MAP kinase activation increases BK polyomavirus replication and facilitates viral propagation in vitro

Journal of Virological Methods 170 (2010) 21–29

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MAP kinase activation increases BK polyomavirus replication and facilitates viral propagation in vitro Mark E. Seamone a , Wenjie Wang a , Philip Acott b , Paul L. Beck a , Lee Anne Tibbles a , Daniel A. Muruve a,∗ a b

Department of Medicine, University of Calgary, Calgary, AB, Canada Department of Pediatrics and Pharmacology, Dalhousie University, Halifax, NS, Canada

a b s t r a c t Article history: Received 3 February 2010 Received in revised form 27 June 2010 Accepted 23 August 2010 Available online 8 September 2010 Keywords: BK polyomavirus Mitogen-activated protein kinase Cell cycle Kidney transplantation

BK polyomavirus causes disease in immunosuppressed patients. BK virus replication was augmented in HEL-299 cells cultured in conditions that activated the MAP kinase, ERK1/2. To determine if MAP kinase activation increased BK virus replication, cells were treated with serum and phorbol 12-myristate 13-acetate (PMA). Serum and PMA stimulated large T-antigen expression and increased BK virus DNA replication. The effects of serum/PMA were directly related to MAP kinase signal activation since viral replication was reduced by the MEK1/2 inhibitor U0126. PMA also increased cyclin D1 expression and inhibition of cyclin D1/CDK4 complex and the cell cycle reduced BK virus infection. The PMA effect occurred independent of direct transcriptional activation of the viral NCCR. In HEL-299 cells, virus infection in high serum and PMA accelerated viral replication that resulted, within 7 days, in the production of high titer infectious BK virus. These results show that MAP kinase signal activation increases BK virus replication. © 2010 Elsevier B.V. All rights reserved.

1. Introduction BK virus is a member of the polyomaviradae family of small dsDNA viruses. The seroprevalence of BK virus infection is high and most individuals are exposed to the virus in childhood. BK virus establishes a latent infection in the kidney and urinary tract (Chesters et al., 1983; Knowles, 2006) but is frequently reactivated following renal transplantation and immunosuppressive therapy. BK virus is recognized as the cause of polyomavirus-associated nephropathy that occurs in approximately 10% of transplant recipients (Comoli et al., 2006; Hirsch et al., 2005). Polyomavirusassociated nephropathy is characterized by BK virus reactivation in the tubular epithelial cells of the transplanted kidney resulting in acute inflammation, interstitial fibrosis and accelerated allograft failure in up to 80% of affected patients (Hirsch et al., 2005). Despite the recent increased recognition of BK virus-associated diseases, relatively little is known regarding the biology of the virus and the factors that regulate viral replication. All members of polyomaviradae have a similar structural configuration consisting of three structural proteins (VP1-3) and a circular dsDNA genome of approximately 5000 bp (Hirsch et al., 2002). The

∗ Corresponding author at: Department of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, T2N 4N1 Canada. Tel.: +1 403 220 2418; fax: +1 403 210 3949. E-mail address: [email protected] (D.A. Muruve). 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.08.014

genome of BK virus is divided into early and late regions. Expression of early genes (large T- and small t-antigen) continues throughout infection and late gene expression (VP1-3) occurs subsequent to viral DNA replication. BK virus also contains a variable bidirectional promoter/enhancer region of about 500 base pairs known as the non-coding control region (NCCR) that regulates bidirectional viral gene transcription (Cubitt, 2006; Hirsch and Steiger, 2003). Although a number of studies show that cytokines and host transcription factors can activate the BK virus NCCR (Abend and Imperiale, 2008; Gorrill and Khalili, 2005; Jordan et al., 2009; Moens et al., 1994), polyomaviruses also require the host cell machinery to mediate their replication. Both Simian Virus 40 (SV40) and BK virus T-antigen interacts with the tumor suppressor protein p53 and retinoblastoma protein (pRb) family members. The interaction of T-antigen with the pRb family members results in an inability to sequester E2F transcription factors that results in G1/S-phase transition and activation of the cellular machinery required to drive virus replication (DeCaprio et al., 1988; Eash et al., 2006; Harris et al., 1996, 1998; Shivakumar and Das, 1996). Accordingly, BK virus infection increases BrdU incorporation and cellular DNA replication (Bernhoff et al., 2008, 2009). Despite this understanding of polyomavirus biology, the effect of potentially synergistic signaling and cell cycle pathways on BK virus replication has not been demonstrated. The mitogen-activated protein (MAP) kinases play significant roles in cellular biology and include the extracellular signal related kinases ERK-1 and ERK-2. These protein kinases are activated by a

