Deching Chang

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

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.

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

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.

High incidence of JC viruria in JC-seropositive older individuals

The prevalence of the human JC virus (JCV) in the general population at various ages was investigated. Polymerase chain reaction was employed to detect viral DNA in the urine. The results showed that the incidence of JC viruria was low in the young population, but it was high in the elderly. Hemagglutination inhibition assay was performed for JCV seroprevalence study. The results showed that the seropositive rate of JCV was lower in children than that in adults. The ratio of viruria to seropositive for JCV increased with age and reached 79.7% for those older than 70 years. The results indicated that aging immunity may correlate with JCV reactivation.

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

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.

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

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.

Different genotypes of human polyomaviruses found in patients with autoimmune diseases in Taiwan

We have assayed for the presence of human polyomaviruses in urine of autoimmune disease patients, such as systemic lupus erythematosus (SLE), Sjogrens syndrome (SS), rheumatoid arthritis (RA), or dermatomyositis/polymyositis (DM/PM), by PCR. The results indicate that approximately 40% of patients were JCV positive and 15% of the JCV positive patients were also infected by BKV at the same time according to Southern blot and DNA sequencing of the PCR products. Interestingly, the JCV present in autoimmune diseases patients were Taiwan‐1, Taiwan‐2, and Taiwan‐3 strains with pentanucleotide‐A (GGGAA) and/or ‐B (AAAGC) deletions within the regulatory region. In addition, BKV found in the examined samples were Taichung‐1 and Taichung‐2 strains. Taichung‐1 had two nucleotide alterations and Taichung‐2 had six nucleotide differences within the regulatory region when compared to WW BKV archetype. Although the examined autoimmune diseases patients included RA, SLE, PM, DM, and SS patients, there appears to be no correlation between disease and virus strains. However, Taiwan‐2 strain JCV with two copies of pentanucleotide‐A deletion was present in the patient with the longest period of immunosuppressive medication.

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

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.

Prevalence and genotype identification of human JC virus in colon cancer in Taiwan.

Although JC virus (JCV), a human polyomavirus, has been detected in colon cancers, the association between JCV and colon cancer remains controversial. In Taiwan, the prevalence of JCV infection in colon cancer patients has not been reported. Thus, the purpose of this study was to investigate JCV infection in colon cancers in Taiwan. Formalin‐fixed, paraffin‐embedded tissues from 22 colon cancer patients were examined in this study. Nested PCR was performed to detect viral genomic DNA. The product of the nested PCR flanking the JCV regulatory region was sequenced further. Viral large tumor protein, LT, and late capsid protein, VP1, were examined by immunohistochemistry (IHC). Nested PCR revealed JCV genomic DNA in 86.4% (19/22) of the colon cancer tissue samples. Both rearranged and archetypal genotypes of JCV were identified. Expression of JCV LT was positive in 63.6% (14/22) of the examined colon cancer tissue samples but not in any adjacent normal region. Expression of viral capsid protein VP1 was not detected in any of the tissues examined. The current study demonstrates that JCV genomic DNA was present in the examined colon cancer tissues. The genotypes of JCV in colon cancer tissues were also identified. Expression of viral early protein but not structural capsid protein was detected in the examined colon cancer tissues. Furthermore, a high prevalence of JCV infection in colon cancer tissues in Taiwan was also demonstrated. J. Med. Virol. 80:1828–1834, 2008.

Distinct DNA methylation epigenotypes in bladder cancer from different Chinese sub-populations and its implication in cancer detection using voided urine.

BackgroundBladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. Previous studies have identified several tumor-related genes that are hypermethylated in bladder cancer; however the DNA methylation profile of bladder cancer in Taiwan is not fully understood.MethodsIn this study, we compared the DNA methylation profile of multiple tumor suppressor genes (APC, DAPK, E-cadherin, hMLH1, IRF8, p14, p15, RASSF1A, SFRP1 and SOCS-1) in bladder cancer patients from different Chinese sub-populations including Taiwan (104 cases), Hong Kong (82 cases) and China (24 cases) by MSP. Two normal human urothelium were also included as control. To investigate the diagnostic potential of using DNA methylation in non-invasive detection of bladder cancer, degree of methylation of DAPK, IRF8, p14, RASSF1A and SFRP1 was also accessed by quantitative MSP in urine samples from thirty bladder cancer patients and nineteen non-cancer controls.ResultsThere were distinct DNA methylation epigenotypes among the different sub-populations. Further, samples from Taiwan and China demonstrated a bimodal distribution suggesting that CpG island methylator phentotype (CIMP) is presented in bladder cancer. Moreover, the number of methylated genes in samples from Taiwan and Hong Kong were significantly correlated with histological grade (P < 0.01) and pathological stage (P < 0.01). Regarding the samples from Taiwan, methylation of SFRP1, IRF8, APC and RASSF1A were significantly associated with increased tumor grade, stage. Methylation of RASSF1A was associated with tumor recurrence. Patients with methylation of APC or RASSF1A were also significantly associated with shorter recurrence-free survival. For methylation detection in voided urine samples of cancer patients, the sensitivity and specificity of using any of the methylated genes (IRF8, p14 or sFRP1) by qMSP was 86.7% and 94.7%.ConclusionsOur results indicate that there are distinct methylation epigenotypes among different Chinese sub-populations. These profiles demonstrate gradual increases with cancer progression. Finally, detection of gene methylation in voided urine with these distinct DNA methylation markers is more sensitive than urine cytology.

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

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.