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Two Major Antigenic Polypeptides of Molluscum Contagiosum Virus

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Two Major Antigenic Polypeptides of Molluscum Contagiosum Virus 284 Two Major Antigenic Polypeptides of Molluscum Contagiosum Virus Takahiro Watanabe, Shigeru Morikawa, Kenji Suzuki, Departments of Virology II, Virology I, and Pathology, National Tatsuo Miyamura, Kunihiko Tamaki, and Yoshiaki Ueda Institute of Infectious Diseases, and Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan A library of molluscum contagiosum virus (MCV) transferred into the cowpox vector expression system was screened with 12 sera from molluscum patients. Two recombinant proteins of 70 and 34 kDa were detected by immunoblotting and mapped to the open-reading frames MC133L and MC084L, respectively. Consensus sites were found between the C-terminus of the 70-kDa MCV protein and the 14-kDa fusion protein of vaccinia and variola virus, and between the 34-kDa MCV protein and the 37.5-kDa viral membrane±associated protein of vaccinia and variola virus. Rabbit Downloaded from https://academic.oup.com/jid/article/177/2/284/925332 by guest on 30 September 2021 antisera against these two proteins were prepared. An immuno¯uorescence study demonstrated that the 70- and 34-kDa proteins were predominantly expressed on the surface of recombinant virus± infected HeLa cells, indicating the potential to be inserted into the membrane. On immunoelectron microscopy, antiserum against 70-kDa protein showed signi®cant labeling of the MCV membrane, while the antiserum against 34-kDa protein failed to do so. After the eradication of variola virus in 1977, molluscum conditions indicated that they were essentially collinear and contagiosum virus (MCV) has been the sole member of the that minor sequence heterogeneity among subtypes contributed poxviruses speci®c for humans. MCV has a linear double- to the loss or gain of individual restriction sites [14]. Further, stranded DNA genome of 185,000±195,000 bp and induces molecular epidemiologic studies have failed to demonstrate any benign and self-limiting skin tumors, preferentially in children correlation of MCV subtype with clinical pro®les of MCV, and young adults [1, 2]. In children, molluscum lesions are including patient age and sex and duration, site, and number scattered haphazardly over the trunk and extremities, whereas of molluscum contagiosum lesions [14, 15]. in young adults the papules occur in the genital area and are The skin lesions induced by MCV usually persist for sexually transmitted [3]. Recently, multiple and extensive mol- months to years until spontaneous resolution occurs, and some luscum lesions were found in up to 20% of AIDS patients, and circumstantial evidence has suggested that it may be associ- much greater importance has been attached to the virus as a ated with the induction of cellular and humoral immune re- cutaneous marker for opportunistic infection of immunocom- sponses [18, 19]. A serial histologic observation revealed promised hosts [4±6]. Although inef®cient replication and dense mononuclear cell in®ltration around the disappearing growth of MCV were demonstrated in human skin grafted to molluscum lesions, indicating that a cell-mediated in¯amma- athymic mice [7, 8], other attempts to propagate the virus in tory reaction plays an important role in exclusion of the virus tissue culture and to transmit it to experimental animals have [20]. In earlier studies, the crude antigen derived from mol- not been successful [9±11]. Once the sterility of the virus can luscum lesions was prepared, and virus-speci®c antibodies be improved, MCV has a great potential as a tissue-restricted were detected by the Ouchterlony method [21], complement vector for expression of foreign genes in human keratinocytes. ®xation test [22], and indirect immuno¯uorescence staining The molluscum skin lesions contain millions of virus parti- [23±25]. Later, an ELISA was developed to measure levels cles that are suf®cient for molecular epidemiologic analyses. of IgG antibodies; a very close relationship was also found Three major subtypes and some variants have been reported, in the antibody levels between different molecular subtypes based on the restriction endonuclease cleavage pattern of viral of MCV on cross-testing patients' sera using DNA-typed puri- DNA [12±17]. Cross-hybridization studies under stringent ®ed virions as antigens [26]. We are interested in which molecules of the MCV are recog- nized in the humoral immune system of molluscum patients. Although all of the genome of MCV, with the exception of Received 27 May 1997; revised 25 August 1997. both terminal hairpin loops, has been cloned [27, 28] and se- Presented in part: annual meeting of the Society for Investigative Dermatol- quenced [29±33], functional studies have not been reported. ogy (Washington, DC, 23±27 April 1997). A full genomic library of MCV DNA contained in the cowpox Financial support: Promotion of Polio Eradication, Ministry of Health and Welfare; grants-in-aid from Ministry of Education, Science, and Culture; Lydia vector expression system was screened with sera from mol- O'Leary Memorial Foundation. luscum patients. The purpose of our study was to map and Reprints or correspondence: Dr. Takahiro Watanabe, Dept. of Dermatology, characterize the sera-reactive polypeptides. Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113, Japan (email: [email protected]). Materials and Methods The Journal of Infectious Diseases 1998;177:284±92 0 q 1998 by The University of Chicago. All rights reserved. Cells and viruses. HeLa and human 143TK cells were grown 0022±1899/98/7702±0003$02.00 as monolayers in Eagle's MEM supplemented with 10% bovine / 9d3f$$fe13 12-23-97 07:32:33 jinfa UC: J Infect JID 1998;177 (February) Antigenic Polypeptides of MCV 285 serum. A white pock variant from pock-puri®ed red cowpox virus Immunoblotting. Puri®ed MCV or cell extracts were dis- strain Brighton (cowpox white variant) was propagated in HeLa rupted in 21 sample buffer containing 100 mM Tris-hydrochlo- cells and puri®ed as described previously [34]. Spodoptera frugi- ride (pH 7.0), 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, perda (Sf) cells were grown in TC-100 medium containing 10% and 0.2% bromophenol blue. Solubilized polypeptides were run fetal bovine serum. Autographa californica nuclear polyhedrosis on 10% SDS-polyacrylamide gels and transferred onto nitrocellu- virus (AcNPV) and a recombinant baculovirus were propagated lose ®lters as described previously [39]. Before transfer, parts of in Sf cells as described previously [35]. each lane were separated, and fractionated polypeptides were Molluscum lesions were obtained from 12 patients attending the visualized by Coomassie brilliant blue staining. The transferred Department of Dermatology in Tokyo University Hospital (aged ®lters were soaked with blocking solution (dried nonfat milk) at 2.3±9.4 years; mean, 5.5). Puri®cation of MCV and DNA extrac- 47C overnight, washed three times with 5% fetal bovine serum tion were done as described previously [16]. Viral DNA was in 10 mM Tris-hydrochloride (pH 7.3), 115 mM sodium chloride, cleaved with restriction endonucleases BamHI, ClaI, and HindIII. and 0.05% Tween-20 (TBS-T), and then incubated with diluted The virus isolates were typed as MCV subtype 1 variant on the serum at 47C overnight. After washing three times with TBS-T, Downloaded from https://academic.oup.com/jid/article/177/2/284/925332 by guest on 30 September 2021 basis of the cleavage pattern as previously described [15, 16]. the ®lters were incubated with anti-human IgG or anti-rabbit IgG MCV DNA fragments cleaved with BamHI, ClaI, or HindIII were conjugated with alkaline phosphatase at 377C for 1 h. The ®lters inserted into plasmids or cosmids, and an overlapping library con- were washed three times with TBS-T and visualized with a mix- taining the entire genome of the MCV 1 variant was established. ture of 0.016% 5-bromo-4-chloro-3-indolyl phosphate and It was possible that the library would form a mosaic of MCV 1 0.033% nitroblue tetrazolium. variant because the MCV DNA was puri®ed from molluscum le- Immuno¯uorescence. Cells growing on coverslips were in- sions from different patients. fected with recombinant cowpox virus at an MOI of 10 pfu/cell. Antibodies. Blood samples were collected from 12 patients At 6 h after infection, cells were washed three times with PBS described above at ®rst consultation. All patients were healthy and ®xed under nonpermeable (4% formaldehyde±0.1% glutaral- young children and had not been immunized with vaccinia virus. dehyde in PBS at 47C for 45 min) or permeable (acetone at 0207C Control sera from 10 healthy infants were drawn from the World for 15 min) conditions. Cells were washed three times in PBS and Health Organization and the National Serum Reference Bank/ then incubated with rabbit antiserum diluted 1:320 in 10% dried Tokyo (National Institute of Infectious Diseases, Japan) (aged 0.8 nonfat milk at 377C for 1 h. After washing three times in PBS, months to 1.1 years; mean, 6.7 months). cells were incubated with anti-rabbit IgG at 377C for 1 h, washed New Zealand White rabbits were immunized with the puri®ed three times, mounted in glycerol-PBS (1:1 [vol/vol]), and visual- MCV and with the recombinant baculovirus±infected Sf cells with ized under a Zeiss ¯uorescence microscope. Freund's complete adjuvant, followed by a booster 3 weeks later. Immunoelectron microscopy. For postembedding labeling for The antisera were collected 1 week after booster injection. immunoelectron microscopy [40], puri®ed MCV was ®xed with 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 8.0). After Production of recombinant virus. Recombinant cowpox vi- dehydration with increasing amounts of ethanol (50%±100%), the ruses were obtained by insertion of MCV DNA fragments into the MCV samples were embedded (Lowicryl K4M; Chemische Werke thymidine kinase (TK) gene of the cowpox virus transfer vector Lowi, Waldkraiburg, Germany) and polymerized by UV irradia- [36].
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