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Joanna L Shisler - One of the best experts on this subject based on the ideXlab platform.

  • a comparison of the effect of Molluscum Contagiosum Virus mc159 and mc160 proteins on vaccinia Virus virulence in intranasal and intradermal infection routes
    Journal of General Virology, 2018
    Co-Authors: Sunetra Biswas, Geoffrey L Smith, Brian M Ward, Joanna L Shisler
    Abstract:

    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 mc159 abrogates ciap1 nemo interactions and inhibits nemo polyubiquitination
    Journal of Virology, 2017
    Co-Authors: Sunetra Biswas, Joanna L Shisler
    Abstract:

    : 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.

  • immune evasion strategies of Molluscum Contagiosum Virus
    Advances in Virus Research, 2015
    Co-Authors: Joanna L Shisler
    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 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.

  • inhibition of interferon gene activation by death effector domain containing proteins from the Molluscum Contagiosum Virus
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Crystal M H Randall, Sunetra Biswas, Catherine V Selen, Joanna L Shisler
    Abstract:

    Apoptosis, NF-κB activation, and IRF3 activation are a triad of intrinsic immune responses that play crucial roles in the pathogenesis of infectious diseases, cancer, and autoimmunity. FLIPs are a family of viral and cellular proteins initially found to inhibit apoptosis and more recently to either up- or down-regulate NF-κB. As such, a broad role for FLIPs in disease regulation is postulated, but exactly how a FLIP performs such multifunctional roles remains to be established. Here we examine FLIPs (MC159 and MC160) encoded by the Molluscum Contagiosum Virus, a dermatotropic poxVirus causing skin infections common in children and immunocompromised individuals, to better understand their roles in viral pathogenesis. While studying their molecular mechanisms responsible for NF-κB inhibition, we discovered that each protein inhibited IRF3-controlled luciferase activity, identifying a unique function for FLIPs. MC159 and MC160 each inhibited TBK1 phosphorylation, confirming this unique function. Surprisingly, MC159 coimmunoprecipitated with TBK1 and IKKe but MC160 did not, suggesting that these homologs use distinct molecular mechanisms to inhibit IRF3 activation. Equally surprising was the finding that the FLIP regions necessary for TBK1 inhibition were distinct from those MC159 or MC160 regions previously defined to inhibit NF-κB or apoptosis. These data reveal previously unappreciated complexities of FLIPs, and that subtle differences within the conserved regions of FLIPs possess distinct molecular and structural fingerprints that define crucial differences in biological activities. A future comparison of mechanistic differences between viral FLIP proteins can provide new means of precisely manipulating distinct aspects of intrinsic immune responses.

  • Molluscum Contagiosum Virus persistence pays off
    Future Virology, 2013
    Co-Authors: Crystal M H Randall, Joanna L Shisler
    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.

Bernard Moss - One of the best experts on this subject based on the ideXlab platform.

  • Molluscum Contagiosum Virus transcriptome in abortively infected cultured cells and a human skin lesion
    Journal of Virology, 2016
    Co-Authors: Jorge D Mendezrios, Jeffrey I. Cohen, Zhilong Yang, Karl J Erlandson, Craig Martens, Daniel Bruno, Stephen F Porcella, Bernard Moss
    Abstract:

    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.

  • Molluscum Contagiosum Virus interleukin 18 il 18 binding protein is secreted as a full length form that binds cell surface glycosaminoglycans through the c terminal tail and a furin cleaved form with only the il 18 binding domain
    Journal of Virology, 2003
    Co-Authors: Yan Xiang, Bernard Moss
    Abstract:

    Molluscum Contagiosum Virus (MCV) and variola Virus are the sole members of the poxVirus family that use humans as exclusive natural hosts (8). Variola Virus belongs to the OrthopoxVirus genus and until recently caused smallpox, an acute infection with a high mortality rate (9). MCV, the only member of the MolluscipoxVirus genus, causes small, benign skin lesions in children and young adults and more extensive disease only when there is a concurrent immunodeficiency such as AIDS (10). Even in immunocompetent individuals, however, the Virus-filled skin lesions frequently persist for many months with few signs of inflammation, suggesting local immune suppression. Several potential immune evasion proteins were discovered after the complete MCV genome sequence was determined (17). One of these is an interleukin-18 (IL-18) binding protein (IL-18BP) that inhibits the gamma interferon-inducing activity of IL-18 (25). IL-18 is a proinflammatory cytokine that enhances innate and acquired immunity and protects against microbial infections and tumors in murine models (6). Excessive IL-18 activity, however, is associated with some autoimmune and inflammatory diseases (13). Regulation of IL-18 activity is mediated by soluble IL-18BPs (14, 24). MC54L, an MCV homolog of mammalian IL-18BP, binds IL-18 with a nanomolar Kd and inhibits the gamma interferon-inducing activity of IL-18 in a dose-dependent manner (22, 25). Functional homologs of IL-18BP are also present in orthopoxViruses, including vaccinia Virus, cowpox Virus, and ectromelia Virus (1, 3, 19), and as yet uncharacterized homologs are encoded by variola Virus and members of other poxVirus genera. Deletion of the IL-18BP gene from ectromelia Virus decreased Virus replication and elevated natural killer cell activity in infected mice, indicating that these proteins contribute to defense against the host immune system (1). The IL-18 binding sites of human IL-18BP and MC54L were determined through site-directed mutagenesis and quantitative binding studies (22, 23). Despite the relatively low overall sequence identity between MC54L and human IL-18BP, their IL-18 binding sites are almost identical. MC54L, however, is unique among poxVirus and mammalian IL-18BPs in having a C-terminal tail that is almost 100 amino acids long and is entirely dispensable for IL-18 binding (22). Our present studies show that the protein is secreted in two forms: a full-length form that binds cell surface glycosaminoglycans with high affinity through the C-terminal tail and a furin-cleaved form consisting solely of the IL-18 binding domain (IL-18BD). The long and short forms of MC54L may inhibit IL-18 near the site of infection and at more distal locations, respectively.

