WO2014008382A1

WO2014008382A1 - Materials and methods for jc polyomavirus propagation and detection

Info

Publication number: WO2014008382A1
Application number: PCT/US2013/049299
Authority: WO
Inventors: Yi Zhou; Cornelis RIJNBRAND; Randal Byrn; James Daniel Frantz
Original assignee: Vertex Pharmaceuticals Incorporated
Priority date: 2012-07-05
Filing date: 2013-07-03
Publication date: 2014-01-09

Abstract

The invention relates to a method of producing John Cunningham (JC) polyomavirus. The method comprises infecting a population of neural stem cells with JC polyomavirus; and culturing the infected neural stem cells under conditions under which JC polyomavirus is produced. The invention also includes methods of assaying an agent for activity against JC polyomavirus and a JC pseudovirus comprising a JC polyomavirus genome that does not encode a functional T antigen, e.g., a polynucleotide sequence encoding JC polyomavirus T antigen is removed and optionally replaced with a polynucleotide sequence encoding a reporter protein. Also provided is a kit comprising a population of cells, optionally infected with JC pseudovirus or JC polyomavirus, and instructions for using the population of cells to assay an agent for activity against JC polyomavirus.

Description

MATERIALS AND METHODS FOR JC POLYOMA VIRUS PROPAGATION

AND DETECTION

TECHNICAL FIELD OF THE INVENTION

[0001] The invention relates to materials and methods for producing and detecting John Cunningham (JC) polyomavirus.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No.

61/668,301, filed July 5, 2013, the disclosure of which is hereby incorporated by reference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0003] Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: ASCII (text) file named "46418A_SeqListing.txt," 24,184 bytes created July 2, 2013.

BACKGROUND OF THE INVENTION

[0004] John Cunningham (JC) polyomavirus is nonenveloped, icosahedral polyomavirus comprising a small, double- stranded, circular DNA genome of approximately 5 kb. The virus is genetically similar to BK virus and SV40 virus. The early region of the genome encodes two viral proteins, large T antigen and small t antigen, which contribute to viral DNA replication and regulate host cell cycle. Alternative splicing of JCV early mRNA also encodes T135', T136' and T149" proteins, the functions of which have not been fully identified. The late region of the genome encodes capsid proteins VP1, VP2 and VP3, as well as a regulatory protein, agnoprotein. Coelho et al., Virology Journal, 7:42 (2010); Safak et al., /. Virol., 75(3), 1476-1486 (2001). Multiple genotypes and subtypes of JC

polyomavirus have been identified in different geographic locations. The length and sequence of the coding regions of different genotypes are highly conserved; a great deal of the variability between JC polyomavirus strains lies in non-coding regulatory regions of the genome. Jobes et al., /. Gen. Virol., 79, 2491-2498 (1998). The polynucleotide sequence of the complete genome of at least 22 different JC polyomavirus strains is available on

Genbank. Jobes et al., supra.

[0005] JC polyomavirus is believed to infect almost 70-90% of the population. Shackelton et al, / Virol, 80(20), 9928-9933 (2006). Upon infection, the virus resides in tonsillar tissue, gastrointestinal tissue, bladder, and renal tissue; approximately 20% of adults excrete JC viral particles in their urine. Id. The virus also crosses the blood-brain barrier and has been detected in brain tissue. While JC polyomavirus infection is largely benign and asymptomatic, the virus has been linked to debilitating diseases in immunocompromised and immune-suppressed patients and patients taking certain medications. For example, JC polyomavirus has been detected in colorectal cancers and is believed to contribute to tumorigenesis. Coelho, supra. JC polyomavirus also is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system causing paralysis, vision loss, cognitive deterioration, and eventually death. There is currently no marketed treatment for PML. Mefloquine, an antimalarial agent, was reported to inhibit the viral infection rate of three JC polyomavirus isolates in vitro, but failed to demonstrate a benefit in HIV/ AIDS patients with active PML. Friedman, Neurology Today, 77(8), 8 (2011).

[0006] Research to identify antiviral agents effective against JC polyomavirus is ongoing, and there exists a need in the art for alternative tools for propagating and detection JC polyomavirus, as well as identifying antiviral agents effective against JC polyomavirus infection.

SUMMARY OF THE INVENTION

[0007] The invention provides materials, methods, and systems for propagating and detecting John Cunningham (JC) polyomavirus. For example, the invention provides a method of producing JC polyomavirus, the method comprising infecting a population of neural stem cells with JC polyomavirus; culturing the infected neural stem cells under conditions under which JC polyomavirus is produced in the neural stem cells; and, optionally, harvesting the JC polyomavirus from the neural stem cells or from culture medium.

[0008] The invention further provides a method of assaying an agent for activity against John Cunningham (JC) polyomavirus. The method comprises culturing a population of neural stem cells infected with JC polyomavirus under conditions permissive of JC polyomavirus replication and in the presence of a candidate agent, and measuring virus replication by the neural stem cells. An increase or decrease in virus replication following exposure to the candidate agent compared to virus replication in the absence of the candidate agent indicates that the candidate agent is a modulator of JC polyomavirus replication. In one aspect, the method further comprises assessing activity of the candidate agent against JC polyomavirus by comparing virus replication in the presence of the candidate agent versus the absence of the candidate agent.

[0009] A JC pseudovirus also is provided. In one aspect, the JC pseudovirus comprises a JC polyomavirus genome that does not encode a functional T antigen. In one aspect, the polynucleotide sequence encoding JC polyomavirus T antigen in the JC polyomavirus genome is removed, and optionally replaced with a polynucleotide sequence encoding a reporter protein. The invention also includes a method of producing a JC pseudovirus. The method comprises transfecting a cell producing polyomavirus T antigen with a JC

polyomavirus genome lacking a polynucleotide sequence encoding JC polyomavirus T antigen. The transfected cell is cultured and JC pseudovirus is harvested. Optionally, the JC polyomavirus genome is operably linked to a polynucleotide sequence encoding a reporter protein.

[0010] Additionally, the invention provides a method of assaying an agent for activity against JC polyomavirus. The method comprises culturing a cell producing polyomavirus T antigen and infected with a JC pseudovirus in the presence of a candidate agent, and measuring JC pseudovirus DNA replication. An absence or reduction of JC pseudovirus DNA replication indicates that the candidate agent is a JC polyomavirus inhibitor. In various aspects, the JC pseudovirus encodes a reporter protein, and reporter activity is quantified to measure DNA replication.

