Molluscum Contagiosum Virus Topoisomerase: Purification, Activities, and Response to Inhibitors

ABSTRACT Molluscum contagiosum virus (MCV), the only member of theMolluscipoxvirus genus, causes benign papules in healthy people but disfiguring lesions in immunocompromised patients. The sequence of MCV has been completed, revealing that MCV encodes a probable type I topoisomerase enzyme. All poxviruses sequenced to date also encode type I topoisomerases, and in the case of vaccinia virus the topoisomerase has been shown to be essential for replication. Thus, inhibitors of the MCV topoisomerase might be useful as antiviral agents. We have cloned the gene for MCV topoisomerase, overexpressed and purified the protein, and begun to characterize its activities in vitro. Like other eukaryotic type I topoisomerases, MCV topoisomerase can relax both positive and negative supercoils. An analysis of the cleavage of plasmid and oligonucleotide substrates indicates that cleavage by MCV topoisomerase is favored just 3′ of the sequence 5′ (T/C)CCTT 3′, resulting in formation of a covalent bond to the 3′ T residue, as with other poxvirus topoisomerases. We identified solution conditions favorable for activity and measured the rate of formation and decay of the covalent intermediate. MCV topoisomerase is sensitive to inhibition by coumermycin A1 (50% inhibitory concentration, 32 μM) but insensitive to five other previously reported topoisomerase inhibitors. This work provides the point of departure for studies of the mechanism of function of MCV topoisomerase and the development of medically useful inhibitors.

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Type I Interferon Counteracts Antiviral Effects of Statins in the Context of Gammaherpesvirus Infection

Journal of Virology ◽

10.1128/jvi.02277-15 ◽

2016 ◽

Vol 90 (7) ◽

pp. 3342-3354 ◽

Cited By ~ 5

Author(s):

Philip T. Lange ◽

Eric J. Darrah ◽

Emily P. Vonderhaar ◽

Wadzanai P. Mboko ◽

Michaela M. Rekow ◽

...

Keyword(s):

Cholesterol Synthesis ◽

Type I Interferon ◽

Antiviral Agents ◽

Statin Treatment ◽

Type I ◽

Type I Ifn ◽

Protein Prenylation ◽

Antiviral Effects

ABSTRACTThe cholesterol synthesis pathway is a ubiquitous cellular biosynthetic pathway that is attenuated therapeutically by statins. Importantly, type I interferon (IFN), a major antiviral mediator, also depresses the cholesterol synthesis pathway. Here we demonstrate that attenuation of cholesterol synthesis decreases gammaherpesvirus replication in primary macrophagesin vitroand reactivation from peritoneal exudate cellsin vivo. Specifically, the reduced availability of the intermediates required for protein prenylation was responsible for decreased gammaherpesvirus replication in statin-treated primary macrophages. We also demonstrate that statin treatment of a chronically infected host attenuates gammaherpesvirus latency in a route-of-infection-specific manner. Unexpectedly, we found that the antiviral effects of statins are counteracted by type I IFN. Our studies suggest that type I IFN signaling counteracts the antiviral nature of the subdued cholesterol synthesis pathway and offer a novel insight into the utility of statins as antiviral agents.IMPORTANCEStatins are cholesterol synthesis inhibitors that are therapeutically administered to 12.5% of the U.S. population. Statins attenuate the replication of diverse viruses in culture; however, this attenuation is not always obvious in an intact animal model. Further, it is not clear whether statins alter parameters of highly prevalent chronic herpesvirus infections. We show that statin treatment attenuated gammaherpesvirus replication in primary immune cells and during chronic infection of an intact host. Further, we demonstrate that type I interferon signaling counteracts the antiviral effects of statins. Considering the fact that type I interferon decreases the activity of the cholesterol synthesis pathway, it is intriguing to speculate that gammaherpesviruses have evolved to usurp the type I interferon pathway to compensate for the decreased cholesterol synthesis activity.

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

Journal of Virology ◽

10.1128/jvi.02911-15 ◽

2016 ◽

Vol 90 (9) ◽

pp. 4469-4480 ◽

Cited By ~ 7

Author(s):

Jorge D. Mendez-Rios ◽

Zhilong Yang ◽

Karl J. Erlandson ◽

Jeffrey I. Cohen ◽

Craig A. Martens ◽

...

