Creation of a mouse model system for JC virus ORF4 protein - Abstract The human neurotropic virus, JC virus (JCV), causes a devastating and fatal disease of the human brain known as progressive multifocal leukoencephalopathy (PML). JCV establishes an asymptomatic infection for more than 70% of the human population during childhood and resides primarily in the kidneys and B cells in a latent form. Every individual with immunocompromised conditions, including AIDS patients and those treated with immunosuppressive drugs, for example, Crohn's disease, multiple sclerosis (MS), psoriasis, and rheumatoid arthritis patients are at risk for reactivating JCV and developing PML. Reactivated virus carries more than 20% fatality rate, and even those who survive the disease are severely disabled due to brain damage caused by the viral infection and inflammation. Currently, there are no effective treatment options available for PML patients, nor are there animal models that manifest the infection and symptoms of PML. The only treatment option is restoring the immune system to clear the infection. Still, even this often leads to increased brain damage, resulting from a condition known as immune reconstruction inflammation syndrome (IRIS). Thus, to address the unmet medical need for the treatment of JCV infection and to improve the management of PML patients, there is an urgent need to understand the disease progression at the molecular level to develop effective treatment options. Such options will only be possible by understanding the molecular mechanisms governing the JCV biology, which is mediated by its critical regulatory proteins. JCV encodes a limited number of such proteins encoded by the alternative splicing of the viral early and late transcripts. We have recently discovered a novel regulatory protein of JCV, ORF4 protein, generated by the viral late coding region, and its expression was validated during the viral infection cycle by proteomics studies. ORF4 is a small protein (173-aa long) found to be the only JCV protein specifically targeting the promyelocytic leukemia nuclear bodies (PML-NBs) and induce their reorganization in the nucleus. PML-NBs are known to play critical roles in intrinsic and innate immune responses against various viral infections, suggesting that ORF4 protein, by altering their reorganization, inhibits the function of PML-NBs and thus significantly contributes to the progression of the JCV life cycle. Our novel discoveries further revealed that ORF4 protein differentially regulates the reorganization pattern of the four permanent members of the PML-NBs. Three members, including PML protein, Daxx, and ATRX, remain together in the same complex after reorganization, while the fourth member, Sp100, behaves differently. Collectively, all these novel findings provide us with a strong rationale to investigate further the functional consequences of this targeting in vitro and in vivo model systems and allow us to formulate our central hypothesis, which is that JCV ORF4 protein inactivates the antiviral response function of the PML-NBs by altering their distribution pattern and thus significantly contributes to the progression of the viral infection cycle . To further examine our hypothesis, we propose two Aims: (i) Investigate the molecular mechanisms by which the ORF4 protein induces reorganization patterns of PML-NBs, employing in vitro tissue culture model systems, and (ii) further evaluate the functional roles of ORF4 protein by creating an innovative mouse model system.