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variety of stimuli such as changes in serum concentration, growth factors and pro-inflammatory cytokines (Gille et al., 1992; Lewis et al., 1998). Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) are potent activators of the ERK1/2 kinases through their ability to activate protein kinase C (PKC) (Gause et al., 1993). Signaling via ERK1/2 is an essential element that stimulates entry into the cell cycle, transition to S-phase and cellular proliferation (Liu et al., 2004; Mansour et al., 1994; Robinson et al., 1998). The activation of ERK1/2 signaling upregulates cyclin D1 which phosphorylates pRb promoting G1/S-phase transition via the release of E2F transcription factors (Lavoie et al., 1996). The relationship between MAP kinase activation, cyclin D1 upregulation and pRb suggests that ERK1/2 signaling may act in synergy with BK virus large T-antigen to promote G1/S-phase transition and enhance BK virus replication. This study demonstrates that MAP kinase signal activation has a significant positive influence on BK virus replication, shedding light on the cellular conditions that promote viral propagation. Furthermore, the activation of MAP kinase signaling can be exploited to increase BK virus replication in vitro and produce rapidly high titer, purified viral stocks. 2. Materials and methods 2.1. Cell lines and culture Cell lines used included HEL-299 and Vero (ATCC, Manassas, VA, USA). HEL-299 cells were cultured in minimal essential medium (MEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 1% non-essential amino acids, 10 mM sodium pyruvate, 10 mM sodium bicarbonate, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS). Medium for Vero cells consisted of Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 1% P/S. Cells were grown to 50% confluence at 37 ◦ C, CO2 5% and infected with BK virus. After 2.5 h at 37 ◦ C, the BK virus-containing medium was removed. Cells were washed with 1x PBS buffer solution and then cultured in virus-free medium for the remainder of the experimental time period. FBS and Phorbol 12-myristate 13-acetate (PMA, 10–80 nM) (Sigma, Oakville, ON, Canada) were added to cells following BK virus infection and maintained in the culture medium throughout the experimental time period. The MEK1/2 inhibitor U0126 (20 ␮M), the protein kinase C inhibitor Ro-318220 (1 ␮M) (EMD-Calbiochem, Brookfield, WI, USA) and the cyclin D1 inhibitor fascaplysin chloride hydrate (FCH, 0.175–0.35 ␮M) (Sigma, Oakville, ON, Canada) were added to the cells 30 min prior to BK virus infection unless otherwise specified.

