Here, we report that the MCV protein MC008 specifically inhibits NF-κB through its interaction with NEMO, disrupting its early ubiquitin-mediated activation and subsequent downstream signaling. MC008 is the third NEMO-targeting inhibitor to be described in MCV to date, with each inhibiting NEMO activation in distinct ways, highlighting strong selective pressure to evolve multiple ways of disabling this key signaling protein. IMPORTANCE Inflammation lies at the heart of most human diseases. Understanding the pathways that drive this response is the key to new anti-inflammatory therapies. Viruses evolve to target inflammation; thus, understanding how they do this reveals how inflammation is controlled and, potentially, how to disable it when it drives disease. Molluscum contagiosum virus (MCV) has specifically evolved to infect humans and displays an unprecedented ability to suppress inflammation in our tissue. We have identified a novel inhibitor of human innate signaling from MCV, MC008, which targets NEMO, a core regulator of proinflammatory signaling. Furthermore, MC008 appears to inhibit early ubiquitination, thus interrupting later events in NEMO activation, thereby validating current models of IκB kinase (IKK) complex regulation.

Molluscum Contagiosum Virus: Biology and Immune Response.

Molluscum contagiosum virus is a poxvirus belonging to the Poxviridae family, which includes Orthopoxvirus, Parapoxvirus, Yantapoxvirus, Molluscipoxvirus, Smallpox virus, Cowpox virus and Monkeypox virus. MCV belongs to the genus Molluscipoxvirus and has a tropism for skin tissue. MCV infects keratinocytes and, after an incubation period of 2 weeks to 6 weeks, causes a breakdown of the skin barrier with the development of papules of variable size depending on the proper functioning of the immune response (both adaptive and acquired). MCV only infects humans and does not cause viraemia. MCV encodes for several inhibitory proteins responsible to circumvent the immune response through different signalling pathways. Individuals who can be infected with MCV are children, immunocompromised individuals such as organ transplant recipients and Human Immunodeficiency Virus (HIV)-infected individuals. Current treatments to manage MCV-induced lesions are different and include the use of immunomodulators, which, however, do not provide an effective response.

The MC160 protein of the molluscum contagiosum virus dampens cGAS/STING-induced interferon-ß activation.

Molluscum contagiosum virus (MCV) is a poxvirus that causes benign, persistent skin lesions. MCV encodes a variety of immune evasion molecules to dampen host immune responses. Two of these proteins are the MC159 and MC160 proteins. Both MC159 and MC160 contain two tandem death effector domains and share homology to the cellular FLIPs, FADD, and procaspase-8. MC159 and MC160 dampen several innate immune responses such as NF-κB activation and mitochondrial antiviral signaling (MAVS)-mediated induction of type 1 interferon (IFN). The type 1 IFN response is also activated by the cytosolic DNA sensors cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Both cGAS and STING play a vital role in sensing a poxvirus infection. In this study, we demonstrate that there are nuanced differences between both MC160 and MC159 in terms of how the viral proteins modulate the cGAS/STING and MAVS pathways. Specifically, MC160 expression, but not MC159 expression, dampens cGAS/STING-mediated induction of IFN in HEK 293 T cells. Further, MC160 expression prevented the K63-ubiquitination of both STING and TBK1, a kinase downstream of cGAS/STING. Ectopic expression of the MC160 protein, but not the MC159 protein, resulted in a measurable decrease in the TBK1 protein levels as detected via immunoblotting. Finally, using a panel of MC160 truncation mutants, we report that the MC160 protein requires both DEDs to inhibit cGAS/STING-induced activation of IFN-ß. Our model indicates MC160 likely alters the TBK1 signaling complex to decrease IFN-ß activation at the molecular intersection of the cGAS/STING and MAVS signaling pathways.

Heterologous effect of influenza vaccination on molluscum contagiosum infection; a case report of siblings.