  • Molluscum Contagiosum Virus inhibitors of apoptosis the mc159 v flip protein blocks fas induced activation of procaspases and degradation of the related mc160 protein
    Virology, 2001
    Co-Authors: Joanna L Shisler, Bernard Moss
    Abstract:

    Abstract Molluscum Contagiosum Virus contains two open reading frames, MC159 and MC160, that encode proteins with death effector domains resembling those of cellular regulators of apoptosis. Previous transfection analyses indicated that the MC159 protein binds to cellular FADD and inhibits Fas-induced cytolysis. For further studies, we inserted the MC159 or MC160 gene into the genome of vaccinia Virus that had its own major anti-apoptosis gene deleted. The MC159-expressing Virus blocked Fas-induced activation of caspase-3 and -8, degradation of PARP, and cleavage of DNA, whereas the parental vaccinia Virus did not. The MC159 protein bound to procaspase-8, in addition to FADD, and was included in a complex with Fas upon receptor activation. Although the MC160 protein associated with FADD and procaspase-8 in co-immunoprecipitation studies, no protection against morphological or biochemical changes associated with Fas-induced apoptosis were discerned and the MC160 protein itself was degraded. Co-expression of MC159, as well as other caspase inhibitors, protected the MC160 protein from degradation, suggesting a functional relationship between the two viral proteins.

  • domain structure intracellular trafficking and β2 microglobulin binding of a major histocompatibility complex class i homolog encoded by Molluscum Contagiosum Virus
    Virology, 1998
    Co-Authors: Tatiana G. Senkevich, Bernard Moss
    Abstract:

    Abstract The MC80R gene of Molluscum Contagiosum Virus (MCV) type 1 encodes a major histocompatibility complex (MHC) class I homolog that lacks several amino-acid residues critical for peptide binding by MHC molecules, contains an unusually long N-terminal hydrophobic domain possibly derived by triplication of a signal peptide, and has a C-terminal transmembrane domain with two glutamate residues. All of these features were present in the orthologous gene of MCV type 2. The MC80R gene was expressed as two glycosylated polypeptides of Mr 47,000 and 42,000. Pulse-chase experiments indicated that the larger polypeptide was a precursor of the shorter one and that the entire N-terminal domain was slowly removed, consistent with its function as a long signal peptide. The protein was largely sequestered in the endoplasmic reticulum and Golgi membranes, remained endoglycosidase-H sensitive, and was not detected on the cell surface. In addition, a genetically modified form of the MC80R protein lacking the transmembrane and cytoplasmic domains was not secreted. The roles of the MC80R protein domains were investigated by constructing chimera between the viral protein and the MHC class I protein HLA-A2. Expression studies confirmed that the N- and C-terminal hydrophobic regions of the MC80R protein served as signal and transmembrane domains, respectively. The central portion of the MC80R protein, corresponding to the α1–α3 extracellular domains of HLA-A2, was largely responsible for sequestering the protein in the endoplasmic reticulum or Golgi compartments. The MC80R protein, as well as HLA-A2 chimera with the central region of MC80R, formed stable intracellular complexes with β2-microglobulin. Complex formation, however, was detected only by overexpression of the MC80R protein or β2-microglobulin.