[0011] A kit comprising the virus, pseudovirus, or cells (or any combination of the foregoing) described herein also is included in the invention. In various embodiments, the kit comprises (a) a population of cells producing polyomavirus T antigen, optionally infected with JC pseudovirus, and (b) instructions for using the population of cells to assay an agent for activity against JC polyomavirus. Alternatively, the kit comprises (a) a population of neural stem cells, optionally infected with JC polyomavirus, and (b) instructions for using the population of cells to assay an agent for activity against JC polyomavirus. [0012] Aspects of the invention are defined or summarized in the following numbered paragraphs:

[0013] 1. A method of producing John Cunningham (JC) polyomavirus, the method comprising (a) infecting a population of neural stem cells with JC polyomavirus; and (b) culturing the infected neural stem cells under conditions under which JC polyomavirus is produced.

[0014] 2. The method of paragraph 1, wherein the population of neural stem cells is an isolated population of neural stem cells.

[0015] 3. The method of paragraph 1, further comprising (c) harvesting the JC polyomavirus from the neural stem cells or from culture medium.

[0016] 4. A method of assaying an agent for activity against John Cunningham (JC) polyomavirus, the method comprising (al) culturing a population of neural stem cells infected with JC polyomavirus under conditions permissive of JC polyomavirus replication and in the presence of a candidate agent, and (bl) measuring virus replication by the neural stem cells, wherein an increase or decrease in virus replication following exposure to the candidate agent compared to virus replication in the absence of the candidate agent indicates that the candidate agent is a modulator of JC polyomavirus replication.

[0017] 5. The method of paragraph 6, wherein step (al) comprises culturing multiple populations of neural stem cells in the presence of different candidate agents.

[0018] 6. The method of paragraph 4 or paragraph 5, wherein the method comprises (a2) culturing a population of neural stem cells infected with JC polyomavirus under conditions permissive of JC polyomavirus replication in the absence of the candidate agent, (b2) measuring virus replication by the neural stem cell cultured in the absence of the candidate agent.

[0019] 7. The method of any one of paragraphs 4-6, wherein the method further comprises (c) assessing activity of the candidate agent against JC polyomavirus by comparing virus replication in the presence of the candidate agent versus the absence of the candidate agent.

[0020] 8. The method of any one of paragraphs 4-7, wherein the population(s) of neural stem cells are cultured for 24 hours to one month prior to measuring virus replication by the neural stem cells. [0021] 9. The method of any one of paragraphs 4-8, wherein virus replication is measured using a bDNA assay.

[0022] 10. The method of any one of paragraphs 1-9, wherein the neural stem cell is a glioma neural stem cell.

[0023] 11. The method of any one of paragraphs 1-9, wherein the neural stem cell is a neural progenitor stem cell.

[0024] 12. The method of any one of paragraphs 1-11, wherein the neural stem cell is an adult neural stem cell.

[0025] 13. The method of any one of paragraphs 1-11, wherein the neural stem cell is a fetal neural stem cell.

[0026] 14. The method of any one of paragraphs 1-9, wherein the neural stem cell is G144 (ATCC Deposit No. PTA-8895).

[0027] 15. A method of producing John Cunningham (JC) pseudovirus, the method comprising (a) transfecting a population of cells producing polyomavirus T antigen with a JC polyomavirus genome that does not encode a functional JC polyomavirus T antigen, (b) culturing the transfected cells, and (c) harvesting JC pseudovirus.

[0028] 16. The method of paragraph 15, wherein the cell producing SV40 T antigen is a Cos-7 cell, 293T cell, or VLC cell.

[0029] 17. The method of paragraph 15 or paragraph 16, wherein the JC polyomavirus genome is operably linked to a polynucleotide sequence encoding a reporter protein.

[0030] 18. The method of paragraph 17, wherein the reporter protein is luciferase.

[0031] 19. A method of assaying an agent for activity against John Cunningham (JC) polyomavirus, the method comprising (a) culturing a population of cells producing polyomavirus T antigen and infected with a JC pseudovirus in the presence of a candidate agent, and (b) measuring JC pseudovirus DNA replication in the population of cells, wherein an absence or reduction of JC pseudovirus DNA replication indicates that the candidate agent is a JC polyomavirus inhibitor.

[0032] 20. The method of paragraph 19, wherein the cells producing polyomavirus T antigen are human brain glioblastoma or astrocytoma. [0033] 21. The method of paragraph 20, wherein the cells producing polyomavirus T antigen are U87-MG (ATCC No. HTB-14™) or U138-MG (ATCC No. HTB-16™).

[0034] 22. The method of any one of paragraphs 19-21, wherein JC pseudovirus DNA replication is measured using a bDNA assay.

[0035] 23. The method of any one of paragraphs 19-22, wherein the JC pseudovirus comprises a polynucleotide sequence encoding a reporter protein that is produced in the cell, and JC pseudovirus DNA replication is measured by detecting reporter protein produced by the cells.

[0036] 24. The method of any one of paragraphs 15-23, wherein the polyomavirus T antigen is JC polyomavirus T antigen, BK polyomavirus T antigen, KI polyomavirus T antigen, WU polyomavirus T antigen, Merkel cell polyomavirus T antigen, Human polyomavirus 6 T antigen, Human polyomavirus 7 T antigen, Trichodysplasia spinulosa associated-polyomavirus T antigen, or human polyomavirus 9 T antigen.

[0037] 25. A JC pseudovirus comprising a JC polyomavirus genome wherein a polynucleotide sequence encoding JC polyomavirus T antigen is replaced with a

polynucleotide sequence encoding a reporter protein.

[0038] 26. A kit comprising (a) a population of cells producing polyomavirus T antigen and infected with John Cunningham (JC) pseudovirus and (b) instructions for using the population of cells to assay an agent for activity against JC polyomavirus.

[0039] 27. The kit of paragraph 26, wherein the polyomavirus T antigen is JC

polyomavirus T antigen, BK polyomavirus T antigen, KI polyomavirus T antigen, WU polyomavirus T antigen, Merkel cell polyomavirus T antigen, Human polyomavirus 6 T antigen, Human polyomavirus 7 T antigen, Trichodysplasia spinulosa associated- polyomavirus T antigen, or human polyomavirus 9 T antigen.

[0040] 28. The kit of paragraph 27, wherein the polyomavirus T antigen is JC

polyomavirus T antigen.

[0041] 29. A kit comprising (a) a population of neural stem cells infected with John Cunningham (JC) polyomavirus and (b) instructions for using the population of cells to assay an agent for activity against JC polyomavirus.

[0042] 30. A kit comprising (a) a population of neural stem cells and (b) John

Cunningham (JC) pseudovirus. [0043] The foregoing summary is not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. In addition, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned herein. With respect to aspects of the invention described as a genus, all individual species are individually considered separate aspects of the invention. If aspects of the invention are described as "comprising" a feature, embodiments also are contemplated "consisting of or "consisting essentially of the feature. With respect to elements described as a selection of one or more (or at least one) within a set, it should be understood that all combinations within the set are contemplated. With respect to aspects of the invention described or claimed with "a" or "an," it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning. The term "or" should be understood to encompass items in the alternative or together, unless context unambiguously requires otherwise.