BACKGROUND: Molluscum contagiosum virus (MCV) is a benign, common cutaneous infection predominantly affecting the younger pediatric population. Traditional treatments may be time consuming with variable efficacy. Time to spontaneous resolution is variable and treatment is often sought to shorten duration of infection, prevent further autoinoculation, prevent infectious spread to others and treat cosmetic intolerability. CASE PRESENTATION: We present the case of two patients with complete, simultaneous clearance of their molluscum contagiosum infections after receiving a routine 2018 quadrivalent influenza vaccination. Neither patient has had recurrence of molluscum contagiosum or permanent scarring. We review trials of intralesional immunotherapy in treatment of cutaneous infections to theorize the mechanism of MCV infection clearance post influenza vaccination. CONCLUSION: We propose a delayed-type hypersensitivity reaction was induced as a heterologous effect of the influenza vaccination, similar to that seen in current immunotherapy treatments. This is the first reported case of MCV-directed immune reaction with infection clearance after influenza vaccination.

Molluscum Contagiosum Virus Evasion of Immune Surveillance: A Review.

BACKGROUND: Molluscum contagiosum (MC) is an acute infection caused by the molluscum contagiosum virus (MCV) with a worldwide incidence of approximately 8,000 cases per 100,000 individuals annually. Greater than 90% of MC cases occur in the pediatric population, and affected adults are more likely to be younger or immunocompromised. MC has minimal inflammation initially; however, a strong inflammatory response can occur during resolution of the infection, termed the beginning of the end (BOTE). MC infections may last months to years, and it is hypothesized that persistent infections may be due to suppression of immunity by MCV proteins, thus affecting MC’s clinical progression. OBJECTIVE: We reviewed the current proposed mechanisms of MCV immune evasion and discuss potential therapeutic options for MC treatment. METHODS: A literature search was conducted using electronic databases (Pubmed, Google Scholar, Medline). RESULTS: We compiled 18 original research articles and identified 11 proteins produced by MCV that are postulated to participate in evasion of host immunity through various molecular pathways. These proteins and/or their downstream pathways may be influenced by MC treatments in phase 3 development, including berdazimer gel 10.3% and VP-102 cantharidin, 0.7%. CONCLUSION: MCV is distinctive in evading immune surveillance by inhibiting or dampening several immune pathways via the production of viral proteins. The result is decreasing local inflammatory response which contributes to the prolonged survival of MCV in the epidermis. Persistent MC can be a nuisance for some patients and treatment may be desired. Currently, no treatment has been approved by the US Food and Drug Administration (FDA). Two approaches in the pipeline may affect the immune avoidance mechanisms; nevertheless, their exact mechanisms between the potential therapeutics and viral proteins remain enigmatic. J Drugs Dermatol. 2023;22(2):182-189. doi:10.36849/JDD.7230.

Molecular and genomic characterization of a novel equine molluscum contagiosum-like virus.

Cases of pox-like lesions in horses and donkeys have been associated with poxviruses belonging to different genera of the family Poxviridae. These include the orthopoxviruses vaccinia virus (VACV), horsepoxvirus (HPXV) and cowpoxvirus (CPXV), as well as a potentially novel parapoxvirus and molluscum contagiosum virus (MOCV). However, with the exception of VACV, HPXV and CPXV, the genomic characterization of the causative agents remains largely elusive with only single short genome fragments available. Here we present the first full-length genome sequence of an equine molluscum contagiosum-like virus (EMCLV) directly determined from skin biopsies of a horse with generalized papular dermatitis. Histopathological analysis of the lesions revealed severe epidermal hyperplasia with numerous eosinophilic inclusion bodies within keratinocytes. Virions were detected in the lesions in embedded tissue by transmission electron microscopy. The genome sequence determined by next- and third-generation sequencing comprises 166 843 nt with inverted terminal repeats (ITRs) of 3473 nt. Overall, 20 of the predicted 159 ORFs have no equivalents in other poxviruses. Intriguingly, two of these ORFs were identified to encode homologues of mammalian proteins involved in immune signalling pathways, namely secreted and transmembrane protein 1 (SECTM1) and insulin growth factor-like family receptor 1 (IGFLR1), that were not described in any virus family so far. Phylogenetic analysis with all relevant representatives of the Poxviridae suggests that EMCLV should be nominated as a new species within the genus Molluscipoxvirus.

The Molluscum Contagiosum Gene MC021L Partially Compensates for the Loss of Its Vaccinia Virus Homolog, F13L.

