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The replication of human polyomavirus JCV, which causes Progressive Multifocal Leukoencephalopathy, is initiated by the virally encoded T-antigen (T-ag). The structure of the JC virus T-ag origin-binding domain (OBD) was recently solved by X-ray crystallography. This structure revealed that the OBD contains a C-terminal pocket, and that residues from the multifunctional A1 and B2 motifs situated on a neighboring OBD molecule dock into the pocket. Related studies established that a mutation in a pocket residue (F258L) rendered JCV T-ag unable to support JCV DNA replication. To establish why this mutation inactivated JCV T-ag, we have solved the structure of the F258L JCV T-ag OBD mutant. Based on this structure, it is concluded that the structural consequences of the F258L mutation are limited to the pocket region. Further analyses, utilizing the available polyomavirus OBD structures, indicate that the F258 region is highly dynamic and that the relative positions of F258 are governed by DNA binding. The possible functional consequences of the DNA dependent rearrangements, including promotion of OBD cycling at the replication fork, are discussed. [ABSTRACT FROM AUTHOR]

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. [ABSTRACT FROM AUTHOR]

JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel “pocket”; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable. [ABSTRACT FROM AUTHOR]

Within immunocompromised populations, the JC polyomavirus is the cause of the often-fatal disease Progressive Multifocal Leukoencephalopathy (PML). JC virus encodes a protein, termed T-antigen (T-ag), which is essential for its replication and pathogenicity. Previous studies of JCV T-ag have, in general, used antibodies raised against SV40 T-ag. Unfortunately, SV40 T-ag is also detected in humans and therefore there have been concerns about cross-reactivity. To address this issue, we have isolated a monoclonal antibody that binds to the JCV, but not the SV40, T-ag origin-binding domain (OBD). Furthermore, the region on the surface of the JCV T-ag OBD that is recognized by the "anti-JCV OBD mAb" has been mapped. We also demonstrate that the "anti-JCV OBD mAb" will be a useful reagent for standard techniques (e.g., Westerns blots and ELISAs). Finally, we note that additional monoclonal Abs that are specific for the T-ags encoded by the other human polyomaviruses could be generated by adopting the approach described herein. [ABSTRACT FROM AUTHOR]

Progressive multifocal leukoencephalopathy (PML) is an often-fatal demyelinating disease of the central nervous system. PML results when oligodendrocytes within immunocompromised individuals are infected with the human JC virus (JCV). We have identified an oligodendrocyte precursor cell line, termed G144, that supports robust levels of JCV DNA replication, a central part of the JCV life cycle. In addition, we have determined that JC virus readily infects G144 cells. Furthermore, we have determined that JCV DNA replication in G144 cells is stimulated by myristoylated (i.e., constitutively active) Akt and reduced by the Akt-specific inhibitor MK2206. Thus, this oligodendrocyte-based model system will be useful for a number of purposes, such as studies of JCV infection, establishing key pathways needed for the regulation of JCV DNA replication, and identifying inhibitors of this process. [ABSTRACT FROM AUTHOR]