[0044] Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a patent office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention. Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figures 1A and IB are line graphs illustrating the results of cellular assays using G144 cells as host cells for JC polyomavirus. Time course is provided on the X-axis, and viral DNA (Figure 1A) and viral RNA (Figure IB) is denoted on the Y-axis. G144 cells cultured in growth medium in T25 flasks were infected with MAD4 virus at MOI=l TCID50 for 24 hours The infected cells were plated into six well plates in differentiation medium, and harvested every day after removal of supernatant. With respect to Figure 1A, both DNA and RNA were isolated with Qiagen kits and quantified using bDNA assays. With respect to Figure IB, supematants were harvested and replaced with fresh medium twice a week. The viral titers in the supematants were determined by infection of COS-7 cells and quantified by bDNA assay.

[0046] Figure 2 is a line graph correlating percent of viral inhibition (y-axis) and concentration of spiperone. G144 cells cultured in T25 flasks in growth medium were infected with MAD4 vims at MOI=l TCID50 for 24 hours. The infected cells were plated into a 96 well plate in differentiation medium and treated with test compound at different concentrations. The plates were harvested for RNA and DNA quantification by bDNA assay. EC50 was calculated by Softmax (Molecular Devices, LLC) program.

[0047] Figure 3 is an illustration of an exemplary method of JC polyomavims replication assay and compound screening.

[0048] Figure 4 is a schematic of the pMAD4-T2Fluc vector. The sequence of pMAD4- T2Fluc is provided as SEQ ID NO: 1.

[0049] Figures 5A and 5B are bar graphs illustrating viral DNA replication (Figure 5A) and luciferase activity (Figure 5B) of reporter pseudovims in COS-7 cells. Plasmid DNA was cut with SanDl, purified, ligated with T4 ligase. The DNA was transfected into COS-7 cells with uncut DNA and no DNA as control. The supematants from transfected COS-7 cells were harvested and used to infect new COS-7 cells. Twenty hours after infection, the cells were washed to remove inocula, and the culture continued for another 48 hours. One set of the cells was harvested to determine JC viral DNA by the bDNA assay (Figure 5A). One set of cells was harvested to determine firefly luciferase activity (Figure 5B).

DETAILED DESCRIPTION OF THE INVENTION

[0050] The invention is predicated, at least in part, on the discovery of neural stem cells that support production of infectious JC virions. Few host cells that efficiently support JC polyomavims production in culture have been identified. Attempts to cultivate JC

polyomavims in primary embryonic kidney, lung, intestine, liver, and amnion; primary human adult testes; human diploid cells; human heteroploid cells; primary African green monkey kidney cells; BSC-1, CV-1, and Vero cells; adult rhesus monkey glial cells; hamster fetal glial cells; adult mink glial cells; and mouse embryo cells have failed, as indicated by lack of cytopathic effect and hemoglutination. See, e.g., Padgett et al., Infection and

Immunity, 15(2), 656-662 (1977). A limited number of cell lines have been reported to support JC polyomavirus production if engineered to express non-JC viral proteins, such as the SV40 T antigen. Expression of non-JC polyomavirus proteins, however, is not preferred in some instances. Exogenous production of non-JC polyomavirus proteins can interfere with screening candidate antiviral agents that target JC polyomavirus-specific proteins, leading to false positive or false negative results. Further, production of exogenous viral proteins can be toxic to host cells, thereby reducing viral production. Primary human fetal glial cells support JC polyomavirus production without requiring exogenous viral protein; however, working with primary cultures can be challenging given the limited life span of primary cells and need for animal sacrifice to obtain fresh cultures. The invention addresses many of the

shortcomings of existing materials and methods for propagating and detecting JC

polyomavirus.

[0051] The invention provides a method of producing John Cunningham (JC)

polyomavirus. The method comprises infecting one or more (e.g., a population of) neural stem cells with JC polyomavirus and culturing the infected neural stem cell(s) under conditions under which JC polyomavirus is produced. In various embodiments, the method further comprises harvesting the JC polyomavirus from the neural stem cell(s) or from culture medium.

[0052] Neural stem cells are self-renewing, multipotent cells that differentiate into cell types of the central nervous system (e.g., neurons, astrocytes, and oligodendrocytes). The neural stem cells of the invention are not primary cells. Neural stem cells are identified by a number of biological markers, i.e., biological molecules whose presence or concentration are used to identify or categorize a cell type. Neural stem cell markers include, but are not limited to, Nestin and SOX2, as well as ABCG2, NeuroDl, ASCLl/Mashl, Noggin, BMI-1, Notch- 1, Brgl, Notch-2, CD15/Lewis X, Nucleostemin, CDCP1, PDGF R alpha, CXCR4, Prominin 2, FABP7/B-FABP, SLAIN 1, FABP8/M-FABP, SOX1, FGF R4, Frizzled-9, SOX9, GFAP, SOX11, Glutl, SOX21, HOXB1, SSEA-1, Musashi-1, TRAF-4, Musashi-2, and Vimentin. See, e.g., Jandial et al., Molecular Therapy, 16(3), 450-457 (2008). Methods of characterizing and isolating neural stem cells are well known in the art and include, for example, genomic analysis, immunocytochemistry, and flow cytometry. Exemplary methods are described in, e.g., Pollard et al., Cell Stem Cell, 4, 568-580 (2009); Pastrana et al., Proc Natl Acad Sci USA, 106, 6387-6392 (2009); and Yuan et al, PLoS One, 6(3), el7540 (2011); and U.S. Patent Nos. 7,981,935 (hereby incorporated by reference in their entirety and particularly with respect to descriptions of methods for characterizing and isolating neural stem cells).

[0053] In one aspect, the neural stem cell is a neural progenitor stem cell, i.e., a stem cell more differentiated than a neural stem cell but not fully differentiated into a target cell type. Markers associated with neural progenitor cells include, e.g., FABP7/B-FABP, S100B, GFAP, SOX2, doublecortin, nucleostemin, Nestin, Vimentin, and Pax6. Alternatively, the neural stem cell is a glioma neural stem cell. Common markers for glioma stem cells include, but are not limited to, CD133, CD15, and A2B5. Gilbert and Ross, "Glioma Stem Cells: Cell Culture, Markers and Targets for New Combination Therapies," Cancer Stem Cells Theories and Practice, Prof. Stanley Shostak (Ed.), ISBN: 978-953-307-225-8, InTech (2011)

(available from: http://www.intechopen.com/books/cancer-stem-cells-theories-and- practice/glioma-stem-cells-cell-culture-markers-and-targets-for-new-combination-therapies). In various aspects, the neural stem cell is an adult neural stem cell. In alternative aspects, the neural stem cell is a fetal neural stem cell. In various embodiments, the neural stem cell is a G144 cell (ATCC Deposit No. PTA-8894), as described in Pollard et al., supra.