299 cells except cells were cultured in the absence of PMA. Cells were incubated for 25 days. Media was changed and saved on a weekly basis. BK virus purification was based on the method described by Jiang et al. (2009). Cells and medium were collected and adjusted to pH 7.4 with HEPES pH 5.0 and centrifuged using a Beckman GH 3.8 rotor at 2800 × g for 15 min. The supernatant was saved. Cell pellets were then suspended in 5 ml of buffer consisting of 10 mM HEPES (pH 7.9) 1 mM CaCl2 1 mM MgCl2 and 5 mM KCl and subject to 6 rounds of freeze/thaw after which the total volume was adjusted to 20 ml. The pH of the cell lysates was then adjusted to 5.4 with HEPES pH 5.0 and subjected to digestion with 1 U/ml of neuraminidase type V (Sigma, Oakville, ON Canada) for 1 h at room temperature. The pH was then corrected to 7.4 and the cell lysate incubated for 5 min at 42◦ . The cell lysate was centrifuged at 2800 × g RPM for 10 min at 4 ◦ C and the supernatant saved. The pellet was resuspended in 2.5 ml of buffer containing 10 mM HEPES (pH 7.9), 1 mM CaCl2 , 1 mM MgCl2 , 5 mM KCl and 0.1% deoxycholate for 15 min followed by centrifugation at 3000 × g for 10 min at 4 ◦ C. The supernatant was then combined with the previously saved supernatants and layered over 10 ml of 20% sucrose in buffer containing 10 mM HEPES (pH 7.9), 1 mM CaCl2 , 1 mM MgCl2 , and 5 mM KCl. The BK virus was pelletted by centrifugation using a Beckman SW-28 rotor for 3 h at 120,000 × g. The pellet was re-suspended in 1.5 ml of buffer containing 10 mM HEPES (pH 7.9), 1 mM CaCl2 , 1 mM MgCl2 , and 5 mM KCl and centrifuged through a 1.2–1.4 g/cm3 CsCl gradient for 16 h at 110,000 × g using a Beckman SW-50Ti rotor. The resulting virus band was then extracted and dialyzed against buffer containing 10 mM HEPES (pH 7.9), 1 mM CaCl2 , 1 mM MgCl2 , and 5 mM KCl, divided into aliquots and stored at −80 ◦ C. 2.3. Real-time PCR To analyze BK virus DNA, infected cells were suspended in 1x PBS buffer, pelletted and subjected to three rounds of freeze–thaw to generate a crude viral lysate for use in real-time PCR. Primers and probes were designed using the Primer3 software (Applied Biosystems, Carlsbad, CA, USA) to amplify BK virus T-antigen as follows: forward primer 5 -CCCTAAAGACTTTCCCTCTGATCTAC-3 , reverse primer 5 -CAAAGCAGGCAAGGGTTCTATT-3 , and probe 6FAM-5 CAGTTTCTTAGTCAAGCTGTAT-3 MGBNFQ. Target gene reactions were performed in a total volume of 25 ␮l consisting of 1 ␮l of crude viral lysate, 12.5 ␮l of 2x Taqman real-time master mix (Applied Biosystems, Carlsbad, CA, USA), 900 nM of the forward and reverse primer and 200 nM probe. Reactions were preformed in MicroAmp optical 96-well reaction plates using an ABI Prism 7000 System (Applied Biosystems, Carlsbad, CA, USA).

2.2. BK virus propagation and purification 2.4. BK virus titration The BK virus-UT strain was obtained from ATCC. Clinical isolates of BK virus (LH and VJ (Acott et al., 2008)) were obtained from the urines of patients with BK polyomavirus infection in accordance with the guidelines set forth by the Health Research Ethics Committee at the IWK Health Centre, Halifax, NS. Canada. Isolates were propagated in HEL-299 cells and confirmed by sequencing. To propagate BK virus, HEL-299 cells were plated at 50% confluence on twenty 150 mm cell culture plates (falcon) in minimal essential medium (supplemented as described above) and incubated with BK virus (MOI:1) for 2.5 h. Virus was then removed from plates and cells were washed with 1x PBS buffer solution. Media was then added to a total volume of 25 ml containing PMA at a final concentration of 80 nM. Cells then incubated at 37 ◦ C for 6 days after which cells were harvested by scraping. A small sample of cells was analyzed by immunoblotting and real-time PCR for BK virus T-antigen and DNA, respectively, to confirm adequate viral replication. Vero cells were infected in the same manner as HEL-