Orthopoxviruses produce two antigenically distinct infectious enveloped virions termed intracellular mature virions and extracellular virions (EV). EV have an additional membrane compared to intracellular mature virions due to a wrapping process at the trans-Golgi network and are required for cell-to-cell spread and pathogenesis. Specific to the EV membrane are a number of proteins highly conserved among orthopoxviruses, including F13, which is required for the efficient wrapping of intracellular mature virions to produce EV and which plays a role in EV entry. The distantly related molluscipoxvirus, molluscum contagiosum virus, is predicted to encode several vaccinia virus homologs of EV-specific proteins, including the homolog of F13L, MC021L. To study the function of MC021, we replaced the F13L open reading frame in vaccinia virus with an epitope-tagged version of MC021L. The resulting virus (vMC021L-HA) had a small-plaque phenotype compared to vF13L-HA but larger than vΔF13L. The localization of MC021-HA was markedly different from that of F13-HA in infected cells, but MC021-HA was still incorporated in the EV membrane. Similar to F13-HA, MC021-HA was capable of interacting with both A33 and B5. Although MC021-HA expression did not fully restore plaque size, vMC021L-HA produced amounts of EV similar to those produced by vF13L-HA, suggesting that MC021 retained some of the functionality of F13. Further analysis revealed that EV produced from vMC021L-HA exhibit a marked reduction in target cell binding and an increase in dissolution, both of which correlated with a small-plaque phenotype.IMPORTANCE The vaccinia virus extracellular virion protein F13 is required for the production and release of infectious extracellular virus, which in turn is essential for the subsequent spread and pathogenesis of orthopoxviruses. Molluscum contagiosum virus infects millions of people worldwide each year, but it is unknown whether EV are produced during infection for spread. Molluscum contagiosum virus contains a homolog of F13L termed MC021L. To study the potential function of this homolog during infection, we utilized vaccinia virus as a surrogate and showed that a vaccinia virus expressing MC021L-HA in place of F13L-HA exhibits a small-plaque phenotype but produces similar levels of EV. These results suggest that MC021-HA can compensate for the loss of F13-HA by facilitating wrapping to produce EV and further delineates the dual role of F13 during infection.

Molluscum contagiosum virus MC80 sabotages MHC-I antigen presentation by targeting tapasin for ER-associated degradation.

The human specific poxvirus molluscum contagiosum virus (MCV) produces skin lesions that can persist with minimal inflammation, suggesting that the virus has developed robust immune evasion strategies. However, investigations into the underlying mechanisms of MCV pathogenesis have been hindered by the lack of a model system to propagate the virus. Herein we demonstrate that MCV-encoded MC80 can disrupt MHC-I antigen presentation in human and mouse cells. MC80 shares moderate sequence-similarity with MHC-I and we find that it associates with components of the peptide-loading complex. Expression of MC80 results in ER-retention of host MHC-I and thereby reduced cell surface presentation. MC80 accomplishes this by engaging tapasin via its luminal domain, targeting it for ubiquitination and ER-associated degradation in a process dependent on the MC80 transmembrane region and cytoplasmic tail. Tapasin degradation is accompanied by a loss of TAP, which limits MHC-I access to cytosolic peptides. Our findings reveal a unique mechanism by which MCV undermines adaptive immune surveillance.

Downregulation of HLA-I by the molluscum contagiosum virus mc080 impacts NK-cell recognition and promotes CD8+ T-cell evasion.

Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.

MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction.

MC159 is a viral FLIP (FLICE inhibitory protein) encoded by the molluscum contagiosum virus (MCV) enabling MCV to evade antiviral immunity and to establish persistent infections in humans. Here, we show that MC159 contains a functional SH3 binding motif, which mediates avid and selective binding to SH3BP4, a signaling protein known to regulate endocytic trafficking and suppress cellular autophagy. The capacity to bind SH3BP4 was dispensable for regulation of NF-κB-mediated transcription and suppression of proapoptotic caspase activation but contributed to inhibition of amino acid starvation-induced autophagy by MC159. These results provide new insights into the cellular functions of MC159 and reveal SH3BP4 as a novel host cell factor targeted by a viral immune evasion protein.IMPORTANCE After the eradication of smallpox, molluscum contagiosum virus (MCV) is the only poxvirus restricted to infecting humans. MCV infection is common and causes benign skin lesions that usually resolve spontaneously but may persist for years and grow large, especially in immunocompromised individuals. While not life threatening, MCV infections pose a significant global health burden. No vaccine or specific anti-MCV therapy is available. MCV encodes several proteins that enable it to evade antiviral immunity, a notable example of which is the MC159 protein. In this study, we describe a novel mechanism of action for MC159 involving hijacking of a host cell protein called SH3BP4 to suppress autophagy, a cellular recycling mechanism important for antiviral immunity. This study contributes to our understanding of the host cell interactions of MCV and the molecular function of MC159.