[0054] The population of neural stem cells is optionally substantially free from other cell types typically found in vivo (e.g., an "isolated" population of neural stem cells). For example, the population of neural stem cells comprises, in various embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80% at least about 90%, or at least about 95% neural stem cells. Put another way, the population of neural stem cells optionally contains fewer than about 50%, or fewer than about 40%, or fewer than about 30% or fewer than about 20%, or fewer than about 10%, or fewer than about 5%, of other cell types, including lineage-committed cells derived from the neural stem cells.

[0055] The neural stem cell(s) are infected with JC polyomavirus and cultured under conditions under which JC polyomavirus is produced in the stem (or progenitor or

differentiated) cell. "Culturing" refers to any method of maintaining cell growth and/or viability such that viral replication occurs. Any physical format of cell culture is appropriate in the context of the invention. Cell cultures may be monolayer or in suspension. If desired, a "continuous flow culture" apparatus is used to provide a continuous flow of fresh medium. Cell culture medium is well known in the art, and generally refers to a solid, liquid, or gaseous mixture of components that support cell growth, e.g., nutrient rich agar, agarose, gelatin, or liquid preparations. Cell culture media generally contains amino acids, nucleotides, salts, vitamins, and/or carbohydrates, and, if appropriate, additional supplements such as serum, growth factors, antibiotics, and lipids. Examples of media include, but are not limited to, Minimum Essential Medium, Dulbecco's Modified Eagle's Medium, Dulbecco's MEM/F-1 2, RPMI 1640, NeuroCult® Proliferation Medium, StemPro® NSC SFM, and Neurobasal™ Medium and Neurobasal™-A Medium. In various aspects, the medium is supplemented with, e.g., growth factors such as epidermal growth factor (EGF) and/or fibroblast growth factor (FGF-2), to promote differentiation of the neural stem cell into target cell types of interest, and/or heparin. Methods of viral purification are known in the art and include, e.g., cell lysis, sedimentation, and centrifugation.

[0056] In addition, the invention includes methods for studying an agent's effect on viral replication. In this regard, a method of assaying an agent for activity against JC

polyomavirus is provided. The method comprises culturing a population of neural stem cells infected with JC polyomavirus in the presence of a candidate agent and under conditions permissive of JC polyomavirus replication. The method further comprises measuring virus replication. In various embodiments, the method comprises comparing virus replication in the presence of the candidate agent versus the absence of the candidate agent. Optionally, viral replication levels in the absence of the candidate agent are determined experimentally by, for example, culturing a population of neural stem cells infected with JC polyomavirus in the absence of the candidate agent and under conditions permissive of JC polyomavirus replication, and measuring virus replication by the neural stem cell cultured in the absence of the candidate agent. Alternatively, viral replication levels observed in response to the candidate agent are compared to previously calculated or reported virus replication levels in the absence of the candidate agent. An increase or decrease in virus replication in the presence of the candidate agent compared to virus replication in the absence of the candidate agent indicates that the candidate agent is a modulator of JC polyomavirus replication.

[0057] In one aspect, the inventive method is employed as a screening assay to identify antiviral agents. In this regard, the method comprises selecting an agent that mediates a decrease in viral replication. Such agents may be included in further screening assays to characterize antiviral activity, including in vivo models of JC polyomavirus infection.

Alternatively, an agent is selected which mediates an increase in viral replication. Such agents may be desirable for, e.g., supplementing culture media to promote robust viral propagation in vitro. [0058] The population of cells is cultured or incubated under conditions and for a time sufficient to complete at least one lifecycle of the virus prior to measuring viral replication (e.g., 24 hours to one month or more). Cell culture conditions are described herein. The population of cells is contacted with the candidate agent before the cells have been infected, during the exponential phase of viral spread through the cell culture, and/or at the time of infection.

[0059] Viral replication or yield is assessed using any of a number of methods. For example, the number of viral particles in a sample can be quantified by plaque-based assays, detection of viral proteins, single radial immunodiffusion, hemagglutination assays, immuno staining, transmission electron microscopy (TEM), flow cytometry, or enzyme- linked immunosorbent assay (ELISA). Nucleotide-based assays for detecting and

quantifying viral replication, include, but are not limited to, DNA hybridization-based methods, branched-DNA methods, and quantitative polymerase chain reactions (qPCR) based on, e.g., VP1 or T antigen genes. See, e.g., McQuaig et al., Appl. Environ. Microbiol., 75(11), 3379-3388 (2009); Randhawa, J Infect Dis., 192(3), 504-509 (2005); Mori et al, Ann Neurol., 29(4), 428-32 (1991).

[0060] Optionally, virus replication is measured using a branched DNA (bDNA) assay, which is premised on signal amplification to allow quantification of small amounts of nucleic acid. In an exemplary version of a bDNA assay, capture probes adhered to a solid support are bound by oligonucleotide capture extenders (CEs), which hybridize to both the capture probes and a target nucleic acid. Target nucleic acids bind the CEs, and are labeled with multiple target- specific label extenders (LEs). In one format, LEs hybridize to a branched DNA amplifier that, in turn, binds to a number of detectable label probes. Alternatively, LEs hybridize to intermediate pre-amplifier molecules, which are bound by multiple amplifiers, each of which are bound by multiple label probes. Examples of label probes include, but are not limited to, alkaline phosphatase-linked oligonucleotides. bDNA materials and use thereof to detect and quantify DNA and RNA are further described in, e.g., Tsongalis, Am J Clin Pathol, 126, 448-453 (2006); Collins et al., Nucleic Acids Research, 25(15), 2979-2984 (1997); Chernoff et al., J. Clin. Microbiol, 35(11), 2740-2744 (1997). bDNA-based assays include the QuantiGene® family of assays from Affymetrix (Santa Clara, CA) and

VERS ANT™ from Siemens (Tarrytown, NY).