The physical titer of BK virus was determined by DNA quantification. BK virus DNA was amplified by conventional PCR employing real-time PCR primers as described above. PCR-generated BK virus DNA was then purified and limiting dilutions of ranging from 10−4 to 10−10 ng/ml were used to generate a standard curve in real-time PCR reactions by plotting the CT value generated for each known DNA concentration using a −Log scale. The equation for the standard curve was then utilized to convert the CT value generated for a specific BK virus preparation into a value corresponding to the DNA concentration. The number of BK virus genomes/ml was then calculated by dividing the amount of BK virus DNA in each preparation by the amount of DNA present in a single BK virus genome (∼5.67 × 10−9 ng). Infectious titer of BK virus was determined using the fluorescent focus assay as described (Abend et al., 2007; Moriyama and Sorokin, 2009) with the following modifications: HEL-299 cells

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were plated over cover slips in 24-well plates (30,000 cells/well). Cells were infected with BK virus preparations in serial dilutions. After 2.5 h, virus was removed and the cells washed with 1x PBS. Media (EMEM, 0.5% FBS, 1% PS) was added and infected cells were incubated for 3 days at 37 ◦ C. Cells were fixed with a 1:1 solution of methanol/acetone, washed with PBS solution and blocked with 5% goat serum/3% bovine serum albumin (BSA) for 1 h at room temperature. Cells were incubated anti-SV40 T(large)antigen (mouse monoclonal PAb416, EMD-Calbiochem, Brookfield, WI, USA) diluted 1:500 in 5% goat serum/3% BSA/0.1% tween-20 in PBS overnight at 4 ◦ C. Cells were washed with 1x PBS solution and incubated with Alexa-488 labeled goat-anti-mouse secondary antibody diluted 1:500 in 5% goat serum/3% BSA/0.1% tween-20 in PBS for 1 h at room temperature. Cells were then washed and mounted onto slides using Prolong Gold containing DAPI. Slides were analyzed by fluorescent microscopy and T-antigen-positive fluorescent foci counted in 10 separate fields of view.

24-well plates using calcium phosphate precipitation. The pTKrenilla luciferase (pTK-Ren) reporter plasmid carrying the basal HSV thymidine kinase promoter was co-transfected (50 ng) and used as a control. Cells were allowed to recover for 16 h and then stimulated with PMA (80 nM) or 5% fetal bovine serum. At 24 h, the cells were collected and the lysates analyzed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) as per the manufacturer’s protocol and a Moonlight 3010 luminometer (BD Pharmingen, San Diego, CA, USA).

2.5. Immunoblotting

3. Results

Total cell protein was harvested at various time-points using a whole cell lysis buffer consisting of 50 mM Tris, pH 6.8, 10% glycerol, 2% SDS and 6 M urea. Proteins were then separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5%-milk proteins in 1x PBS/TBS and 0.5% tween-20 depending on the antibody specifications. Membranes were probed with the following primary antibodies at 4 ◦ C overnight: mouse monoclonal anti-SV40 T(large)-antigen (clone PAb416) (EMD-Calbiochem, Brookfield, WI, USA), mouse monoclonal anti-␤-tubulin (clone D66) (Sigma, Oakville, ON, Canada), rabbit polyclonal anti-human ERK1/2, antihuman phosphor-specific ERK1/2 (Cell Signaling, Danvers, MA, USA), mouse monoclonal anti-cyclin D1 (clone HD 11) (Santa Cruz, Santa Cruz, CA, USA). Appropriate horseradish peroxidaseconjugated secondary antibodies were used and proteins were detected using the ECL reagent (GE-Amersham, Piscataway, NJ, USA). Protein expression was quantified by luminescence captured using a Fluor-S Max device and the Quantity-One software (BioRad, Mississauga, ON, Canada). Protein expression was normalized to endogenous controls (total ERK1/2 or tubulin) within the same sample.

