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Journal of General Virology | 1997

SELF-ASSEMBLY OF THE JC VIRUS MAJOR CAPSID PROTEIN, VP1, EXPRESSED IN INSECT CELLS

Deching Chang; Chiung-Yau Fung; Wei-Chih Ou; Pei-Chi Chao; Shuan-Yow Li; Meilin Wang; Yih-Leh Huang; Tsair-Yuh Tzeng; Rong-Tai Tsai

The major capsid protein of human polyomavirus JC virus, VP1, has been cloned into a baculovirus genome and expressed in insect cells. The VP1 protein was expressed in the cytoplasm and transported into the nucleus. It was then purified by a sucrose cushion and CsCI density gradient centrifugation to near homogeneity. Electron microscopy showed that isolated recombinant VP1 protein self-assembled into a capsid-like structure similar to the natural empty capsid. Both chelator (EDTA) and reducing agent (DTT) are required to disrupt the capsid structure into the pentameric capsomeres, as demonstrated by haemagglutination assay and electron microscopy. These results suggest that JC virus VP1 can be transported into the nucleus and self-assembled to form capsid-like particles without the involvement of the viral minor capsid proteins, VP2 and VP3. In addition, metal ions and disulphide bonds appear to be important in maintaining the integrity of the viral capsid structure.


Journal of General Virology | 1999

The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.

Wei-Chih Ou; Meilin Wang; Chiung-Yau Fung; Rong-Tai Tsai; P.-C. Chao; Tzong-Hsiung Hseu; Deching Chang

The full-length major capsid protein, VP1, of the human polyomavirus JC virus was cloned and expressed in Escherichia coli. VP1 protein expressed in E. coli self-assembled into capsid-like particles and caused haemagglutination of human O-type red blood cells. Caesium chloride density-gradient centrifugation analysis revealed that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations. The morphology of pseudo-virion and pseudocapsid particles was observed under the electron microscope. The pseudovirions contained DNA and RNA molecules but the pseudocapsids did not contain any nucleic acid, as analysed by DNA extraction. DNA-binding activity of VP1 was also demonstrated by the South-Western probing method in vitro. Furthermore, pseudocapsids were able to deliver exogenous DNA into human foetal kidney epithelial cells. These results indicate that recombinant JC virus VP1 is able to self-assemble into capsid-like particles and to package DNA in the absence of the minor capsid proteins, VP2 and VP3. This prokaryotic assembly system may facilitate the investigation of maturation mechanism(s) of polyomaviruses. Furthermore, capsid-like particles of JC virus VP1 generated in E. coli potentially could be used as a human gene transfer vector.


Journal of Medical Virology | 1996

Genotypes of human polyomaviruses in urine samples of pregnant women in Taiwan

Deching Chang; Meilin Wang; Wei-Chih Ou; Mou-Shern Lee; Hong-Nerng Ho; Rong-Tai Tsai

The viral DNA of human polyomaviruses JC virus (JCV) and BK virus (BKV) was detected by the polymerase chain reaction (PCR) in urine samples from 31 pregnant women in Taiwan. A pair of appropriate primers amplified both JCV and BKV DNA of the regulatory region simultaneously in PCR. An oligonucleotide probe homologous to both JCV and BKV regulatory region was used subsequently to detect the viral DNA by Southern blotting after PCR amplification. Approximately 36% of the examined urine samples were human polyomavirus positive. The genotypes of JCV and BKV were determined by DNA sequencing of their regulatory regions. Besides CY archetype, a new strain (Taiwan‐1) of JCV with a pentanucleotide (GGGAA) deletion and a new strain (Taichung‐1) of BKV with two nucleotide alterations within the regulatory region were found in the urine samples. Eight of the examined samples were JCV infected, one was BKV infected, and two were JCV and BKV mix‐infected. The JCV positive individuals were infected by CY archetype and Taiwan‐1 strain equally. However, Taichung‐1 strain was the only BKV strain found in the BKV positive individuals.


FEBS Letters | 2001

Disulfide bonds stabilize JC virus capsid-like structure by protecting calcium ions from chelation.

Pei-Lain Chen; Meilin Wang; Wei-Chih Ou; Chong-Kuei Lii; Li-Sheng Chen; Deching Chang

To investigate the role of disulfide bonds in the capsid structure, a recombinant JC virus‐like particle (VLP) was used. The major capsid protein, VP1, of the JC virus was expressed in yeast cells. The yeast‐expressed VP1 was self‐assembled into a VLP. Disulfide bonds were found in the VLP which caused dimeric and trimeric VP1 linkages as demonstrated by non‐reducing SDS–PAGE. The VLP remained intact when disulfide bonds were reduced by dithiothreitol. The VLP without disulfide bonds could be disassembled into capsomeres by EGTA alone, but those with disulfide bonds could not be disassembled by EGTA. Capsomeres were reassembled into VLPs in the presence of calcium ions. Capsomeres formed irregular aggregations instead of VLPs when treated with diamide to reconstitute the disulfide bonds. These results indicate that disulfide bonds play an important role in maintaining the integrity of the JC VLP by protecting calcium ions from chelation.


