Identification of a DNA encapsidation sequence for human polyomavirus pseudovirion formation
ABSTRACT Human polyomavirus is a naked capsid virus containing a closed circular double-stranded DNA genome. The mechanism of DNA encapsidation for the viral progeny formation is not fully understood. In this study, DNA encapsidation domain of the major capsid protein, VP1, of the human polyomavirus JCV was investigated. When the first 12 amino acids were deleted, the E. coli expressed VP1 (ΔN12VP1) failed to encapsidate the host DNA although the integrity of the capsid-like structure was maintained. In addition, capsid-like particles of ΔN12VP1 did not package exogenous DNA in vitro, which is in contrast to that of the full-length VP1 protein. These findings suggest that the N-terminal of the first 12 amino acids of VP1 were responsible for DNA encapsidation. The importance of amino acids in the DNA encapsidation domain was determined further using site-directed mutagenesis. All of the positively charged amino acids at the N-terminal region of VP1 were essential for DNA encapsidation. The results indicate that the N-terminal region of the human polyomavirus major capsid protein VP1 may be involved in viral genome encapsidation during progeny maturation. J. Med. Virol. 64:366–373, 2001. © 2001 Wiley-Liss, Inc.
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ABSTRACT: To investigate the DNA binding activity of the JC virus minor capsid protein, VP2, both wild-type and mutant VP2 were cloned and expressed in Escherichia coli. Southwestern blotting was employed for the DNA-binding assay. The results showed that VP2 was able to bind to DNA, except when either the last 13 or the last 29 amino acids were truncated. The results indicate that the DNA-binding domain of VP2 is located within the last 13 amino acids. Furthermore, we also demonstrated that Lys(332) and Lys(336) within the DNA-binding domain are crucial for DNA binding. The findings may provide further information for understanding the mechanism of virion assembly of the JC virus.Journal of NeuroVirology 02/2003; 9 Suppl 1:21-4. · 2.85 Impact Factor
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ABSTRACT: 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.Gene therapy 08/2010; 17(8):1033-41. · 4.75 Impact Factor
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ABSTRACT: Human JC virus (JCV) is a neurotropic virus, and the etiological agent of progressive multifocal leukoencephalopathy (PML), a fatal neurological disease. Because of its natural infection tropism, it is possible to use the JCV capsid as a gene-transducing vector for therapeutic purposes in neurological disorders. In the current study, a recombinant JCV virus-like particle (VLP) was generated and purified from yeast. VLP was able to accommodate and protect DNA molecules of up to approximately 2000 bp in length. VLP was able to package and deliver an antisense oligodeoxynucleotide (AS-ODN) against simian virus 40 (SV40) large tumor antigen (LT) into SV40-transformed human fetal glial (SVG) cells in order to inhibit expression of the oncoprotein. Subsequently, apoptosis of VLP-AS-ODN-treated cells was demonstrated after the blocking of LT expression. In addition, JCV VLP was able to deliver ODN into human astrocytoma, neuroblastoma, and glioblastoma cells with high efficiency. In vivo delivery of ODN into a human neuroblastoma tumor nodule by VLP was also demonstrated. These findings suggest that JCV VLP is a gene delivery vector with potential therapeutic use for human neurological disorders.Human Gene Therapy 12/2004; 15(11):1077-90. · 4.02 Impact Factor