  • chemokine homolog of Molluscum Contagiosum Virus sequence conservation and expression
    Virology, 1998
    Co-Authors: Joachim Jakob Bugert, Inger K. Damon, Bernard Moss, Claudia Lohmuller, Gholamreza Darai
    Abstract:

    An analysis of the complete Molluscum Contagiosum Virus (MCV-1) genome sequence revealed a 104-amino-acid open reading frame (MC148R) that is structurally related to the β (CC) family of chemokines. The predicted MCV chemokine homolog (MCCH) has a deletion in the NH2-terminal activation domain, suggesting the absence of chemoattractant activity. The principal objectives of the present study were to determine whether: (i) MCCH is conserved in independent isolates of MCV-1 and MCV-2; (ii) MCCH mRNA is expressedin vivo;and (iii) the MCCH protein is secreted from mammalian cells. The nucleotide sequence of the MCCH gene locus was determined for 27 isolates of MCV-1 and 2 of MCV-2 obtained from 29 MCV-infected individuals. In each case, the characteristic CC sequence, the NH2-terminal deletion, and the length of the open reading frame were conserved, although there were some, mostly conservative, amino acid substitutions. Since MCV cannot be propagated in cell culture, mRNA was synthesizedin vitroby the early transcription apparatus in purified MCV virions. MCCH RNA was amplified by RT–PCR; the sequence included the complete open reading frame and extended 40 to 50 nucleotides past the first poxviral termination signal (TTTTTNT). Similar RT–PCR results were obtained using total cellular RNA derived from MCV-infected tissue specimens. Finally, the MCCH open reading frame was expressed in a vaccinia Virus vector and the predicted size polypeptide was secreted into the medium, as determined by Western blotting. Taken together, our data support the prediction that MCV expresses a secreted chemokine homolog that could antagonize the inflammatory responsein vivo.

Joachim Jakob Bugert - One of the best experts on this subject based on the ideXlab platform.

  • elisa for Molluscum Contagiosum Virus
    Current protocols in microbiology, 2017
    Co-Authors: Subuhi Sherwani, Mohammed Chowdhury, Joachim Jakob Bugert
    Abstract:

    Molluscum Contagiosum Virus (MCV) is a common skin pathogen of children and young adults. Infection with MCV causes benign skin tumors in children and young adults and is mostly self-limiting. In contrast to orthopoxViruses, MCV infections tend to take a subacute clinical course but may persist for up to 12 months. Current numbers for MCV seroprevalence in different geographical areas are based on a variety of historical serological methods from complement fixation assays to MCV ELISAs based on purified MCV virions and MC133 antigen expressed in a Semliki Forest Virus expression system. A standardized ELISA for the assessment of MCV seroprevalence would be useful to determine global MCV seroprevalence. The methods described show that polypeptides derived from MCV open reading frames MC084 (residues V123 to R230 and V33 to G117), mc133 (residues M1 to N370), and glutathione S-transferase (GST)-H3L (residues I142 to W251) expressed in E. coli RIL+ as GST fusion proteins can be used to assess antibody binding in a GST capture ELISA. We show how the ELISA can be used to screen a panel of patient sera previously characterized with the mc084 V123-R230 ELISA. © 2017 by John Wiley & Sons, Inc.

  • the Molluscum Contagiosum Virus protein mc163 localizes to the mitochondria and dampens mitochondrial mediated apoptotic responses
    Virology, 2017
    Co-Authors: Jesse Coutu, Joachim Jakob Bugert, Melissa R Ryerson, Daniel Brian Nichols
    Abstract:

    Abstract Apoptosis is a powerful host cell defense to prevent Viruses from completing replication. PoxViruses have evolved complex means to dampen cellular apoptotic responses. The poxVirus, Molluscum Contagiosum Virus (MCV), encodes numerous host interacting molecules predicted to antagonize immune responses. However, the function of the majority of these MCV products has not been characterized. Here, we show that the MCV MC163 protein localized to the mitochondria via an N-terminal mitochondrial localization sequence and transmembrane domain. Transient expression of the MC163 protein prevented mitochondrial membrane permeabilization (MMP), an event central to cellular apoptotic responses, induced by either Tumor Necrosis Factor alpha (TNF-α) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP). MC163 expression prevented the release of a mitochondrial intermembrane space reporter protein when cells were challenged with TNF-α. Inhibition of MMP was also observed in cell lines stably expressing MC163. MC163 expression may contribute to the persistence of MCV lesions by dampening cellular apoptotic responses.

  • Seroprevalence of Molluscum Contagiosum Virus in German and UK Populations
    2016
    Co-Authors: Subuhi Sherwani, Laura Farleigh, Nidhi Agarwal, Samantha Loveless, Neil Robertson, Eva Hadaschik, Paul Schnitzler, Joachim Jakob Bugert
    Abstract:

    Molluscum Contagiosum Virus (MCV) is a significant but underreported skin pathogen for children and adults. Seroprevalence studies can help establish burden of disease. Enzyme linked immunosorbent assay (ELISA) based studies have been published for Australian and Japanese populations and the results indicate seroprevalences between 6 and 22 percent in healthy individuals, respectively. To investigate seroprevalence in Europe, we have developed a recombinant ELISA using a truncated MCV virion surface protein MC084 (V123-R230) expressed in E. coli. The ELISA was found to be sensitive and specific, with low inter- and intra-assay variability. Sera from 289 German adults and children aged 0–40 years (median age 21 years) were analysed for antibodies against MC084 by direct binding ELISA. The overall seropositivity rate was found to be 14.8%. The seropositivity rate was low in children below the age of one (4.5%), peaked in children aged 2– 10 years (25%), and fell again in older populations (11–40 years; 12.5%). Ten out of 33 healthy UK individuals (30.3%; median age 27 years) had detectable MC084 antibodies. MCV seroconversion was more common in dermatological and autoimmune disorders, than in immunocompromised patients or in patients with multiple sclerosis. Overall MCV seroprevalence is 2.1 fold higher in females than in males in a UK serum collection. German seroprevalences determined in the MC084 ELISA (14.8%) are at least three times higher than incidence of MC in a comparable Swiss population (4.9%). While results are not strictly comparable, this is lower than Australian seroprevalence in a virion based ELISA (n = 357; 23%

  • seroprevalence of Molluscum Contagiosum Virus in german and uk populations
    PLOS ONE, 2014
    Co-Authors: Subuhi Sherwani, Laura Farleigh, Nidhi Agarwal, Samantha Loveless, Neil Robertson, Eva Hadaschik, Paul Schnitzler, Joachim Jakob Bugert
    Abstract:

    Molluscum Contagiosum Virus (MCV) is a significant but underreported skin pathogen for children and adults. Seroprevalence studies can help establish burden of disease. Enzyme linked immunosorbent assay (ELISA) based studies have been published for Australian and Japanese populations and the results indicate seroprevalences between 6 and 22 percent in healthy individuals, respectively. To investigate seroprevalence in Europe, we have developed a recombinant ELISA using a truncated MCV virion surface protein MC084 (V123-R230) expressed in E. coli. The ELISA was found to be sensitive and specific, with low inter- and intra-assay variability. Sera from 289 German adults and children aged 0-40 years (median age 21 years) were analysed for antibodies against MC084 by direct binding ELISA. The overall seropositivity rate was found to be 14.8%. The seropositivity rate was low in children below the age of one (4.5%), peaked in children aged 2-10 years (25%), and fell again in older populations (11-40 years; 12.5%). Ten out of 33 healthy UK individuals (30.3%; median age 27 years) had detectable MC084 antibodies. MCV seroconversion was more common in dermatological and autoimmune disorders, than in immunocompromised patients or in patients with multiple sclerosis. Overall MCV seroprevalence is 2.1 fold higher in females than in males in a UK serum collection. German seroprevalences determined in the MC084 ELISA (14.8%) are at least three times higher than incidence of MC in a comparable Swiss population (4.9%). While results are not strictly comparable, this is lower than Australian seroprevalence in a virion based ELISA (n = 357; 23%; 1999), but higher than the seroprevalence reported in a Japanese study using an N-terminal truncation of MC133 (n = 108, 6%; 2000. We report the first large scale serological survey of MC in Europe (n = 393) and the first MCV ELISA based on viral antigen expressed in E. coli.

  • Molluscum Contagiosum Virus infection
    Lancet Infectious Diseases, 2013
    Co-Authors: X. Chen, Alexander Vincent Anstey, Joachim Jakob Bugert
    Abstract:

    Summary Molluscum Contagiosum Virus is an important human skin pathogen: it can cause disfigurement and suffering in children, in adults it is less common and often sexually transmitted. Extensive and persistent skin infection with the Virus can indicate underlying immunodeficiency. Traditional ablative therapies have not been compared directly with newer immune-modulating and specific antiviral therapies. Advances in research raise the prospect of new approaches to treatment informed by the biology of the Virus; in human skin, the infection is localised in the epidermal layers, where it induces a typical, complex hyperproliferative lesion with an abundance of Virus particles but a conspicuous absence of immune effectors. Functional studies of the viral genome have revealed effects on cellular pathways involved in the cell cycle, innate immunity, inflammation, and cell death. Extensive lesions caused by Molluscum Contagiosum can occur in patients with DOCK8 deficiency—a genetic disorder affecting migration of dendritic and specialised T cells in skin. Sudden disappearance of lesions is the consequence of a vigorous immune response in healthy people. Further study of the unique features of infection with Molluscum Contagiosum Virus could give fundamental insight into the nature of skin immunity.

Gholamreza Darai - One of the best experts on this subject based on the ideXlab platform.

  • targeting the retinoblastoma protein by mc007l gene product of the Molluscum Contagiosum Virus detection of a novel Virus cell interaction by a member of the poxViruses
    Journal of Virology, 2008
    Co-Authors: Stefan Mohr, Gholamreza Darai, Stephanie Grandemange, Paola Massimi, Lawrence Banks, Jeanclaude Martinou, Martin Zeier, Walter Muranyi
    Abstract:

    The human pathogenic poxVirus Molluscum Contagiosum Virus (MCV) is the causative agent of benign neoplasm, with worldwide incidence, characterized by intraepidermal hyperplasia and hypertrophy of cells. Here, we present evidence that the MC007L protein of MCV targets retinoblastoma protein (pRb) via a conserved LxCxE motif, which is present in many viral oncoproteins. The deregulation of the pRb pathway plays a central role in tumor pathogenesis. The oncoproteins of small DNA Viruses contain amino acid sequences that bind to and inactivate pRb. Isolated expression of these oncoproteins induces apoptosis, cell proliferation, and cellular transformation. The MC007L gene displays no homology to other genes within the poxVirus family. The protein anchors into the outer mitochondrial membrane via an N-terminal mitochondrial targeting sequence. Through the LxCxE motifs, MC007L induces a cytosolic sequestration of pRb at mitochondrial membranes, leading to the inactivation of the protein by mislocalization. MC007L precipitates the endogenous pRb/E2F-1 complex. Moreover, MC007L is able to cooperate to transform primary rat kidney cells. The interaction between MC007L and pRb provides a novel mechanism by which a Virus can perturb the cell cycle.

  • mapping of mrna transcripts in the genome of Molluscum Contagiosum Virus transcriptional analysis of the viral slam gene family
    Virus Genes, 2000
    Co-Authors: Joachim Jakob Bugert, Nadja V Melquiot, Gholamreza Darai
    Abstract:

    Molluscum Contagiosum Virus (MCV) is a member of the poxVirus family and causes benign skin tumors in children and immunocompromised individuals. The primary structure and coding capacity of MCV was previously determined by DNA nucleotide sequencing (Senkevich et al., Science 273, 813-816, 1996). Hypothetical genes were predicted based on (i) amino acid homologies with known genes, (ii) presence or absence of conserved transcription regulation signals, and (iii) algorithms based on learning sets of coding sequences. These methods provide a rational basis for the prediction of MCV coding sequences. However, the existence and exact size of MCV open reading frames and the precise position of transcription regulation signals can only be determined by MCV mRNA transcript mapping experiments. We developed methods for the characterization of the mRNA transcripts of MCV genes in infected skin tissue and abortively infected human fibroblast cell cultures. Using these methods the properties of the mRNA transcripts of the MCV SLAM (signaling lymphocytic activating molecule) gene family (mc002L, mc161R, and mc162R) were analyzed. The mRNA start site found for the mc161R transcript suggests that a second start codon is used leading to a mc161R open reading frame that is nine amino acid residues shorter than predicted.

  • characterization of early gene transcripts of Molluscum Contagiosum Virus
    Virology, 1999
    Co-Authors: Joachim Jakob Bugert, Claudia Lohmuller, Gholamreza Darai
    Abstract:

    Molluscum Contagiosum Virus (MCV), a member of the family Poxviridae, replicates wellin vivobut cannot be propagated in cell culture. The coding capacity of the MCV genome was previously determined by DNA nucleotide sequence analysis. The objective of the present study was to establish experimental systems for the identification and characterization of early MCV gene transcripts. MCV mRNA was obtained in three ways: (1) MCV early mRNA was synthesizedin vitrousing permeabilized virions, (2) MCV mRNA was extracted from MCV-infected skin tissue, and (3) MCV mRNA was extracted from MCV-infected human embryonic fibroblasts. RNA/DNA hybridization experiments showed significant early transcriptional activity in two parts of the MCV genome. Transcripts of 11 early MCV genes located in these parts of the genome, including two subunits of the MCV DNA-dependent RNA polymerase (mc077R and mc079R), the MCV poly(A)+polymerase gene (mc076R), and the MCV MHC class I homolog (mc080R), were detected in reverse transcription-polymerase chain reaction experiments. Total RNA obtained from MCV-infected skin tissue was used to confirm these results. Three MCV early transcripts, mc002L, mc004.1L, and mc005L, produced distinct bands on rapid amplification of their 3′ ends (3′ RACE). The 5′ mapping of transcription start sites of MCV open reading frames (ORFs) mc002L, mc004.1L, mc005L, and mc148R revealed that the MCV RNA polymerase transcription start sites are consistently located between 11 and 13 nucleotides downstream of the early MCV consensus promoter signal. When cDNA from both 5′ and 3′ mapping experiments was analyzed, MCV ORFs mc004.1L and mc005L were found to be transcribed as a single bicistronic mRNA. The transcript from MCV ORF mc066L, encoding a glutathione peroxidase, was detected inin vitrosynthesized MCV mRNA as well as in total RNA from MCV-infected human embryonic fibroblasts and MCV-infected skin. This indicates that despite the lack of an early MCV consensus promoter signal immediately proximal to the start codon, this particular gene is transcribed early during MCV infection.