3.1. Characterization of BK virus infection in human cells

2.6. BK virus NCCR cloning and luciferase assay The 421 bp NCCR of the BK virus-UT strain (GenBank accession number M34049) was amplified using PCR then cloned into the pGEM-T Easy plasmid (Promega, Madison, WI, USA). The BK virus-UT NCCR was confirmed by sequencing and the EcoRI DNA fragment was subcloned into pBluescriptII. The BK virus-UT NCCR DNA fragment was digested from pBluescriptII with BamHI-KpnI and cloned into the BglII-KpnI site of the pGL3 luciferase reporter plasmid (Promega, Madison, WI, USA). For luciferase assays, 500 ng of pGL3-BKV-UT was transfected into HEL-299 or 293 cells in

2.7. Statistical analysis Statistical analyses were performed using GraphPad Instat version 3.01. All data were expressed as mean ± standard deviation (SD). The results were analyzed for statistical variance using an unpaired student’s t-test or one-way ANOVA as appropriate. Results were considered significant when p < 0.05.

In order to efficiently study BK virus biology, a consistent and reliable cell culture system is required for viral propagation. Vero cells are green monkey kidney cells that are permissive to BK virus and often used for this purpose (Acott et al., 2006; Eash and Atwood, 2005; Jiang et al., 2009; Moriyama and Sorokin, 2009). However, since polyomaviruses in general, replicate in a speciesrestricted manner, optimization of BK virus replication in a human cell culture system is desirable. HEL-299 cells are human embryonic lung fibroblasts that support BK virus replication and were compared to Vero cells. HEL-299 and Vero cells were infected with BK virus (UT strain, MOI 0.5) and analyzed for their ability to support early BK virus replication. BK virus T-antigen expression over 7 days was similar in both HEL-299 and Vero cells as determined by immunoblotting (Fig. 1A). Consistent with this observation, early cellular BK virus DNA levels were comparable between the two cells line at 3 days post-infection as determined by real-time quantitative PCR (Fig. 1B). Therefore, at baseline, HEL-299 and Vero cells are similar in their ability to support early BK virus infection. Polyomaviruses utilize host cellular machinery for their replication (Bernhoff et al., 2008, 2009; Eash et al., 2006; Harris et al., 1996, 1998; Shivakumar and Das, 1996). Thus experiments were performed to investigate the effects of serum and cell confluency on BK virus replication; conditions that would increase cell growth and stimulate the cell cycle. To determine the effect of cell confluency on BK virus growth, HEL-299 cells were cultured in 6-well plates at a range of 5 × 104 to 5 × 105 cells/well. Cells were then infected with BK virus at an MOI of 0.5 and incubated for 3 days. Not surprisingly, a marked decrease in T-antigen expression occurred with increasing cell number and confluency. Almost no BK virus replication was detectable at a density of 5 × 105 cells/well (>90% confluency) (Fig. 2A). Consistent with this observation, serum had

Fig. 1. BK virus infection in Vero and HEL-299 cells. (A) HEL 299 and Vero cells were infected with BK virus (UT strain). Whole cell lysates were harvested at time-points ranging from 1 to 7 days. Lysates were analyzed by immunoblotting for the BK virus large T-antigen. ␤-Tubulin was used as the endogenous controls. (B) BK virus DNA content in Vero and HEL 299 cells at 3 days following infection (real-time PCR).

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a substantial effect on BK virus replication. Infections performed in low serum or serum free conditions that induce growth arrest were associated with less T-antigen expression and BK virus DNA (determined by real-time PCR) compared to high serum conditions (Fig. 2B and C). These data show that conditions that promote cell growth and division increase BK virus replication in vitro. 3.2. MAP kinase activation increases BK virus replication

Fig. 2. Effect of serum and cell division in MAP kinase activation and BK virus replication. (A) HEL-299 cells plated in 6-well plates at numbers ranging from 5 × 104 to 5 × 105 cells/well. Cells were infected with BK virus (UT strain). Whole cell lysates at 3 days were analyzed by immunoblotting for the BK virus large T-antigen and phosho-ERK1/2. ERK1/2 was used as an endogenous control. (B) HEL 299 cells were infected with BK virus and incubated in increasing concentration of fetal bovine serum. Whole cell lysates were harvested at 3 days post-infection and analyzed by immunoblotting for BK virus large T-antigen, phospo-ERK1/2 and ERK1/2. (C) Effect of serum on BK virus DNA replication in HEL-299 cells at 3 days (real-time PCR; 0% serum vs. 5% serum, ***p < 0.001, n = 3).