Journal of Medical Virology | 1997

Incidence of JC viruria is higher than that of BK viruria in Taiwan

Rong-Tai Tsai; Meilin Wang; Wei-Chih Ou; Yao-Ling Lee; Shuan-Yow Li; Chiung-Yau Fung; Yih-Leh Huang; Tsair-Yuh Tzeng; Yueh Chen; Deching Chang

To investigate the prevalence of human polyomaviruses in Taiwan, urine samples from immunocompetent (healthy), transient immunocompromised (pregnant), and prolonged immunosuppressed (autoimmune disease) individuals were collected throughout the island. The viral DNA in the urine was detected by the polymerase chain reaction (PCR) and Southern blot. The viral genotypes were determined by DNA sequencing within the regulatory region. The overall results, including cases reported previously, show that 13.3% (10/75) of immunocompetent individuals, 26.0% (20/77) of pregnant women, and 37.5% (18/48) of autoimmune disease patients are JCV positive. All of the immunocompetent individuals are BKV negative, but 3.9% (3/77) of the pregnant women and 6.2% (3/48) of autoimmune disease patients are BKV positive. Twenty‐four percent (48/200) of the examined urine samples were JCV positive, but only 3% (6/200) were BKV positive. JCV positive individuals were mainly infected with CY (42%) and TW‐1 (52%) subtypes. These results suggest that the incidence of urinary excretion of human polyomaviruses in immunosuppressed individuals is higher than that of immunocompetent individuals. The prevalence of JCV appears to be higher than that of BKV in Taiwan. In addition, CY and TW‐1 are the predominant subtypes of JCV prevalent in the Taiwanese population. J. Med. Virol. 52:253–257, 1997.


Gene Therapy | 2010

Efficient gene transfer using the human JC virus-like particle that inhibits human colon adenocarcinoma growth in a nude mouse model

Li-Sheng Chen; Meilin Wang; Wei-Chih Ou; Chiung-Yau Fung; Pei-Lain Chen; Chi-Fang Chang; Wei Huang; Jaw-Yuan Wang; Paul Y. Lin; Deching Chang

The JC virus (JCV) may infect human oligodendrocytes and consequently cause progressive multifocal leukoencephalopathy (PML) in patients with immune deficiency. In addition, the virus has also been detected in other human tissues, including kidney, B lymphocytes, and gastrointestinal tissue. The recombinant major structural protein, VP1, of JCV is able to self-assemble to form a virus-like particle (VLP). It has been shown that the VLP is capable of packaging and delivering exogenous DNA into human cells for gene expression. However, gene transfer is not efficient when using in vitro DNA packaging methods with VLPs. In this study, a novel in vivo DNA packaging method using the JCV VLP was used to obtain high efficiency gene transfer. A reporter gene, the green fluorescence protein, and a suicide gene, the herpes simplex virus thymidine kinase (tk), were encapsidated into VLPs in Escherichia coli. The VLP was used to specifically target human colon carcinoma (COLO-320 HSR) cells in a nude mouse model. Intraperitoneal administration of ganciclovir in the tk-VLP-treated mice greatly reduced tumor volume. These findings suggest that it will be possible to develop the JCV VLP as a gene delivery vector for human colon cancer therapy in the future.


Journal of Virological Methods | 1996

Production of the antigen and the antibody of the JC virus major capsid protein VP1

Deching Chang; Zeu-Maun Liou; Wei-Chih Ou; Kai-Ziu Wang; Meilin Wang; Chiung-Yau Fung; Rong-Tai Tsai

The DNA of the major capsid protein VP1 of the human polyomavirus JC virus (JCV), Taiwan-3 strain, was generated from the urine of an autoimmune disease patient by polymerase chain reaction (PRC). The VP1 DNA was cloned into a prokaryotic expression vector, pGEX-4T-1, for expression in E. coli. The nucleotide sequences and the deduced amino acid sequences were determined and compared with the JC virus prototype, Mad-1. Thirty nucleotides were different between these two strains. Six of the altered nucleotides affected amino acid coding and ten of them caused changes in endonuclease recognition sites. The recombinant VPI protein was purified and used to raise monospecific antiserum in rabbit. Recombinant JCV VP1 protein and its monospecific antiserum are important clinical reagents and could possibly be developed as a subunit vaccine and as a serological diagnostic antigen in the future. In addition, the region between amino acid residues 40 and 80 of JCV VP1 is predicted to be an antigenic epitope on the basis of its hydropathy plot and comparison with the VP1 sequences of SV40 and BK virus.