Keyword(s):

Gene Expression ◽

Skin Lesion ◽

Human Skin ◽

Cultured Cells ◽

Skin Lesions ◽

Molluscum Contagiosum ◽

Rna Seq ◽

Molluscum Contagiosum Virus ◽

Human Specific

ABSTRACTMolluscum 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 duringin vitroinfections 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 anin vitroreplication system.IMPORTANCEThe 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 thein vitroreplication 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 replicationin vitro.

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Mechanism-Based Covalent Neuraminidase Inhibitors with Broad-Spectrum Influenza Antiviral Activity

Science ◽

10.1126/science.1232552 ◽

2013 ◽

Vol 340 (6128) ◽

pp. 71-75 ◽

Cited By ~ 135

Author(s):

Jin-Hyo Kim ◽

Ricardo Resende ◽

Tom Wennekes ◽

Hong-Ming Chen ◽

Nicole Bance ◽

...

Keyword(s):

Broad Spectrum ◽

Antiviral Agents ◽

Neuraminidase Inhibitor ◽

New Class ◽

Development Of Resistance ◽

Spectrum Activity ◽

Future Utility ◽

Drug Resistant Strains ◽

Covalent Intermediate

Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here on a new class of specific, mechanism-based anti-influenza drugs that function through the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and we confirm this mode of action with structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad-spectrum activity against drug-resistant strains in vitro. The similarity of their structure to that of the natural substrate and their mechanism-based design make these attractive antiviral candidates.

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Antiviral activities of aerial subsets of Artemisia species against Herpes Simplex virus type 1 (HSV1) in vitro

Asian Biomedicine ◽

10.5372/1905-7415.0501.007 ◽

2011 ◽

Vol 5 (1) ◽

pp. 63-68 ◽

Cited By ~ 12

Author(s):

Mehrangiz Khajeh Karamoddini ◽

Seyed Ahmad Emami ◽

Masoud Sabouri Ghannad ◽

Esmaeel Alizadeh Sani ◽

Amirhossein Sahebkar

Keyword(s):

Herpes Simplex ◽

Antiviral Activity ◽

Antiviral Agents ◽

Positive Control ◽

Type I ◽

Viral Agent ◽

Dye Absorption ◽

Antiviral Activities ◽

Simplex Virus

Abstract Background: Drug resistance to current anti-herpetic drugs has been increasingly reported. Therefore, there is a need for finding new antiviral agents, in particular from natural sources. Objective: In the present study, antiviral activity of subset extracts obtained from aerial parts of Artemisia including A. incana, A. chamaemelifolia, A. campesteris, A. fragrans, A. annua, A. vulgaris, and A. persica were investigated against Herpes Simplex type I (HSV1). Methods: Different concentrations of extracts (400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 μg/mL) were obtained from subset of each plant separately, and used against KOS strain of HSV1 in HeLa cells. After 24 hours incubation, tetrazolium dye (MTT), was added. The dye absorption by viable cells was measured and compared to the positive control (extract-untreated cells) and acyclovir (as anti-viral agent). Results: The extracts obtained from A. annua had the highest antiviral activity while those of A. chamaemelifolia showed the lowest activity. Conclusion: Subset extracts of A. annua may be an appropriate candidate for further development of anti HSV1 infection.

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A comparison of topoisomerase I activity in normal and transformed cells

Bioscience Reports ◽

10.1007/bf01115159 ◽

1986 ◽

Vol 6 (3) ◽

pp. 301-307 ◽

Cited By ~ 4

Author(s):

W. H. Colledge ◽

M. Edge ◽

J. G. Foulkes

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Topoisomerase I ◽

Transformed Cells ◽

Type I ◽

Experimental Conditions ◽

Viral Oncogenes ◽

Type I Topoisomerases

Many viral oncogenes encode protein—yrosine kinase activities. However, important in vivo substrates of these enzymes have yet to be identified. Recently, type I topoisomerases were shown to be in vitro substrates for two tyrosine kinases. Following tyrosine phosphorylation, topoisomerase I activity was reduced 10-fold (Tse-Dinh et al. Nature312:785–786, 1984). To determine whether topoisomerase I activity was modulated by tyrosine phosphorylation in vivo, we have measured topoisomerase I activity in nuclear lysates prepared from both normal fibroblasts and cells transformed by two different viral oncogenes (v-abl, v-src). Under a variety of experimental conditions, we have found no evidence to support the notion that type I topoisomerase activity is modulated by tyrosine phosphorylation in vivo.