A case of intracranial molluscum contagiosum virus infection diagnosed by metagenomic sequencing of cerebrospinal fluid.

Molluscum contagiosum is a common, self-limiting infectious disease of the skin caused by molluscum contagiosum virus (MCV). The disease primarily affects children, sexually active adults, and immunocompromised individuals. Transmission of the virus occurs by direct skin contact. Therefore, the virus is usually detected in the skin and genitals of patients. However, the diagnosis of intracranial infection by the virus is difficult if the skin/mucosa lessons are atypical or absent, and the presence of the virus in the cerebrospinal fluid has not been reported. We report a very rare case of intracranial infection by molluscum contagiosum virus. A 25-year-old girl was admitted to our hospital due to severe headache but no fever or other symptoms. Upon examination, some small flesh-colored flattened papules on both arms were noticed. Blood tests showed slightly reduced levels of CD3 and CD4 T lymphocytes. Three-dimensional time-of-flight magnetic resonance angiography (3D-TOF-MRA) and head magnetic resonance (MR) were both normal. Lumbar puncture was performed, and metagenomic sequencing was applied to the spinal fluid. The unique sequences of the molluscum contagiosum virus were identified in the fluid. The patient was then diagnosed with intracranial molluscum contagiosum virus infection. No special treatment was given. The headache gradually disappeared, and the patient was discharged. During our quarterly follow-up, the girl appeared normal, and her skin lesions disappeared. However, her CD3 and CD4 T lymphocyte counts were still slightly lower than the normal level. Our case shows that the application of metagenomic sequencing to cerebrospinal fluid is a sensitive and powerful means to detect pathogens causing intracranial infection. Keywords: Molluscum contagiosum; intracranial infection; metagenomics sequencing.

A comparison of the effect of molluscum contagiosum virus MC159 and MC160 proteins on vaccinia virus virulence in intranasal and intradermal infection routes.

Molluscum contagiosum virus (MCV) causes persistent, benign skin neoplasm in children and adults. MCV is refractive to growth in standard tissue culture and there is no relevant animal model of infection. Here we investigated whether another poxvirus (vaccinia virus; VACV) could be used to examine MCV immunoevasion protein properties in vivo. The MCV MC159L or MC160L genes, which encode NF-κB antagonists, were inserted into an attenuated VACV lacking an NF-κB antagonist (vΔA49), creating vMC159 and vMC160. vMC160 slightly increased vΔA49 virulence in the intranasal and intradermal routes of inoculation. vMC159 infection was less virulent than vΔA49 in both inoculation routes. vMC159-infected ear pinnae did not form lesions, but virus replication still occurred. Thus, the lack of lesions was not due to abortive virus replication. This system provides a new approach to examine MCV immunoevasion proteins within the context of a complete and complex immune system.

Molluscum Contagiosum Virus Protein MC005 Inhibits NF-κB Activation by Targeting NEMO-Regulated IκB Kinase Activation.