[0061] The method described herein is suitable for assaying a wide range of candidate agents, including, but not limited to, polypeptides (e.g., antibodies), peptides, nucleic acids (e.g., antisense oligonucleotides), carbohydrates, lipids, synthetic or semi- synthetic chemicals, and purified natural products. Agents may be assayed alone or in mixtures with other agents. The candidate agent also may be part of a library of agents. There are a number of different libraries used for the identification of modulators of viral infection and replication, including, but not limited to, chemical libraries, natural product libraries, and combinatorial libraries comprising peptides and/or organic molecules. A chemical library, in some aspects, consists of structural analogs of known compounds or compounds that are identified as "hits" or "leads" via other screening methods. Natural product libraries are collections of substances isolated from or produced by microorganisms, animals, plants, or marine organisms. Combinatorial libraries are composed of large numbers of peptides or organic compounds, typically as a mixture. In various aspects, the method comprises culturing multiple populations of neural stem cells in the presence of different candidate agents (i.e., individual populations of neural stem cells are cultured in the presence of a different candidate agent), which allows parallel screening of several candidate agents. The invention embraces high throughput screening methods involving automated procedures that allow rapid processing of multiple (e.g., tens to hundreds of thousands of) candidate agents.

[0062] The candidate agent may target any viral component associated with viral infection or replication. For instance, the methods described herein are suitable for identifying agents that target the JC polyomavirus large T antigen to, e.g., inhibit the protein's DNA binding and helicase activities required for replication of the viral genome; the JC polyomavirus small T antigen; and/or agnoprotein. Alternatively, the agent may target cellular machinery that plays a role in viral replication. It will be appreciated that the methods described herein also may be used to assay an agent for the ability to promote (i.e., increase or enhance) viral replication.

[0063] The invention also includes a JC pseudovirus and cell system useful for, e.g., studying viral replication, such as replication in response to candidate agents. The JC pseudovirus comprises a JC polyomavirus genome wherein the polynucleotide sequence encoding JC polyomavirus large and/or small T antigen is removed or disrupted. The JC polyomavirus genome is well characterized, and an exemplary genome sequence is set forth in, e.g., Frisque et al., /. Virol., 51(2), 458-469 (1984), which identifies the position of the large T antigen coding region as nucleotides 2603-4426 and the small T antigen coding region as nucleotides 4495-5013. The genomes of other types and strains of JC polyomavirus are described in Agostini et al., /. Gen. Virol., 79, 801-805 (1998) (strains #124 (Type 1A; GenBank accession no. AF0155526) and #123 (Type IB; GenBank accession no.

AF015527)); Agostini et al., J. Gen. Virol, 79, 1143-1151 (1998) (strains Tokyo-1 (GenBank accession no. AF030085), #223- #230 (GenBank accession nos. AF015529-AF015536), and GS/B (GenBank accession no. M20322) (Type 2A-2C)); Agostini et al., Archives of Virology, 142, 637-655 (1997) (strains #308-311 (Type 3A and 3B)); Agostini et al., J. Clin.

Microbiology, 34, 159-164 (1996) (Type 4); and Ou et al., Journal of the Formosan Medical Association, 96, 511-516 (1997) (strain Tai-3 (Type 7)), as well as GenBank accession no. J02227 (JCVMad-1). The aforementioned references are hereby incorporated by reference in their entirety and particularly with respect to the disclosure regarding JC polyomavirus genome sequences; the sequences set forth in the aforementioned GenBank accession entries are hereby incorporated by reference. In various aspects, all or part of the T antigen coding sequence is replaced with a polynucleotide sequence encoding a reporter protein. Suitable reporter proteins include, for example, luciferase, secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), β-galactosidase, green fluorescent protein (GFP), and horseradish peroxidase. Alternatively or in addition, coding sequences of one or more of the JC polyomavirus structural genes are disrupted or removed. The pseudovirus is unable to produce infectious progeny in the absence a complementing cell line that is permissive of JC polyomavirus particle production and complements the function of the JC polyomavirus T antigen. Accordingly, use of the pseudovirus in screening assays that do not employ complementing cell lines avoids the risk of unwanted infection often associated with viral research.

[0064] A method of producing JC pseudovirus is provided. The method comprises transfecting a cell producing polyomavirus T antigen with a JC polyomavirus genome that does not encode functional JC polyomavirus T antigen. In one aspect, the genome lacks all or part of the polynucleotide sequence encoding JC polyomavirus T antigen, such as T antigen exon 2, such that a functional JC polyomavirus is not produced when the genome is transcribed and translated. Optionally, the JC polyomavirus genome is operably linked to a polynucleotide sequence encoding a reporter protein. In various embodiments,

polynucleotide sequence replaces all or part of the T antigen coding sequence, producing a modified JC polyomavirus genome of appropriate size for packaging into viral particles. The polyomavirus T antigen produced by the cell complements the deficiency of the JC polyomavirus T antigen, allowing the production of infectious JC pseudovirus particles. The method further comprises culturing the transfected cell under conditions that allow virus production, and harvesting JC pseudovirus. Optionally, the polyomavirus T antigen is JC polyomavirus T antigen, BK polyomavirus T antigen, KI polyomavirus T antigen, WU polyomavirus T antigen, Merkel cell polyomavirus T antigen, Human polyomavirus 6 T antigen, Human polyomavirus 7 T antigen, Trichodysplasia spinulosa associated- polyomavirus T antigen, or human polyomavirus 9 T antigen. In various embodiments, the cell is a COS-7 cell, SVG cell, 293T cell, or pOJ-19 cell.

[0065] The invention further includes a method of assaying an agent for activity against JC polyomavirus using the pseudovirus. The method comprises culturing a cell producing polyomavirus T antigen and infected with a JC pseudovirus in the presence of a candidate agent, and measuring JC pseudovirus replication. An absence or reduction of JC pseudovirus replication indicates that the candidate agent is a JC polyomavirus inhibitor. In various embodiments, the cell is not permissive for production of infectious JC virions. Exemplary cell lines include, for instance, human brain glioblastoma or astrocytoma, such as U87-MG (ATCC No. HTB-14™) or U138-MG (ATCC No. HTB-16™) engineered to produce polyomavirus T antigen. Optionally, the polyomavirus T antigen is JC polyomavirus T antigen (e.g., UniProtKB/Swiss-Prot no. P03072), BK polyomavirus T antigen (e.g.,

UniProtKB/Swiss-Prot no. P14999), KI polyomavirus T antigen (e.g., UniProtKB/Swiss-Prot no. A3R4N4), WU polyomavirus T antigen (e.g., UniProtKB/Swiss-Prot no. A5HBG1), Merkel cell polyomavirus T antigen (e.g., UniProtKB/Swiss-Prot no. D2X5N8), human polyomavirus 6 T antigen, human polyomavirus 7 T antigen, Trichodysplasia spinulosa associated-polyomavirus T antigen (e.g., UniProtKB/Swiss-Prot no. E2ESL8), or human polyomavirus 9 T antigen. See, e.g., Chuke et al., / Virol., 60(3), 960-71 (1986). In these circumstances, particle quantification is not possible, and JC pseudovirus DNA replication or JC viral protein production is measured. For example, JC pseudovirus DNA replication is measured using a bDNA assay. Alternatively, the JC pseudovirus comprises a polynucleotide sequence encoding a reporter protein, which is produced in the cell and measured as a surrogate for JC pseudovirus DNA replication. Methods of quantifying reporter protein activity are well known in the art.

