Interferon-α and -β Restrict Polyomavirus JC Replication in Primary Human Fetal Glial Cells: Implications for Progressive Multifocal Leukoencephalopathy Therapy

Juliene K. G. Co 1 2 Saguna Verma 1 2 Ulziijargal Gurjav 0 1 2 Laarni Sumibcay 1 2 Vivek R. Nerurkar () 0 1 2 0 Molecular Biosciences and Bioengineering Graduate Program, University of Hawai'i at Manoa , Honolulu, Hawai'i 1 Received 4 February 2007; accepted 28 March 2007; electronically published 20 July 2007. Potential conflicts of interest: none reported. Financial support: Collaborative Neurological Sciences Program (US Public Health Service grant S11 NS041833); Specialized Neurosciences Research Program (US Public Health Service grant U54 NS039406); National Institute of Neurological Disorders and Stroke; Research Centers in Minority Institutions Program (grant G12 RR003061); Centers of Biomedical Research Excellence (grant P20 RR018727); National Center for Research Resources, National Institutes of Health. Honolulu , HI 96813 2 Retrovirology Research Laboratory, Department of Tropical Medicine, Medical Microbiology and Pharmacology, Asia-Pacific Institute of Tropical Medicine and Infectious Diseases, John A. Burns School of Medicine One of the major limitations of highly active antiretroviral therapy is its inability to inhibit the replication of polyomavirus JC (JCV), the etiologic agent of progressive multifocal leukoencephalopathy (PML), an acquired immunodeficiency syndrome-defining illness. We previously demonstrated the induction of interferon (IFN)stimulated genes (ISGs) by JCV. In the present study, we characterize the specific viral events required to induce ISGs and the potential antiviral effects of type I IFN on JCV replication in human fetal glial cells in the presence and absence of type I IFNs. Productive JCV replication was essential for the induction of the antiviral host response. JCV replication at all steps was significantly inhibited in the presence of IFN, and neutralizing anti-IFN antibody rescued the inhibitory effect of IFN. These results support the use of IFN as an adjunct therapy for patients with PML. Because IFN cannot cross the blood-brain barrier to achieve its direct antiviral effect, intrathecal administration of IFN is warranted. - Because of the lack of a PML-specific therapy, patients with AIDS-associated PML die of the disease within 6 months after diagnosis [2, 4]. Although highly active antiretroviral therapy (HAART) has significantly reduced PML-associated mortality, its complications include the development of immune reconstitution inflammatory syndrome [5] and no clear survival benefit in patients with high baseline JCV loads in cerebrospinal fluid (CSF) [6, 7]. Type I interferons (IFN-a and -b), best known for their antiviral and immunomodulatory properties, have been administered during both the pre- and post-HAART eras to patients with PML [811]. However, the outcomes have been controversial [8, 9, 11]. At present, there are no in vitro studies evaluating the direct antiviral effect of IFN-a or -b on JCV in infected target cells, and such studies are warranted [12]. IFN-stimulated genes (ISGs) such as myxovirus (influenza virus) resistance 1 (Mx1), 2,5 -oligoadenylate synthase 1 40/46 kDa (OAS1), and radical S-adenosyl methionine domain containing 2 (RSAD2) are induced by either IFN, virus infection, or dsRNA, and efficient production of ISGs is an important component of the host antiviral defense response [13]. Using JCV-permissive primary human fetal glial (PHFG) cells, we previously demonstrated marked up-regulation of several ISGs [14]. The increase in ISG expression coincided with the sharp increase in JCV replication and paralleled the inoculum dose. We speculated that antiviral products, along with JCV early proteins, might modulate the severity of infection and limit the spread of JCV, thereby explaining the slow replication and subtle cytopathogenic effects in PHFG cells. Because IFN-a and -b are potent inducers of ISGs [13] and are common therapeutic options for viral diseases [1517], the present study was designed to analyze the specific viral events associated with the induction of ISGs and to characterize the effect of IFN-a and -b on JCV replication in PHFG cells. We demonstrate that ISG induction in JCV-infected PHFG cells is a virus-specific event that requires virus replication. Moreover, JCV replication was significantly restricted in the presence of IFN-a and -b, and this effect was reversed in the presence of neutralizing IFN-a antibody. MATERIALS AND METHODS PHFG cell culture and virus. PHFG cells were obtained as described elsewhere [18] from the Kapiolani Medical Center for Women and Children (KMCWC), after we received approval from the KMCWC Institutional Review Board and the University of Hawaii Committee on Human Studies. Fetal brain tissues were processed as described elsewhere [18]. Fifty hemagglutination units (HAU) of JCV(Mad1) was used for infection of PHFG cells throughout the study [14, 18, 19]. JCV infection of PHFG cells. PHFG cell cultures at 60% 70% confluency plated on 35-mm plates were infected for 2 h with JCV(Mad1), UV-inactivated JCV (UV-JCV), or heat- and UV-inactivated JCV (HA-UV-JCV) or were transfected with UV-inactivated non-JCV plasmid (pUC18) DNA (UV-DNA), washed 3 times with serum-free Dulbeccos modified Eagle medium, and thereafter maintained for 15 days in culture medium with 3% bovine calf serum. JCV was UV inactivated by exposing 50 HAU of JCV to UV using a UV-Stratalinker 2400 device (Stratagene) for 3 min. Heat inactivation was performed by heating UV-JCV at 75 C for 45 min. Pretreatment of PHFG cells with IFN-a and -b and with neutralizing IFN-a antibody. PHFG cells grown on 35-mm plates were treated with 100 U/mL of IFN-a (Sigma) or IFNb (PBL Laboratories) 24 h before JCV inoculation. In few experiments, cells were incubated with neutralizing antiIFN-a (1:2000 dilution; PBL Laboratories) for 1 h before treatment with IFN-a. After infection, cells were grown for 15 days in the continuous presence or absence of antiIFN-a, IFN-a, or IFN-b. The medium was changed every 4 days with fresh anti IFN-a, IFN-a, or IFN-b. Cell toxicity assay. PHFG cells were grown to 70% confluency in 96-well plates and treated with antiIFN-a and/or IFN-a or IFN-b as described above. Cell proliferation and cytotoxicity were assessed on days 0, 3, 5, 8, and 15 after infection in JCV-infected and -uninfected cells, using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega). The absorbance in each well was measured in accordance with the manufacturers protocol at 490 nm using a multiplate reader (Victor3; PerkinElmer). Nucleic acid extraction. Viral DNA and total RNA were extracted from mock- and JCV-infected PHFG cells on days 0 (2 h after infection), 3, 5, 8, and 15 after infection, using the QIAprep Spin Miniprep Kit and Qiagen RNeasy Mini Total RNA Isolation Kit (Qiagen), respectively, in accordance with the manufacturers protocol. Genomic DNA contamination of RNA samples was eliminated by digesting the RNA twice on the column with RNase-free DNase ( (...truncated)