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Current and Past Grants

The PML Consortium has sponsored four rounds of research grants, which include a total of 17 grants. The following grantees have provided brief summaries of their work which is being funded by the PML Consortium.

 

Role of immature/transitional B cells and plasma cells/plasma blasts as reservoirs for JC virus

Principal Investigator: Raphael P Viscidi

Institution: Johns Hopkins University School of Medicine

 

Project Description: Whether B lymphocytes are a reservoir for JC virus (JCV) is controversial.  This might be because of the heterogeneity of the B cell lineage and the possibility that cells capable of supporting JCV replication constitute a small subset of B cells, of which only a small fraction are infected.

 

We hypothesize that immature or transitional B cells, as compared to naïve or memory B cells, are susceptible to JCV infection and support generation of viral genomes with variant regulatory regions.  Alternatively, activated B cells or plasmabalsts may be susceptible to JCV infection.  We further hypothesize that JCV is detectable in transitional B cells and/or circulating plasmablasts/plasma cells from MS patients on natalizumab.

 

To test these hypotheses, we propose to: (i) recover pure populations of immature/transitional B cells from donor blood by flow cytometry cell sorting or Miltenyi microbead separation technology and determine the ability of these cells to support JCV gene expression following transfection with re-ligated JCV genomic DNA or regulatory region bidirectional reporter plasmid DNA and to support infection with viable JC virus; (ii) activate memory B cells in vitro from healthy donors and determine the ability of these cells to support JCV gene expression following transfection with re-ligated JCV genomic DNA or regulatory region bidirectional reporter plasmid DNA and to support infection with viable JC virus; and (iii) Using samples from MS  patients treated with natalizumab and JCV seropositive MS patients never treated with natalizumab, JCV viral load will be measured in enriched populations of immature/transitional B cells and plasmablasts/plasma cells, as well as the frequency of archetype and rearranged regulatory regions by cloning and sequencing of regulatory region PCR products.

 

Genomewide association study of anti-JC-virus antibodies in patients with multiple Sclerosis

Principal Investigator: Dorothea Buck

Co-Investigator: Bernhard Hemmer

Institution: Department of Neurology, Klinikum rechts der Isar, Technische Universität München

 

Project Description: Identification of patients at risk to develop PML is a major goal of MS research. Since a positive anti-JCV antibody titer indicates infection with JC-virus and increases the risk to develop PML, measurement of JC-virus antibodies in serum and plasma of MS patients is a promising tool for further risk stratification.

 

Besides age and gender no other host-factors have so far been identified, that predispose to antibody development as a surrogate parameter of JC-virus infection. Interestingly prior immunosuppressant use, a risk factor for PML development, seems not to be a risk factor for anti-JC-virus antibody development. Therefore, genetic background may be pivotal for infection with the JC-virus. Since latent JC-virus infection seems to be a prerequisite for PML development, understanding individual risk factors will help further elucidate PML pathogenesis and risk stratification of PML development.

 

The aim of this study is to identify host factors that predispose to JC-virus infection and that influence antibody development. The JC-virus antibody status will serve as a surrogate parameter of latent JC-virus infection and will be tested for association with common genetic variants and HLA alleles in a genome wide association analysis (GWAS) on 1000 patients with Multiple Sclerosis (MS).

 

Mechanisms of PML Pathogenesis: Contributions of JC Virus Genomic Variation

Principal Investigator: Richard J. Frisque

Institution: The Pennsylvania State University

Co-Principal Investigator: Vivek R. Nerurkar

Institution: University of Hawaii

 

Project Description: Three types of sequence variation have been identified in the JCV genome, and each has been hypothesized to enhance viral virulence and influence PML development.  The best available evidence supports the clinical importance of two of these forms of variation: one that affects the viral promoter – enhancer signals and a second that alters the VP1 gene encoding the major capsid protein.  These two types of variation, unlike genotypic variation that has occurred during long-term association of JCV with its host, are thought to arise anew in each infected individual; the differences are observed in kidney vs. brain forms of the virus.  Questions regarding the mechanisms by which JCV sequence variations influence disease potential either remain unanswered or have given rise to speculations that are highly controversial.  Gaps in our understanding are due, in part, to the (i) limited and difficult culture systems available for propagating JCV variants; (ii) reliance by most labs upon the use of JCV-SV40 constructs and T antigen (Tag) – expressing cells and (iii) failure to evaluate JCV variation in the context of multiple sequence alterations.

 

In this proposal, the propagation of archetype JCV in biologically-relevant cells (e.g. Primary Human Fetal Glial (PHFG) cells and Human Brain Microvascular Endothelial (HBMVE) cells) and PML-type rearrangement of its non-coding control region will be investigated.  The biological relevance of VP1 variants detected in PML tissue will also be examined.  The project will also attempt to determine if early attempts to isolate JCV from PML patients are influenced by the presence of VP1 variants in the diseased brain tissues.

 

Modulation of JC virus (JCV) DNA replication by Nuclear Factor I (NFI/CTF), c-Jun and c-Fos, via interactions with viral T antigen (TAg) and cellular DNA polymerase-α primase (Pol-primase)

Principal Investigator: William R. Folk

Institution: University of Missouri

Co-Principal Investigator: Heinz Peter Nasheuer

Institution: National University of Ireland, Galway

 

Project Description: Despite considerable recent progress in our understanding of PML, we lack understanding of why and how JCV DNA replication is activated from its low level persistent state. Concomitantly, we lack efficient and selective means to block JCV replication when activated from its latent state.
In this proposal, we hypothesized that JCV DNA replication is directly modulated by distinct NFI/CTF isoforms and by AP1 (c-Jun and c-Fos), interacting with the viral TAg (helicase domain) and the cellular Pol-primase subunits.  Defining these interactions and their effects upon viral DNA replication may yield targets for blocking JCV replication and progressive multifocal leukoencephalopathy (PML).

 

To test this hypothesis, we propose to: (i) express and purify proteins required for JCV DNA replication by the in vitro monopolymerase system: JCV TAg, human DNA polymerase-α primase (Pol-primase), Topoisomerase I (TopoI), Replication Protein A (RPA), Nuclear Factor I (NFI) isoforms and AP1 (c-Jun and c-Fos); (ii) characterize the modulation of in vitro JCV DNA replication by NFI isoforms and c-Jun and c-Fos with the monopolymerase system (iii) characterize the modulation in vivo of JCV replication in haematopoietic progenitor KG1 cells and IMR-32 neuroblastoma cells, and (iv) identify the interaction domains between NFI isoforms, c-Jun, c-Fos, JCV TAg and Pol-primase subunits.

 

Adaptive Mechanism of DNA Replication-driven Recombination Alters JCV Control Region Sequences

Principal Investigator: Edward M. Johnson

Institution: Eastern Virginia Medical School

 

Project Description: By what mechanism does the JCV DNA control region become rearranged so that it is able to drive infection of oligodendroglial cells of the CNS? Our hypothesis is that this neurovirulent transformation is due to blocked DNA replication at preferred sequences leading to double-strand breaks followed by homologous recombination within the JCV non-coding control region (NCCR). This recombination facilitates the JCV ability to adapt its replication to different cell types. We further hypothesize that early NCCR sequence changes occur that may predispose a subset of individuals to ultimate PML.

 

To pursue this hypothesis we shall identify the NCCR sequences at which initial alterations conducive to recombination occur and the cell types most prone to such changes. We shall then, using recently developed methods such as DNA replication analysis followed by double-ChIP, identify proteins that are bound to these sites and are essential for NCCR recombination. This will allow the identification of specific proteins as potential biomarkers and pharmacological targets at the sites of their activities.