Virology | 2010

Global analysis of modifications of the human BK virus structural proteins by LC-MS/MS.

Chiung-Yao Fang; Hsiang-Ying Chen; Meilin Wang; Pei-Lain Chen; Chi-Fang Chang; Li-Sheng Chen; Cheng-Huang Shen; Wei-Chih Ou; Ming-Daw Tsai; Pang-Hung Hsu; Deching Chang

BK virus, a human polyomavirus, may cause nephritis and urological disorders in patients who have undergone renal transplantation. Little is known about the characteristics of the BK viral proteins. In the current study, BK viral proteins were characterized by immunoblotting and LC-MS/MS. The results revealed that BK virus is composed of three structural proteins, VP1, VP2, and VP3 and four cellular histones, H2A, H2B, H3, and H4. The major structural protein, VP1, can be divided into 16 subspecies by two-dimensional gel electrophoresis. Modifications of VP1, VP2, and VP3 were comprehensively identified by LC-MS/MS. The presence of acetylation, cysteinylation, carboxymethylation, carboxyethylation, formylation, methylation, methylthiolation, oxidation, dioxidation, and phosphorylation could be identified. This is the first report providing an analysis of the global modifications present on polyomavirus structural proteins. The identification of these modifications of VP1, VP2, and VP3 should facilitate an understanding of the physiology of BKV during its life cycle.


Expert Opinion on Biological Therapy | 2011

Human JC virus-like particles as a gene delivery vector

Chi-Fang Chang; Meilin Wang; Wei-Chih Ou; Pei-Lain Chen; Cheng-Huang Shen; Paul Y. Lin; Chiung-Yao Fang; Deching Chang

Introduction: As a viral gene delivery vector, the recombinant JC virus-like particles (VLPs) can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLP without the involvement of viral genetic material and then delivered into any tissue susceptible to JC virus (JCV) to allow gene transduction. Therefore, it should be possible in the future to develop a gene delivery vector using the human JC VLPs that will allow gene therapy. Areas covered: Development of a gene delivery vector using the polyomavirus VLPs is reviewed in this article. The advantages and disadvantages of using JC VLP for gene delivery are discussed. Expert opinion: Human JC VLPs are readily produced and can be engineered with ease; they allow specific targeting without the presence of any viral genetic material. For therapeutic purposes, gene(s) of interest or other compounds can be packaged into the VLP and delivered to JCV-susceptible cells at high efficiency.


Journal of Medical Virology | 2000

Treatment with cytotoxic immunosuppression agents increases urinary excretion of JCV in patients with autoimmune disease.

Meilin Wang; Rong-Tai Tsai; Wei-Chih Ou; Chih-Kuang Lin; Gregory J. Tsay; Han Chang; Deching Chang

Human JC virus is ubiquitous in human populations and is reactivated frequently in immunosuppressed patients. Fifty‐one patients with autoimmune disease receiving immunomodulating therapy were evaluated to study the possible relationship between immunosuppression and JCV viruria. Patients were divided into cytotoxic and noncytotoxic treatment groups based on their prescription. The incidence of JCV viruria in the cytotoxic treatment group was significantly higher than that in the noncytotoxic group (67% vs. 28%; P < 0.05). Most patients with JCV viruria were receiving corticosteroid (P = 0.03 for any dose and P < 0.001 for higher‐dose treatments) and cytotoxic agents (P = 0.02). Age, disease duration, and medication duration appeared not to be the precipitating factors of JCV viruria in this study. The results of clinical evaluation indicate that cytotoxic immunosuppression may play an important role in JC virus reactivation. J. Med. Virol. 62:505–510, 2000.

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Deching Chang

National Chung Cheng University

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Meilin Wang

Chung Shan Medical University

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Chiung-Yau Fung

National Chung Cheng University

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Rong-Tai Tsai

Chung Shan Medical University

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Pei-Lain Chen

Central Taiwan University of Science and Technology

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Chiung-Yao Fang

National Chung Cheng University

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Cheng-Huang Shen

National Chung Cheng University

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Chi-Fang Chang

National Chung Cheng University

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Li-Sheng Chen

National Chung Cheng University

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Tzong-Hsiung Hseu

National Tsing Hua University

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