  • chemokine homolog of Molluscum Contagiosum Virus sequence conservation and expression
    Virology, 1998
    Co-Authors: Joachim Jakob Bugert, Inger K. Damon, Bernard Moss, Claudia Lohmuller, Gholamreza Darai
    Abstract:

    An analysis of the complete Molluscum Contagiosum Virus (MCV-1) genome sequence revealed a 104-amino-acid open reading frame (MC148R) that is structurally related to the β (CC) family of chemokines. The predicted MCV chemokine homolog (MCCH) has a deletion in the NH2-terminal activation domain, suggesting the absence of chemoattractant activity. The principal objectives of the present study were to determine whether: (i) MCCH is conserved in independent isolates of MCV-1 and MCV-2; (ii) MCCH mRNA is expressedin vivo;and (iii) the MCCH protein is secreted from mammalian cells. The nucleotide sequence of the MCCH gene locus was determined for 27 isolates of MCV-1 and 2 of MCV-2 obtained from 29 MCV-infected individuals. In each case, the characteristic CC sequence, the NH2-terminal deletion, and the length of the open reading frame were conserved, although there were some, mostly conservative, amino acid substitutions. Since MCV cannot be propagated in cell culture, mRNA was synthesizedin vitroby the early transcription apparatus in purified MCV virions. MCCH RNA was amplified by RT–PCR; the sequence included the complete open reading frame and extended 40 to 50 nucleotides past the first poxviral termination signal (TTTTTNT). Similar RT–PCR results were obtained using total cellular RNA derived from MCV-infected tissue specimens. Finally, the MCCH open reading frame was expressed in a vaccinia Virus vector and the predicted size polypeptide was secreted into the medium, as determined by Western blotting. Taken together, our data support the prediction that MCV expresses a secreted chemokine homolog that could antagonize the inflammatory responsein vivo.

  • the genome of Molluscum Contagiosum Virus analysis and comparison with other poxViruses
    Virology, 1997
    Co-Authors: Tatiana G. Senkevich, Joachim Jakob Bugert, Eugene V Koonin, Gholamreza Darai, Bernard Moss
    Abstract:

    Analysis of the Molluscum Contagiosum Virus (MCV) genome revealed that it encodes approximately 182 proteins, 105 of which have direct counterparts in orthopoxViruses (OPV). The corresponding OPV proteins comprise those known to be essential for replication as well as many that are still uncharacterized, including 2 of less than 60 amino acids that had not been previously noted. The OPV proteins most highly conserved in MCV are involved in transcription; the least conserved include membrane glycoproteins. Twenty of the MCV proteins with OPV counterparts also have cellular homologs and additional MCV proteins have conserved functional motifs. Of the 77 predicted MCV proteins without OPV counterparts, 10 have similarity to other MCV proteins and/or distant similarity to proteins of other poxViruses and 16 have cellular homologs including some predicted to antagonize host defenses. Clustering poxVirus proteins by sequence similarity revealed 3 unique MCV gene families and 8 families that are conserved in MCV and OPV. Two unique families contain putative membrane receptors; the third includes 2 proteins, each containing 2 DED apoptosis signal transduction domains. Additional families with conserved patterns of cysteines and putative redox active centers were identified. Promoters, transcription termination signals, and DNA concatemer resolution sequences are highly conserved in MCV and OPV. Phylogenetic analysis suggested that MCV, OPV, and leporipoxViruses radiated from a common poxVirus ancestor after the divergence of avipoxViruses. Despite the acquisition of unique genes for host interactions and changes in GC content, the physical order and regulation of essential ancestral poxVirus genes have been largely conserved in MCV and OPV.

Jeffrey I. Cohen - One of the best experts on this subject based on the ideXlab platform.

  • Molluscum Contagiosum Virus transcriptome in abortively infected cultured cells and a human skin lesion
    Journal of Virology, 2016
    Co-Authors: Jorge D Mendezrios, Jeffrey I. Cohen, Zhilong Yang, Karl J Erlandson, Craig Martens, Daniel Bruno, Stephen F Porcella, Bernard Moss
    Abstract:

    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.

  • Investigation of Molluscum Contagiosum Virus, orf and other parapoxViruses in lymphomatoid papulosis
    Journal of the American Academy of Dermatology, 2013
    Co-Authors: Kristen Heins Fernandez, Hui Zhao, Matthew Bream, Mir A. Ali, Tammy Krogmann, Jeffrey I. Cohen, Inger K. Damon, Vincent Liu
    Abstract:

    Lymphomatoid papulosis (LyP) is a primary cutaneous CD30+ lymphoproliferative disorder with large, atypical CD-30+ cells, which despite their ominous appearance, also occur in reactive processes, such as CD30+ cutaneous lymphoid hyperplasia. As most causes of CD30+ cutaneous lymphoid hyperplasia are viral,1 we explored the possibility that poxVirus or parapoxVirus could be detected in lesions of LyP. Following University of Iowa Institutional Review Board approval, nine patients with a diagnosis of LyP that had available skin tissue blocks were included. For negative controls, two scar tissue samples were included. DNA was extracted from formalin-fixed, paraffin-embedded slides. Tissue was dewaxed by addition of xylene, vortexing, and centrifuging; xylene was removed and the pellet was rehydrated in 100% ethanol. After vortexing, centrifugation, and air drying, the sample was resuspended in digestion buffer and was incubated at 55°C for 4–6 hrs. Chelex-100 (BioRad) was added to a final concentration of 5%. The mixture was boiled 8 min, chilled 2 min, and centrifuged for 5 min at 4°C. The supernatant was transferred to a fresh centrifuge tube as template. Quantitative real-time polymerase chain reaction (q-PCR) was performed to detect Molluscum Contagiosum Virus p43K gene2; 10 ul of DNA was used in each assay. To ensure sufficient DNA was present and amplification was not due to presence of an inhibitor, the single copy RNaseP gene was detected by amplification of the DNA sample using a TaqMan RNaseP detection reagent kit (Applied Biosystems). A plasmid containing MCV p43K gene served as a positive control2. ParapoxVirus generic and orf Virus specific q-PCR assays were also performed on the extracted DNA3, with positive controls using nucleic acid from Ovine Ecthyma Vaccine (Colorado Serum Company) and orf Virus. No Molluscum Contagiosum, parapoxVirus generic, or orf Virus DNA was detected the LyP samples or scars. Of the many infectious conditions associated with CD30+ cutaneous lymphoid hyperplasias, the most commonly associated Viruses have been parapoxViruses (28.5%), herpes simplex and varicella zoster Viruses (25%), and Molluscum Contagiosum Virus (10.7%)1. Extrapolating these findings to true CD30+ lymphoproliferative disorders, previous studies have explored the possibility that LyP may represent a virally-induced cutaneous lymphoid hyperplasia. One analysis of LyP specimens by PCR demonstrated one out of nine LyP samples with detectable HHV 6 DNA, but no detectable HSV 1 and 2 or EBV DNA. 4. Recently, biopsy specimens of cutaneous lymphomas (including 3 cases of LyP) were assayed for Merkel cell polyoma Virus, human polyomaVirus type 6, human polyoma Virus type 7, and trichodysplasia spinulosa-associated polyoma Virus DNA by q- PCR; the cases of LyP were negative for all viral types5. As LyP and parapoxVirus and poxVirus virally-mediated CD30+ cutaneous lymphoid hyperplasias share overlapping clinical features (papular lesions, generally indolent course) and pathologic features (large, atypical CD30+ lymphoid infiltrates), LyP appears an intriguing candidate for induction by viral infection. However, the lack of detection of viral DNA in our LyP specimens argues against a link between parapoxViruses or Molluscum Contagiosum Virus and LyP, although the role of sampling and stage of lesions should be considered.

  • detection of Molluscum Contagiosum Virus mcv dna in the plasma of an immunocompromised patient and possible reduction of mcv dna with cmx 001
    The Journal of Infectious Diseases, 2012
    Co-Authors: Jeffrey I. Cohen, Wilmer F Davila, Siuping Turk, Edward W Cowen, Alexandra F Freeman, Kening Wang
    Abstract:

    Molluscum Contagiosum Virus (MCV) is a poxVirus that causes localized papules in healthy persons. We evaluated a woman with severe immunodeficiency and disseminated MCV. During treatment with CMX-001, an antiviral with activity against other poxViruses, MCV DNA was detected in 20% of plasma samples. When the patient was not receiving CMX-001, MCV DNA was detected in 50% of samples. We also noted improvement in warts on her fingers during CMX-001 therapy. Although MCV is caused by direct inoculation of Virus into skin in healthy persons, in a severely immunocompromised person MCV DNA was present in blood and may spread by viremia.

  • the death effector domains deds of the Molluscum Contagiosum Virus mc159 v flip protein are not functionally interchangeable with each other or with the deds of caspase 8
    Virology, 2002
    Co-Authors: Tara L Garvey, John Bertin, Richard M Siegel, Michael J Lenardo, Jeffrey I. Cohen
    Abstract:

    The Molluscum Contagiosum Virus (MCV) MC159 protein contains two death effector domains (DEDs) that bind to the DEDs of caspase-8 and FADD and inhibit apoptosis. We constructed MC159 truncation mutants and found that the amino-terminal region before the first DED and nearly all the carboxyl terminus after the second DED were dispensable for the antiapoptotic activity of MC159. We also engineered tandem repeats of two identical MC159 DEDs, MC159 DEDs in the reverse orientation, and MC159-caspase-8 chimeras in which a DED of MC159 was replaced with the corresponding DED of caspase-8. Each of these constructs bound to caspase-8, but was unable to bind to FADD or block apoptosis. In addition, we constructed mutants containing only a single DED of MC159. These mutants bound to both FADD and caspase-8, but could not block apoptosis or the formation of death effector filaments. Thus, the DEDs of MC159 are not functionally interchangeable with each other or with those of caspase-8.

