Purified JC virus T antigen derived from insect cells preferentially interacts with binding site II of the viral core origin under replication conditions

Brigitte Bollag, Patricia C. Mackeen, Richard John Frisque

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The human polyomavirus JC virus (JCV) establishes persistent, asymptomatic infections in most individuals, but in severely immunocompromised hosts it may cause the fatal demyelinating brain disease progressive multifocal leukoencephalopathy. In cell culture JCV multiplies inefficiently and exhibits a narrow host range. This restricted behavior occurs, in part, at the level of DNA replication, which is regulated by JCV's multifunctional large tumor protein (TAg). To prepare purified JCV TAg (JCT) for biochemical analyses, the recombinant baculovirus B-JCT was generated by cotransfection of insect cells with wild-type baculovirus and the vector pVL-JCT(Int-) containing the JCT-coding sequence downstream of the efficient polyhedrin promoter. JCT expressed in infected cells was immunoaffinity purified using the anti-JCT monoclonal antibody PAb 2000. Characterization of the viral oncoprotein indicated that it exists in solution as a mixture of monomeric and oligomeric species. With the addition of ATP, the population of monomers decreased and that of hexamers and double hexamers increased. A DNA mobility shift assay indicated that origin binding occurred primarily with the double-hexamer form. A comparison of the specific DNA-binding activities of JCT and SV40 TAg (SVT) revealed that JCT generally exhibited greater affinity for binding site II relative to binding site I (B.S.I) of both viral origin regions, whereas SVT preferentially bound B.S.I. Furthermore, JCT bound nonviral DNA more efficiently than did SVT. These functional differences between the two TAgs may contribute to the reduced DNA replication potential of JCV in vitro, and to the virus' ability to establish persistent infections in vivo.

Original languageEnglish (US)
Pages (from-to)81-93
Number of pages13
JournalVirology
Volume218
Issue number1
DOIs
StatePublished - Apr 1 1996

Fingerprint

JC Virus
Replication Origin
Viral Tumor Antigens
Insects
Binding Sites
Baculoviridae
DNA Replication
DNA
Progressive Multifocal Leukoencephalopathy
Asymptomatic Infections
Aptitude
Host Specificity
Oncogene Proteins
Immunocompromised Host
Brain Diseases
Demyelinating Diseases
Electrophoretic Mobility Shift Assay
Cell Culture Techniques
Adenosine Triphosphate
Monoclonal Antibodies

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

Bollag, Brigitte ; Mackeen, Patricia C. ; Frisque, Richard John. / Purified JC virus T antigen derived from insect cells preferentially interacts with binding site II of the viral core origin under replication conditions. In: Virology. 1996 ; Vol. 218, No. 1. pp. 81-93.
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Purified JC virus T antigen derived from insect cells preferentially interacts with binding site II of the viral core origin under replication conditions. / Bollag, Brigitte; Mackeen, Patricia C.; Frisque, Richard John.

In: Virology, Vol. 218, No. 1, 01.04.1996, p. 81-93.

Research output: Contribution to journalArticle

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