Generation and characterization of JCV permissive hybrid cell lines

Abstract

JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous system induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV particles have been detected primarily in oligodendrocytes and astrocytes of the brains of patients with PML and in the laboratory its propagation is limited to primary cultures of human fetal glial cells. In this short communication, the development of a new cell culture system is described through the fusion of primary human fetal astrocytes with the human glioblastoma cell line, U-87MG. The new hybrid cell line obtained from this fusion has the capacity to support efficiently expression of JCV and replication of viral DNA in vitro up to 16 passages. This cell line can serve as a reliable culture system to study the biology of JCV host-cell interaction, determine the mechanisms involved in cell type specific replication of JCV, and provide a convenient cell culture system for high throughput screening of anti-viral agents.

Figure 1. Morphological features of parental (U-87MG-HPRT-deficient and PHFA) and hybrid clones (HC-7 and HC-15) before and after the cell fusion process

Phase contrast images of hypoxanthine phosphoribosyl transferase (HPRT)-deficient U-87MG cell line (U-87MG-HPRT-deficient) and PHFA before PEG-mediated fusion (upper panels). The hypoxanthine phosphoribosyl transferase (HPRT)-deficient U-87MG cell line was created from the parental U-87MG cell line by negative selection with 8-azaguanine (Sigma, #A5284) as described previously (Amano et al., 1974). HPRT-deficient U-87MG cells were isolated by selection with 8-azaguanine (Sigma, #A5284) and used as a fusion partner with PHFA cultures which were prepared and maintained as described previously (Radhakrishnan et al., 2003). Polyethylene glycol (PEG) mediated fusion between U-87MG HPRT-deficient and PHFA cells was performed by combining equal numbers of U-87MG HPRT-deficient and PHFA cells in serum free medium containing 50% PEG 1500 (PEG 1500, Roche) (Beggs et al., 1988). PEG mediated fusion resulted in double nucleated cells 2h after fusion while at 24h after fusion, most of the cells showed a single nucleated phenotype. Fused cells were grown in HAT selection medium for 72h and clonal cell lines were isolated by limiting dilution. Isolated clones HC-1 through HC-32 were further analyzed. Phase contrast images of representative hybrid clones HC-7 and HC-15 at passage 6 show a phenotype distinct from their parental cells (bottom panels).

Figure 2. Marker protein expression and flow cytometric analyses of hybrid clones

(A) Western blot analysis of GFAP expression in parental and hybrid cells. Whole-cell extracts were prepared from U-87MG-HPRT-deficient, PHFA, HC-7, and HC-15 cells, resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Expression of the cellular protein, GFAP, was determined using an anti-GFAP antibody. Grb2 was probed as a loading control. (B) Flow cytometric analysis of the parental (U-87MG HPRT-deficient and PHFA) and hybrid clones (HC-7 and HC-15). Cells were fixed in 70% ethanol, stained with Guava Cell Cycle Reagent (Guava Technologies), and DNA content determined with the Guava Easycycle Mini machine according to the manufacturer's instructions (Guava Cell Cycle Reagent, Guava Technologies).

Figure 3. JCV replication and gene expression in hybrid clones (HC-7 and HC-15) and their parental cells

Southern blot analysis of viral DNA isolated from infected cells (U-87MG HPRT-deficient, PHFA, HC-7 and HC-15 cells) (upper panel). Cells were infected with Mad-1 strain of JC virus and maintained in DMEM supplemented with 2% FBS and antibiotics at 37°C until collected for protein extraction or DNA isolation. Transfection/infections were performed as described previously (Sariyer et al., 2006). Low molecular weight viral DNA was purified from cells infected with the Mad-1 strain of JC virus at the indicated time points post-infection using a QIAprep-spin miniprep kit as described previously (Ziegler et al., 2004 and Sariyer et al., 2008). Purified low molecular weight DNA was digested with DpnI/BamHI and resolved by 1% agarose gel electrophoresis. Replicated viral genome was analyzed by Southern blotting using the entire Mad1 genome radiolabled with [³²P]α-dCTP by Klenow reaction as probe as described previously (Sariyer et al., 2008). In parallel, Western blot analysis was performed on whole cell extracts prepared from U-87MG-HPRT-deficient, PHFA, HC-7 and HC-15 cells uninfected or infected with JC virus. Expression of JCV proteins was detected with antibodies recognizing large T-antigen (pAb416, Oncogene Research Products), and VP1 (pAb597, kindly provided by Walter Atwood). DNA or proteins isolated from uninfected cells served as negative controls (i.e., 0 days). Western blotting with anti-Grb2 antibody (Santa Cruz, C-19) demonstrates the equal loading of the samples (bottom panel).

Figure 4. Immunocytochemistry for viral proteins in hybrid clones and parental cells infected with JCV

U-87MG HPRT-deficient, PHFA and the hybrid clones HC-7 and HC-15 were infected with Mad-1 strain of JCV, seeded onto chamber slides, and fixed with ice-cold acetone 8 days after infection. Immunocytochemistry to detect viral proteins was performed as described previously (Safak et al., 2002: Sariyer et al., 2006) with anti-large T-antigen (pAb416) and anti-VP1 (pAb597) monoclonal antibodies and anti-agnoprotein polyclonal antibody (Del Valle et al, 2002) for 8 h. Cells were then washed with PBS, incubated with FITC-conjugated secondary antibodies for 2 h, then mounted with aqueous mounting medium and examined by indirect immunofluorescence microscopy. Results revealed expression of T-antigen, agnoprotein and VP1 in PHFA, HC-7, and HC-15 cells but not in U-87MG cells.

Figure 5. JCV replication is restricted at later passages of the hybrid clones

Different passage numbers of the hybrid clone HC-15 (P6, P11, P16 and P26) were infected with the Mad-1 strain of JCV and viral DNA was isolated by QIAprep-spin miniprep kit at 8 days post infection. A. Purified low molecular weight DNA was digested with DpnI/BamHI and analyzed by Southern blotting to detect replicated viral DNA. In lane 1, 2 ng of Mad-1 genome digested with BamH1 was loaded as a positive control. In lane 2, a DNA sample isolated from uninfected cells was processed as in lanes 3-6 and loaded as a negative control. B. Whole cell extracts were prepared and analyzed for VP1 expression by Western blotting using anti-VP1 antibody. Reprobing of the blot with an anti-Grb2 antibody demonstrated the equal loading of the samples. In lane 1 protein samples prepared from uninfected cells was loaded as a negative control. C. Hybrid clones transfected with JCV Mad-1 genome (10 μg/flask) using lypofectamine (GibcoBRL). Eight days posttransfection, viral DNA was isolated and analyzed by Southern blotting as described for Panel A. D. In parallel, whole cell protein extracts were prepared and analyzed by Western blotting as described for Panel B. Trxn: Transfection, Infx: Infection.