Molluscum contagiosum virus (MCV), the only known extant human-adapted poxvirus, causes a long-duration infection characterized by skin lesions that typically display an absence of inflammation despite containing high titers of live virus. Despite this curious presentation, MCV is very poorly characterized in terms of host-pathogen interactions. The absence of inflammation around MCV lesions suggests the presence of potent inhibitors of human antiviral immunity and inflammation. However, only a small number of MCV immunomodulatory genes have been characterized in detail. It is likely that many more remain to be discovered, given the density of such sequences in other poxvirus genomes. NF-κB activation occurs in response to both virus-induced pattern recognition receptor (PRR) signaling and cellular activation by virus-induced proinflammatory cytokines like tumor necrosis factor and interleukin-1. Activated NF-κB drives cytokine and interferon gene expression, leading to inflammation and virus clearance. We report that MC005, which has no orthologs in other poxvirus genomes, is a novel inhibitor of PRR- and cytokine-stimulated NF-κB activation. MC005 inhibited NF-κB proximal to the IκB kinase (IKK) complex, and unbiased affinity purification revealed that MC005 interacts with the IKK subunit NEMO (NF-κB essential modulator). MC005 binding to NEMO prevents the conformational priming of the IKK complex that occurs when NEMO binds to ubiquitin chains during pathway activation. These data reveal a novel mechanism of poxvirus inhibition of human innate immunity, validate current dynamic models of NEMO-dependent IKK complex activation, and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity and suppress inflammation to persist in human skin lesions.IMPORTANCE Poxviruses adapt to specific hosts over time, evolving and tailoring elegantly precise inhibitors of the rate-limiting steps within the signaling pathways that control innate immunity and inflammation. These inhibitors reveal new features of the antiviral response, clarify existing models of signaling regulation while offering potent new tools for approaching therapeutic intervention in autoimmunity and inflammatory disease. Molluscum contagiosum virus (MCV) is the only known extant poxvirus specifically adapted to human infection and appears adept at evading normal human antiviral responses, yet it remains poorly characterized. We report the identification of MCV protein MC005 as an inhibitor of the pathways leading to the activation of NF-κB, an essential regulator of innate immunity. Further, identification of the mechanism of inhibition of NF-κB by MC005 confirms current models of the complex way in which NF-κB is regulated and greatly expands our understanding of how MCV so effectively evades human immunity.

Molluscum Contagiosum Virus MC159 Abrogates cIAP1-NEMO Interactions and Inhibits NEMO Polyubiquitination.

Molluscum contagiosum virus (MCV) is a dermatotropic poxvirus that causes benign skin lesions. MCV lesions persist because of virally encoded immune evasion molecules that inhibit antiviral responses. The MCV MC159 protein suppresses NF-κB activation, a powerful antiviral response, via interactions with the NF-κB essential modulator (NEMO) subunit of the IκB kinase (IKK) complex. Binding of MC159 to NEMO does not disrupt the IKK complex, implying that MC159 prevents IKK activation via an as-yet-unidentified strategy. Here, we demonstrated that MC159 inhibited NEMO polyubiquitination, a posttranslational modification required for IKK and downstream NF-κB activation. Because MCV cannot be propagated in cell culture, MC159 was expressed independent of infection or during a surrogate vaccinia virus infection to identify how MC159 prevented polyubiquitination. Cellular inhibitor of apoptosis protein 1 (cIAP1) is a cellular E3 ligase that ubiquitinates NEMO. Mutational analyses revealed that MC159 and cIAP1 each bind to the same NEMO region, suggesting that MC159 may competitively inhibit cIAP1-NEMO interactions. Indeed, MC159 prevented cIAP1-NEMO interactions. MC159 also diminished cIAP1-mediated NEMO polyubiquitination and cIAP1-induced NF-κB activation. These data suggest that MC159 competitively binds to NEMO to prevent cIAP1-induced NEMO polyubiquitination. To our knowledge, this is the first report of a viral protein disrupting NEMO-cIAP1 interactions to strategically suppress IKK activation. All viruses must antagonize antiviral signaling events for survival. We hypothesize that MC159 inhibits NEMO polyubiquitination as a clever strategy to manipulate the host cell environment to the benefit of the virus.IMPORTANCE Molluscum contagiosum virus (MCV) is a human-specific poxvirus that causes persistent skin neoplasms. The persistence of MCV has been attributed to viral downregulation of host cell immune responses such as NF-κB activation. We show here that the MCV MC159 protein interacts with the NEMO subunit of the IKK complex to prevent NEMO interactions with the cIAP1 E3 ubiquitin ligase. This interaction correlates with a dampening of cIAP1 to polyubiquitinate NEMO and to activate NF-κB. This inhibition of cIAP1-NEMO interactions is a new viral strategy to minimize IKK activation and to control NEMO polyubiquitination. This research provides new insights into mechanisms that persistent viruses may use to cause long-term infection of host cells.

Recombination events and variability among full-length genomes of co-circulating molluscum contagiosum virus subtypes 1 and 2.

Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and causes a highly prevalent human disease of the skin characterized by the formation of a variable number of lesions that can persist for prolonged periods of time. Two major genotypes, subtype 1 and subtype 2, are recognized, although currently only a single complete genomic sequence corresponding to MCV subtype 1 is available. Using next-generation sequencing techniques, we report the complete genomic sequence of four new MCV isolates, including the first one derived from a subtype 2. Comparisons suggest a relatively distant evolutionary split between both MCV subtypes. Further, our data illustrate concurrent circulation of distinct viruses within a population and reveal the existence of recombination events among them. These results help identify a set of MCV genes with potentially relevant roles in molluscum contagiosum epidemiology and pathogenesis.

Molluscum Contagiosum Virus Transcriptome in Abortively Infected Cultured Cells and a Human Skin Lesion.

UNLABELLED: Molluscum contagiosum virus (MOCV), the only circulating human-specific poxvirus, has a worldwide distribution and causes benign skin lesions that may persist for months in young children and severe infections in immunosuppressed adults. Studies of MOCV are restricted by the lack of an efficient animal model or a cell culture replication system. We used next-generation sequencing to analyze and compare polyadenylated RNAs from abortive MOCV infections of several cell lines and a human skin lesion. Viral RNAs were detected for 14 days after MOCV infection of cultured cells; however, there was little change in the RNA species during this time and a similar pattern occurred in the presence of an inhibitor of protein synthesis, indicating a block preventing postreplicative gene expression. Moreover, a considerable number of MOCV RNAs mapped to homologs of orthopoxvirus early genes, but few did so to homologs of intermediate or late genes. The RNAs made during in vitro infections represent a subset of RNAs detected in human skin lesions which mapped to homologs of numerous postreplicative as well as early orthopoxvirus genes. Transfection experiments using fluorescent protein and luciferase reporters demonstrated that vaccinia virus recognized MOCV intermediate and late promoters, indicating similar gene regulation. The specific recognition of the intermediate promoter in MOCV-infected cells provided evidence for the synthesis of intermediate transcription factors, which are products of early genes, but not for late transcription factors. Transcriptome sequencing (RNA-seq) and reporter gene assays may be useful for testing engineered cell lines and conditions that ultimately could provide an in vitro replication system. IMPORTANCE: The inability to propagate molluscum contagiosum virus, which causes benign skin lesions in young children and more extensive infections in immunosuppressed adults, has constrained our understanding of the biology of this human-specific virus. In the present study, we characterized the RNAs synthesized in abortively infected cultured cells and a human skin lesion by next-generation sequencing. These studies provided an initial transcription map of the MOCV genome, suggested temporal regulation of gene expression, and indicated that the in vitro replication block occurs prior to intermediate and late gene expression. RNA-seq and reporter assays, as described here, may help to further evaluate MOCV gene expression and define conditions that could enable MOCV replication in vitro.

The molluscum contagiosum virus death effector domain containing protein MC160 RxDL motifs are not required for its known viral immune evasion functions.

The molluscum contagiosum virus (MCV) uses a variety of immune evasion strategies to antagonize host immune responses. Two MCV proteins, MC159 and MC160, contain tandem death effector domains (DEDs). They are reported to inhibit innate immune signaling events such as NF-κB and IRF3 activation, and apoptosis. The RxDL motif of MC159 is required for inhibition of both apoptosis and NF-κB activation. However, the role of the conserved RxDL motif in the MC160 DEDs remained unknown. To answer this question, we performed alanine mutations to neutralize the arginine and aspartate residues present in the MC160 RxDL in both DED1 and DED2. These mutations were further modeled against the structure of the MC159 protein. Surprisingly, the RxDL motif was not required for MC160's ability to inhibit MAVS-induced IFNß activation. Further, unlike previous results with the MC159 protein, mutations within the RxDL motif of MC160 had no effect on the ability of MC160 to dampen TNF-α-induced NF-κB activation. Molecular modeling predictions revealed no overall changes to the structure in the MC160 protein when the amino acids of both RxDL motifs were mutated to alanine (DED1 = R67A D69A; DED2 = R160A D162A). Taken together, our results demonstrate that the RxDL motifs present in the MC160 DEDs are not required for known functions of the viral protein.