  • binding of fadd and caspase 8 to Molluscum Contagiosum Virus mc159 v flip is not sufficient for its antiapoptotic function
    Journal of Virology, 2002
    Co-Authors: Tara L Garvey, John Bertin, Guanghua Wang, Richard M Siegel, Michael J Lenardo, Jeffrey I. Cohen
    Abstract:

    Death receptors constitute a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which are defined by the presence of a signaling domain with six α-helices in the cytoplasmic region that is termed the death domain (13). These receptors function to maintain homeostasis in the immune system by eliminating autoreactive cells, antigen-reactive T cells following an immune response, and Virus-infected or malignant cells. In humans, six death receptors have been identified: Fas (also called CD95 and APO-1), TNF receptor 1 (TNFR1; also called TNFRSF1A and CD120a p55-R), DR3 (also called APO3, Wsl-1, TRAMP. and LARD), DR4 (also called TRAIL-R1 and Apo-2), DR5 (also called TRAIL-R2, KILLER, and TRICK2), and DR6 (17, 19). Binding of a death receptor to its ligand initiates a change in the receptor complex, resulting in signaling through a series of protein-protein interactions that culminate in apoptosis (4, 22). Fas binding to its ligand (FasL) leads to the recruitment of the adapter molecule FADD (Fas-associated death domain) through interactions of the death domains in Fas and FADD. FADD contains another region known as the death effector domain (DED). The FADD DED also possesses six α-helices in a folded region similar to the death domains, but it forms distinct contacts only with other DED-containing proteins, whereas death domain-containing proteins interact chiefly with other death domain-containing adapter proteins. Once FADD is recruited to Fas, its DED binds to the DEDs in the prodomain of caspase-8 or caspase-10 (1, 28). The complex containing Fas, FADD, and caspase-8 or caspase-10 is termed the death-inducing signaling complex (DISC). Recruitment of caspase-8 or caspase-10 into the DISC results in autocatalytic cleavage of the caspase into its active subunits and subsequent cleavage and activation of substrates, including other caspases, ultimately leading to apoptosis (15, 16). FADD also serves as an adapter molecule in other death receptor pathways (3, 5, 9, 10, 12, 24). Hence, death receptors may all work by a common mechanism involving recruitment of DED-containing caspases to form an active signaling complex. Apoptosis affords the host a defense mechanism to eliminate Virus-infected cells. This must have proven sufficiently effective, because Viruses in turn evolved mechanisms to interfere with host apoptosis pathways. A family of proteins known as the viral FLICE-inhibitory proteins (v-FLIPs) inhibit the signaling pathways in death receptor-induced apoptosis. These include the Molluscum Contagiosum Virus (MCV) MC159 protein, the equine herpesVirus 2 (EHV-2) E8 protein, and the bovine herpesVirus E2 protein (2, 25). v-FLIPs block apoptosis induced through the Fas, TNFR1, DR3, DR4, and DR5 pathways. Apoptosis inhibition by the v-FLIPs stems from their two DEDs that can interact with the DEDs of FADD and caspase-8 (2, 11, 25, 27). The MCV MC159 protein is present in a complex with Fas when the receptor is activated (21). The EHV-2 E8 protein can be recruited to the DISC and may prevent activation of caspase-8 by blocking its recruitment to the DISC (25). v-FLIPs also inhibit formation of a cytoplasmic structure termed the death effector filament (18, 23). High-level expression of FADD or the prodomain of caspase-8 in cells results in their oligomerization as death effector filaments that recruit and activate caspases. Coexpression of v-FLIPs with FADD or the prodomain of caspase-8 blocks death effector filament formation and subsequent apoptosis. Despite these correlations, the precise inhibitory mechanism of v-FLIP, especially MC159, in the Fas signaling complex is unknown. The MC159 v-FLIP protein contains a six-amino-acid N-terminal sequence followed by a 74-amino-acid DED, a 14-amino-acid linker region, an 83-amino-acid DED, and a 64-amino-acid carboxy-terminal tail. An RXDL motif, conserved among other DED-containing proteins, is present at the carboxy end of each DED. MC159 DEDs are homologous to other DED-containing proteins, including FADD. Alignment of the MC159 DED sequences onto the nuclear magnetic resonance (NMR)-determined structure of the FADD DED suggests that each MC159 DED consists of six α-helices and contains a highly conserved hydrophobic patch on its surface (6). The role that each of these protein motifs might play in apoptosis inhibition has not yet been defined. Based on prior studies, v-FLIP is thought to prevent apoptosis by binding to FADD and caspase-8 (2, 21, 25, 26). In order to identify functionally important regions of MC159, we constructed a series of mutations in which charged amino acids were changed to alanines. Since charged amino acids are often on the surface of proteins, they are likely to participate in protein-protein interactions. Surprisingly, we found that the majority of the MC159 mutants that lost the ability to block apoptosis induced by Fas, TNF, and TNF-related apoptosis-inducing ligand (TRAIL) still bound FADD and caspase-8. The predicted hydrophobic patch 1 and α2 regions were important for FADD and caspase-8 binding. We also found that the conserved RXDL motif within the predicted α6 region is critical for protection from apoptosis and for inhibition of death effector filament formation.