The previous studies suggested that signaling pathways acting to positively regulate the cell cycle may increase BK virus replication. The MAP kinases are key signaling molecules that drive cell cycle entry and cell division (Lavoie et al., 1996; Liu et al., 2004; Pages et al., 1993). In the cell culture conditions that promoted BK virus replication, ERK1/2 phosphorylation increased in parallel with T-antigen expression indicating that this pathway might directly impact the virus life cycle (Fig. 2A and B). To further examine the link between the activation of MAP kinase signaling and BK virus replication, infections were performed in the presence of the phorbol ester, PMA, a potent mitogen and activator of MAP kinase signaling cascades (Gause et al., 1993). HEL-299 cells were plated at 5 × 104 cells/well (∼30–40% confluency) and incubated with BK virus (MOI 0.5) in the presence of PMA (10–80 nM). Cells were harvested at 3 days and analyzed by immunoblotting for T-antigen expression (Fig. 3A and C). PMA significantly increased T-antigen expression in HEL-299 cells at all serum levels, but the effect was most pronounced in 5% FBS suggesting the effects of PMA and serum were synergistic. As expected, a dose-dependent increase in ERK1/2 phosphorylation also occurred in PMA-treated cells, an effect that was not induced by BK virus infection alone (Fig. 3A, C and D). Viral DNA levels increased in parallel with T-antigen expression, confirming that PMA enhances viral replication in vitro (Fig. 3B and E). PMA also augmented BK virus replication in Vero cells however the effect was substantially less. In HEL-299 cells, PMA increased BK virus DNA levels 10–20 fold, where as only a 2-fold increase was observed in Vero cells (Fig. 4A). Finally, PMA also increased the

Fig. 3. PMA enhances BK virus T-antigen expression and DNA replication. HEL-299 cells were infected with BK virus (UT strain) and incubated with PMA in concentrations ranging from 10 to 80 nM in serum free (A) or 5% serum (C) conditions. Whole cell lysates were harvested at 3 days and analyzed by immunoblotting for BK virus large T-antigen and phospho-ERK1/2. Total ERK1/2 was used as an endogenous control. (D) Quantification of phosphorylated ERK1/2 following PMA (80 nM) stimulation in 5% serum conditions (% increase over uninfected cells, BK virus alone p = NS, BK virus + PMA, **p < 0.01, n = 4). Quantification of BK virus DNA in HEL-299 cells treated with PMA (80 nM) in serum free (B) or 5% serum (E) conditions. Cells were harvested at 3 days post-infection and total BK virus genomic DNA was quantified by real-time PCR using a standard curve of BK virus DNA (HEL-299 cells untreated vs. PMA, *p < 0.05, n = 4).

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Fig. 4. PMA effects on BK virus replication. (A) Vero cells were infected with BK virus (UT strain) in the presence of PMA (80 nM) and harvested at 3 days post-infection. Total BK virus DNA was quantified by real-time PCR (Vero cells untreated vs. PMA, p = NS, n = 4). Experiments were conducted in 5% FBS. HEL-299 cells were incubated with LH (B) and VJ (C) clinical BK virus isolates in the presence of PMA (80 nM) and 5% FBS. Cells were harvested at 3 days post-infection and fold induction of BK virus DNA was quantified by real-time PCR (untreated vs. PMA, *p < 0.05 for both isolates, n = 3–6).