[0066] Methods of constructing expression vectors, including expression vectors comprising all or part of a viral genome or coding sequences for viral proteins, and use thereof to produce exogenous proteins in host cells are well known to those skilled in the art. A viral or non- viral vector may be used to recombinantly produce, e.g., JC polyomavirus T antigen, BK polyomavirus T antigen, KI polyomavirus T antigen, WU polyomavirus T antigen, Merkel cell polyomavirus T antigen, Human polyomavirus 6 T antigen, Human polyomavirus 7 T antigen, Trichodysplasia spinulosa associated-polyomavirus T antigen, or human polyomavirus 9 T antigen in a host cell. Exemplary viral vectors include, but are not limited to, retroviral vectors, including lentivirus vectors; parvoviral vectors, such as adeno- associated viral (AAV) vectors; adenoviral vectors; adenoviral adeno-associated chimeric vectors; vaccinia viral vectors; and herpesviral vectors. Any of these expression vectors can be prepared using standard recombinant DNA techniques described in, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994).

[0067] The expression of, e.g., JC polyomavirus T antigen, BK polyomavirus T antigen, KI polyomavirus T antigen, WU polyomavirus T antigen, Merkel cell polyomavirus T antigen, Human polyomavirus 6 T antigen, Human polyomavirus 7 T antigen,

Trichodysplasia spinulosa associated-polyomavirus T antigen, or human polyomavirus 9 T antigen may be transient, induced or constitutive, or stable. Expression control sequences include promoters, enhancers, and operators, and are generally selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression. Exemplary expression control sequences include promoter/enhancer sequences, e.g., cytomegalovirus promoter/enhancer; Rous sarcoma virus promoter; SV40 promoter; and albumin promoter, the promoter being operatively linked to a polypeptide coding sequence (e.g., the polyomavirus T antigen coding sequence). In various aspects of the invention, the T antigen coding sequence is operably linked to an inducible promoter. Inducible promoter systems include, but are not limited to, the IL-8 promoter, the metallothionine inducible promoter system, the bacterial lacZYA expression system, the tetracycline expression system, and the T7 polymerase systems. Expression constructs may also optionally include a suitable polyadenylation sequence (e.g., the SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (i.e., 3') of the polypeptide coding sequence.

Expression constructs may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct.

[0068] The invention also includes kits comprising the virus, pseudovirus, and/or cells described herein. For example, the invention includes a kit comprising (a) a population of neural stem cells infected with JC polyomavirus and (b) instructions for using the population of cells to assay an agent for activity against JC polyomavirus. Additionally, a kit comprising (a) a population of cells producing JC polyomavirus T antigen and infected with JC pseudovirus and (b) instructions for using the population of cells to assay an agent for activity against JC polyomavirus also is provided. Optionally, the population of cells is not pre- infected with JC polyomavirus or a pseudovirus.

[0069] The invention also encompasses agents identified by the methods described herein as having activity against JC polyomavirus in pharmaceutical compositions. In this regard, the agent is formulated with a physiologically-acceptable (i.e., pharmacologically-acceptable) carrier, buffer, excipient, or diluent, as described further herein. Optionally, the agent is in the form of a physiologically acceptable salt, which is encompassed by the invention.

"Physiologically acceptable salts" means any salts that are pharmaceutically acceptable. Some examples of appropriate salts include acetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, and oxalate. If desired, the composition comprises one or more additional pharmaceutically-effective agents, such as one or more additional antiviral agents, antibiotics, anti-inflammatory agents (e.g., Non-Steroidal Anti-Inflammatory Drugs

(NSAIDs) or steroidal anti-inflammatory substances), and pain relievers.

[0070] Additionally, the invention is directed to a method of treating a JC polyomavirus infection or complication thereof. The method comprises administering to subject in need thereof the agent in an amount effective to treat JC polyomavirus infection. The dose of agent administered to the subject is, in some instances, about 0.1 mg/kg to about 100 mg/kg. The agent is administered to the subject in an amount and for a time to, e.g., reduce the viral load in the subject by at least 10% (for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% (i.e., virus is below the level of detection)). The course of viral infection can be monitored using any of the techniques described herein, such as, e.g., detecting antibodies against the virus (using, for example, an ELISA based assay), detecting viral antigens, or detecting polyomavirus DNA (using, e.g., PCR; exemplary kit provided by Quest Diagnostics ("JC Polyoma Virus DNA, Qualitative Real-Time PCR")). Suitable methods of administering a physiologically- acceptable composition, such as a pharmaceutical composition comprising a peptide described herein, are well known in the art. Depending on the circumstances, a

pharmaceutical composition is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation. In one aspect, a composition comprising an anti-JC polyomavirus agent is administered intravenously, intraarterially, or intraperitoneally to introduce the agent into circulation. In certain circumstances, it is desirable to deliver a pharmaceutical composition orally, topically, sublingually, vaginally, rectally; through injection by intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intranasal, urethral, or enteral means; by sustained release systems; or by implantation devices.

[0071] The invention is further illustrated and described with reference to the following Example.

EXAMPLE

[0072] This example describes materials and methods for propagating JC polyomavirus, studying JC polyomavirus replication and life cycle, and identifying or characterizing antiviral agents.

Materials and Methods

[0073] G144 cells, a neural stem cell line (University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK) (Pollard et al., 2009) was used to propagate virus. G144 cell expansion was carried out using serum-free media supplemented with N2, B27 (Invitrogen), EGF, and FGF-2 (PeproTech, Rocky Hill, NJ USA), as previously described (Pollard et al., 2009). To differentiate G144 cells, the cells were cultured in the differentiation medium without EGF and FGF-2 growth factors. COS-7 cells obtained from the ATCC (CRL-1651) were maintained in DMEM with 10% FBS, lx NEAA, and lx penicillin/streptomycin.

