DNA replication control in eukaryotic cells and human viruses
In eukaryotes chromosomal DNA replication is highly coordinated and tightly controlled.
The replication proteins ORC (origin recognition complex), CDC6, CDT1, MCM2 to MCM7 (minichromosome maintenance), CDC45, DNA polymerase alpha-primase, and RPA (replication protein A, Fig 1) co-operate during the eukaryotic cell cycle and their activity is controlled by posttranslational modifications and protein-protein interactions.
In order to analyze these replication proteins and cellular signaling pathways, we use a variety of techniques such as: co-immunoprecipitation, a modified ELISA technique, fluorescence (cross) correlation spectroscopy (FC(C)S), and fluorescence recovery after photobleaching (FRAP).
In living cells we have expressed fusion proteins with the green fluorescent protein (GFP) and carried out FCS, FCCS and FRAP.
The human polyoma viruses BKV and JCV are small, non-enveloped, icosahedral viruses and their genomes are organized in circular double-stranded DNA.
BKV is often activated after kidney transplantations and in patients with bone marrow or hematopoietic stem cell transplantations causing Polyomavirus-Associated Nephropathy (PVAN, also called BKV-Associated Nephropathy (BKAN)) of kidneys, and hemorrhagic cystitis (BKV -Associated Hemorrhagic Cystitis, BKV-HC), respectively.
In contrast, the closely related JCV causes Progressive Multifocal Leukoencephalopathy (PML) a demyelating disease of brain in immunosuppressed humans such as AIDS patients (Fig 2A). The viral replication is usually restricted to glial cells in these patients (Fig 2B).
The risk of developing PML is about 1/500 for patients treated with “Tysabri” (a blockbuster drug to treat MS) but it rises to 1/85 in case of additional risk factors.
Therapies to inhibit BKV and JCV replication are still lacking. Therefore, to study the life cycle of these viruses we have established cell-free BKV and JCV DNA replication systems.