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A Novel Target and Approach for Identifying Antivirals against Molluscum Contagiosum Virus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03660-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7383-7389 ◽

Cited By ~ 3

Author(s):

Hancheng Guan ◽

Manunya Nuth ◽

Natalia Zhukovskaya ◽

Yih Ling Saw ◽

Edward Bell ◽

...

Keyword(s):

Dna Synthesis ◽

Sexual Transmission ◽

Skin Lesions ◽

Molluscum Contagiosum ◽

Health Concern ◽

Target System ◽

Molluscum Contagiosum Virus ◽

Dermatological Disease ◽

Novel Target

ABSTRACTThe dermatological disease molluscum contagiosum (MC) presents as lesions restricted solely to the skin. The poxvirus molluscum contagiosum virus (MCV) is responsible for this skin disease that is easily transmitted through casual contact among all populations, with greater frequency in children and immunosuppressed individuals. In addition, sexual transmission of MCV in adolescents and adults is a health concern. Although the skin lesions ultimately resolve in immunocompetent individuals, they can persist for extended periods, be painful, and result in scarring. Treatment is problematic, and there is no drug that specifically targets MCV. The inability of MCV to propagate in cell culture has impeded drug development. To overcome these barriers, we integrated three new developments. First, we identified a new MCV drug target (mD4) that is essential for processive DNA synthesisin vitro. Second, we discovered a small chemical compound that binds to mD4 and prevents DNA synthesisin vitro. Third, and most significant, we engineered a hybrid vaccinia virus (mD4-VV) in which the natural vaccinia D4 (vD4) gene is replaced by the mD4 target gene. This hybrid virus is dependent on mD4 for viral growth in culture and is inhibited by the small compound. This target system provides, for the first time, a platform and approach for the discovery and evaluation of new therapeutics that can be used to treat MC.

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Collagen fibrillogenesis in vitro: interaction of types I and V collagen

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142670 ◽

1986 ◽

Vol 44 ◽

pp. 210-211

Author(s):

Arthur J. Wasserman ◽

Kathy C. Kloos ◽

David E. Birk

Keyword(s):

Type I Collagen ◽

Corneal Stroma ◽

Minor Component ◽

Homogeneous Distribution ◽

Type I ◽

Type V Collagen ◽

Fibril Morphology ◽

Type V ◽

In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

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Plasminogen Activation in Diabetes Mellitus: Normalization of Blood Sugar Levels Improves Impaired Enzyme Kinetics In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657861 ◽

1985 ◽

Vol 54 (02) ◽

pp. 413-414 ◽

Cited By ~ 10

Author(s):

Margarethe Geiger ◽

Bernd R Binder

Keyword(s):

Plasminogen Activator ◽

Blood Sugar ◽

Metabolic Disorders ◽

Normal Values ◽

Diabetic Patients ◽

Type I ◽

Plasminogen Activation ◽

Lag Period ◽

Sugar Levels

SummaryWe have demonstrated previously that fibrin enhanced plasmin formation by the vascular plasminogen activator was significantly impaired, when components isolated from the plasma of three uncontrolled diabetic patients (type I) were used to study plasminogen activation in vitro. In the present study it can be demonstrated that functional properties of the vascular plasminogen activators as well as of the plasminogens from the same three diabetic patients are significantly improved after normalization of blood sugar levels and improvement of HbAlc values. Most pronounced the Km of diabetic vascular plasminogen activator in the presence of fibrin returned to normal values, and for diabetic plasminogen the prolonged lag period until maximal plasmin formation occurred was shortened to almost control values. From these data we conclude that the observed abnormalities of in vitro fibrinolysis are not primarily associated with the diabetic disease, but might be secondary to metabolic disorders caused by diabetes.

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