[0074] pMAD4-T2Fluc (illustrated in Figure 4) was constructed by modification of pBR322-MAD4 purchased from ATCC (ATCC No. 45027; Howley, et al., "Cloned human polyomavirus JC DNA can transform human amnion cells," /. Virol. 36: 878-882 (1980)) and fusion of firefly luciferase (Promega, Madison, WI) in-frame into large T Antigen 2^nd Exon. pBR322-MAD4 (ATCC No. 45027) contains the JC polyomaviral genome isolated from human brain with progressive multifocal leukoencephalopathy (PML), which was linearized with BamHl before cloning into the BamHl site of pBR322. The BamHl segment was removed from pBR322-MAD4 and inserted into the pUC19 SanDl site. Using PCR, the firefly luciferase gene was fused in-frame into the large T Antigen 2^nd Exon, and then cloned into the construct. [0075] SanDl digestion and ligation removed the bacterial vector from pMAD4-T2Fluc and generated the pseudovirus construct with the same size as the original viral genome. The digested DNA was purified with Qiagen gel extraction kit, ligated with T4 ligase, and then re-purified with Qiagen gel extraction kit to obtain ligated DNA. The ligated DNA was transfected into COS-7 cells with TransIT as described by the manufacturer (Minis Bio LLC, Madison, WI, USA). Twenty four hours after transfection, culture medium was replaced, and the cells were cultured for an additional 48 hours. The supernatants were harvested to test viral infectivity on new COS-7 cells.

[0076] MAD4 viruses purchased from ATCC (ATCC No. VR-1583; Major et al., "Owl monkey astrocytoma cells in culture spontaneously produce infectious JC virus which demonstrates altered biological properties," J. Virol. 61: 1435-1441 (1987); Frye et al., "Efficient production of JC virus in SVG cells and the use of purified viral antigens for analysis of specific humoral and cellular immune response," /. Virol. Methods 63: 81-92 (1997)) were used to infect COS-7 cells overnight. The infected cells were maintained in 2% FCS medium. One week after infection, the supernatants were harvested twice a week and kept at -80 °C. High titer supernatants (>lxl0⁵ TCJX>50/ml) were pooled and used as virus stock.

[0077] An end-point dilution assay was used to measure virus titer, TCJD50/ml. Since JC polyomavirus does not cause significant cytopathic effect in COS-7 cells, a bDNA assay was used to determine viral infection instead of cytopathic effect. Briefly, culture supernatants were diluted serially in complete DMEM and used to infect lxlO⁴ COS-7 cells/well in 96- well plates. Three days after infection, the cells were harvested for bDNA assay. Wells with bDNA counts above 1.5-fold of the average of uninfected controls were defined as positives and used for TCID50 calculation.

[0078] For an RNA bDNA assay, JC polyomavirus T antigen and VP1 specific RNA bDNA probes were synthesized. An RNA bDNA assay for purified RNA or whole cell lysate was performed as recommended by Quantigene vl.O kit (Affymatrix, Santa Clara, CA). For a DNA bDNA assay, JC polyomavirus VP1 specific DNA bDNA probes were synthesized and a bDNA assay was developed. Briefly, cultured cells were lysed by addition of 1/3 volume of lysis mixture from the Affymetrix Quantigene v2.0 kit. Twenty microliters of the completely mixed lysate were mixed with 20 20 mM EDTA, ΙΟμί 2.5 N NaOH and 60 μL dH₂0, and incubated at 52 °C for 30 minutes to denature DNA. Fifty microliters of 2.0 M HEPES was added, mixed, and then 20 μΐ of mixture was transferred into bDNA capture plates containing 80 μΐ of JC VP1 specific DNA bDNA probes (50 μΐ of lysis mixture, 28.7 μΐ of dH₂0, 1 μΐ of blocking solution, and 0.3 μΐ of probe stock). Purified DNA is applied to the assay when the DNA was suspended into lysis buffer (1:2 diluted lysis mixture).

[0079] The EC50 values of candidate antiviral agents were determined in a 96 hour assay using G144 cells. Briefly, 500,000 G144 cells/well were plated in a six-well plate pre-coated with poly-Ornithine (0.005%) and Laminie (10 μg/ml) in growth medium for three hours to overnight. The cells were infected with MAD4 virus at MOI = 1 TCID50 for 24 hours. The infected cells were re -plated into 96 well plates pre-coated with poly-Ornithine (0.005%) and Laminie (10 μg/ml) at 5,000 cells/well in 100 μΐ of differentiation medium. Compounds diluted into DMSO were used to treat the infected cells at final concentration of 0.5%

DMSO. After 72 hours of incubation, the viral RNA and DNA levels were determined. The EC50 values of the antiviral agents were calculated using a four-parameter curve fitting method in the Softmax Pro program (Molecular Devices Corporation, Sunnyvale, CA).

Results

[0080] The Neural Stem Cell Line, G144, Is Highly Permissive for JC Virus Replication. To determine whether G144 cells can support JC polyomavirus replication, total RNA and DNA were isolated at different time points following infection of G144 cells, and quantified by the bDNA assay. Large T-antigen RNA was detected as early as 48 hours post-infection, while the late gene VP1 transcript was only detectable at 72 hours post-infection (Figure 1A). Early gene expression plateaued around 72 hours post-infection, while late gene expression continued to increase exponentially. Viral DNA replication was detected at 48 hours postinfection and continued to increase exponentially (Figure 1A). Infectious virus was detected in the supernatants at day 7 post-infection and peaked between day 14 to day 21 postinfection (Figure IB), indicating that the virus was efficiently propagated in G144 cells. In addition, MAD4 viruses replicated in G 144 cells cultured in growth medium, but the level of RNA and DNA was much lower than that observed using differentiation medium. Different passages of G 144 cells also were tested for their ability to support JC virus replication, and no significant difference was observed between early and late passage cells.

[0081] JC Polyomavirus Inhibition Assay. JC polyomaviral inhibition assays were developed using MAD4 infected G144 cells. A 96 well format assay suitable for compound screening was validated with a Z' >0.6. To confirm that the assay can be used to identify anti-JC polyomavirus inhibitors, EC50 of a published JCV inhibitor, spiperone, was determined in the assay. Good dose response curves for both viral RNA and DNA were observed. A EC50 of about 8 μΜ was observed, which is similar to reported EC50 values for the compound (Goodwin, Atwood et al., J. Virol, 83(11):5630 (2009)). Meanwhile, cell viability also was tested, and the CC50 value in G144 cells was greater than 50 μΜ. The results are illustrated in Figure 2.

[0082] JC Polvomaviral Reporter Pseudo virus. Reporter pseudoviruses produced in COS- 7 cells were used to infect fresh COS-7 cells to determine whether infectious particles were produced. pMAD4-T2Fluc was cut with SanDl, purified, and ligated with T4 ligase to create a pseudovector genome. The DNA was transfected into COS-7 cells with uncut DNA (containing the vector insert) and no DNA as control. The supernatant obtained from cells transfected with pseudovector genome (SanDl -cut and ligated DNA) demonstrated a significantly higher level of luciferase, while the supernatant resulting from transfection using uncut DNA only demonstrated counts similar to the no DNA control (Figure 5A). To confirm that the pseudovirus DNA can replicate in second-round infection, viral DNA levels were also measured. DNA replication was detected in COS-7 cells after infection with the supernatant from cells transfected with pseudovirus genome (SanDl -cut and relegated DNA), while the supernatant from cells transfected with uncut DNA only mediated slightly higher DNA level than the negative control (Figure 5B).

[0083] The results described herein establish that a neural stem cell line, G144, supports viral RNA transcription, DNA replication, late gene expression, and production of infectious viruses. The full virus life cycle is easily studied in this infectious system. Virus replication in G144 cells is similar to the natural host cell for the virus, oligodendrocytes, and, therefore, JC polyomavirus replication in the system provides valuable insight into virus replication in vivo. Additionally, cell propagation is efficient and JC polyomavirus replication in G144 cells is robust, providing an excellent high throughput viral replication system and assay, which is useful for screening compound libraries for drug discovery.

[0084] The results described above also demonstrate that the reporter pseudovirus described herein propagates in host cells (e.g., COS-7 cells expressing T antigen), and reinfects host cells to produce high reporter activity. The pseudovirus system is a tool for predictably analyzing JC polyomavirus activity and identifying antiviral agents.

Claims

CLAIMS What is claimed is the following:

1. A method of producing John Cunningham (JC) polyomavirus, the method comprising (a) infecting a population of neural stem cells with JC polyomavirus; and (b) culturing the infected neural stem cells under conditions under which JC polyomavirus is produced.

2. The method of claim 1, wherein the population of neural stem cells is an isolated population of neural stem cells.

3. The method of claim 1, further comprising (c) harvesting the JC polyomavirus from the neural stem cells or from culture medium.

4. A method of assaying an agent for activity against John Cunningham (JC) polyomavirus, the method comprising

(al) culturing a population of neural stem cells infected with JC polyomavirus under conditions permissive of JC polyomavirus replication and in the presence of a candidate agent, and

(bl) measuring virus replication by the neural stem cells,

wherein an increase or decrease in virus replication following exposure to the candidate agent compared to virus replication in the absence of the candidate agent indicates that the candidate agent is a modulator of JC polyomavirus replication.

5. The method of claim 6, wherein step (al) comprises culturing multiple populations of neural stem cells in the presence of different candidate agents.

6. The method of claim 4 or claim 5, wherein the method comprises

(a2) culturing a population of neural stem cells infected with JC polyomavirus under conditions permissive of JC polyomavirus replication in the absence of the candidate agent,

(b2) measuring virus replication by the neural stem cell cultured in the absence of the candidate agent.

7. The method of any one of claims 4-6, wherein the method further comprises (c) assessing activity of the candidate agent against JC polyomavirus by comparing virus replication in the presence of the candidate agent versus the absence of the candidate agent.

8. The method of any one of claims 4-7, wherein the population(s) of neural stem cells are cultured for 24 hours to one month prior to measuring virus replication by the neural stem cells.

9. The method of any one of claims 4-8, wherein virus replication is measured using a bDNA assay.

10. The method of any one of claims 1-9, wherein the neural stem cell is a glioma neural stem cell.

11. The method of any one of claims 1-9, wherein the neural stem cell is a neural progenitor stem cell.

12. The method of any one of claims 1-11, wherein the neural stem cell is an adult neural stem cell.

13. The method of any one of claims 1-11, wherein the neural stem cell is a fetal neural stem cell.

14. The method of any one of claims 1-9, wherein the neural stem cell is G144 (ATCC Deposit No. PTA-8895).

15. A method of producing John Cunningham (JC) pseudo virus, the method comprising

(a) transfecting a population of cells producing polyomavirus T antigen with a JC polyomavirus genome that does not encode a functional JC polyomavirus T antigen,

(b) culturing the transfected cells, and

(c) harvesting JC pseudovirus.

16. The method of claim 15, wherein the cell producing SV40 T antigen is a COS- 7 cell, 293T cell, or VLC cell.

17. The method of claim 15 or claim 16, wherein the JC polyomavirus genome is operably linked to a polynucleotide sequence encoding a reporter protein.

18. The method of claim 17, wherein the reporter protein is luciferase.

19. A method of assaying an agent for activity against John Cunningham (JC) polyomavirus, the method comprising

(a) culturing a population of cells producing polyomavirus T antigen and infected with a JC pseudovirus in the presence of a candidate agent, and

(b) measuring JC pseudovirus DNA replication in the population of cells, wherein an absence or reduction of JC pseudovirus DNA replication indicates that the candidate agent is a JC polyomavirus inhibitor.

20. The method of claim 19, wherein the cells producing polyomavirus T antigen are human brain glioblastoma or astrocytoma.

21. The method of claim 20, wherein the cells producing polyomavirus T antigen are U87-MG (ATCC No. HTB-14™) or U138-MG (ATCC No. HTB-16™).

22. The method of any one of claims 19-21, wherein JC pseudovirus DNA replication is measured using a bDNA assay.

23. The method of any one of claims 19-22, wherein the JC pseudovirus comprises a polynucleotide sequence encoding a reporter protein that is produced in the cell, and JC pseudovirus DNA replication is measured by detecting reporter protein produced by the cells.

24. The method of any one of claims 15-23, wherein the polyomavirus T antigen is JC polyomavirus T antigen, BK polyomavirus T antigen, KI polyomavirus T antigen, WU polyomavirus T antigen, Merkel cell polyomavirus T antigen, Human polyomavirus 6 T antigen, Human polyomavirus 7 T antigen, Trichodysplasia spinulosa associated- polyomavirus T antigen, or human polyomavirus 9 T antigen.

25. A JC pseudovirus comprising a JC polyomavirus genome wherein a polynucleotide sequence encoding JC polyomavirus T antigen is replaced with a

polynucleotide sequence encoding a reporter protein.

26. A kit comprising (a) a population of cells producing polyomavirus T antigen and infected with John Cunningham (JC) pseudovirus and (b) instructions for using the population of cells to assay an agent for activity against JC polyomavirus.

27. The kit of claim 26, wherein the polyomavirus T antigen is JC polyomavirus T antigen, BK polyomavirus T antigen, KI polyomavirus T antigen, WU polyomavirus T antigen, Merkel cell polyomavirus T antigen, Human polyomavirus 6 T antigen, Human polyomavirus 7 T antigen, Trichodysplasia spinulosa associated-polyomavirus T antigen, or human polyomavirus 9 T antigen.

28. The kit of claim 27, wherein the polyomavirus T antigen is JC polyomavirus T antigen.

29. A kit comprising (a) a population of neural stem cells infected with John Cunningham (JC) polyomavirus and (b) instructions for using the population of cells to assay an agent for activity against JC polyomavirus.

30. A kit comprising (a) a population of neural stem cells and (b) John

Cunningham (JC) pseudovirus.