replication of different clinical BK virus isolates (LH and VJ (Acott et al., 2008)) suggesting that increased BK virus replication in association with MAP kinase signaling likely occurs in a strain independent manner (Fig. 4B and C). Phorbol esters such as PMA are potent activators of MAP kinase signaling via protein kinase C (Gause et al., 1993; Liu and Heckman, 1998). Serum, on the other hand, activates MAP kinases via a number of mechanisms including growth factors and receptor tyrosine kinases. To confirm that the effects of serum and PMA on BK virus replication were directly due to MAP kinase and ERK1/2 activation, experiments were performed using the MEK1/2 inhibitor U0126. At 3 days post-infection, U0126 effectively blocked the induction of BK virus T-antigen expression induced by serum and PMA (Fig. 5A). BK virus DNA levels were also diminished by U0126 confirming the reduction in viral replication (Fig. 5B). To verify that

the effect of U0126 was not the result of an effect on viral entry or trafficking, U0126 was added at 4 and 48 h following BK virus infection. U0126 inhibited PMA-enhanced T-antigen expression supporting the premise that MAP kinase signaling could directly impact BK virus replication (Fig. 5C). Taken together, these results show ERK1/2 signaling induced by serum and PMA increase BK virus replication in vitro. To assess the possibility that PMA-induced BK virus large T-antigen expression and viral replication were a result of transcriptional regulation of the BK virus NCCR, a luciferase reporter assay was employed. The BK virus-UT strain NCCR was amplified by PCR and cloned into the luciferase reporter construct pGL3. Following transfection in HEL-299, stimulations with serum or PMA (80 nM) were performed. Neither serum nor PMA activated the BK virus-UT NCCR at 24 h (Fig. 5D and data not

Fig. 5. Role of MAP kinase activation in serum and PMA-induced BK virus replication. (A) HEL-299 cells were pre-incubated with the MEK 1/2 inhibitor U0126 and then infected with BK virus (UT strain). Cells were harvested at 3 days and analyzed by immunoblotting for large T-antigen and total ERK1/2 as a loading control. (B) Quantification of BK virus DNA by real-time PCR in the presence of U0126 (BK virus ± PMA, untreated vs. U0126, ***p < 0.001, n = 3). (C) HEL-299 cells were incubated with U0126 30 min prior or 4 and 48 h post-infection with BK virus. Cellular lysates were analyzed by immunoblotting for T-antigen. (D) HEL-299 cells were transfected with a pGL3 expression vector containing the BK virus-UT strain NCCR (pGL3-BKV-UT) or the control reporter pTK-Ren. Cells were stimulated with PMA (80 nM) for 24 h and analyzed for luciferase activity (pGL3-BKV-UT, untreated vs. PMA, p = NS, n = 6; pTK-Ren, untreated vs. PMA, ***p < 0.001, n = 6). (E) 293 cells were transfected with pGL3-BKV-UT. Cells were then incubated in serum free, 5% FBS or PMA (80 nM) for 24 h and analyzed by luciferase assay for transcriptional activation of the BK virus NCCR (serum free vs. 5% FBS, *p < 0.05, n = 3; serum free vs. PMA, p = NS, n = 3). (F) HEL 299 cells were pre-incubated in the PKC inhibitor RO-31-8220 in the presence of 5% serum. Cells were then infected with BK virus. Whole cell lysates were harvested at 3 days and analyzed by immunoblotting for large T-antigen. Total ERK1/2 was used as an endogenous control.

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Fig. 6. PMA-induced BK virus replication is dependent on cyclin D1. (A) HEL-299 cells were infected with BK virus (UT strain) in the presence or absence of U0126. At 3 days post-infection, whole cell lysates were harvested and analyzed for cyclin D1 expression by immunoblotting. Total ERK1/2 is used as an endogenous control. (B) Quantification of cyclin D1 protein (immunoblotting) following BK virus infection in the presence or absence of PMA (% increase over uninfected cells: BK virus vs. BK virus + PMA, **p < 0.01, n = 4). (C) HEL-299 cells were pre-incubated with the cyclin D1/CDK4 inhibitor fascasplysin chloride hydrate (FCH, 17–35 ␮M) and infected with BK virus. Whole cell lysates were analyzed by immunoblotting at 3 days for large T-antigen. (D) Effect of FCH (35 ␮M) on BK virus DNA replication in serum free and 5% serum conditions as determined by real-time PCR (BK virus ± PMA, untreated vs. FCH, **p < 0.01, n = 3 or ***p < 0.001, n = 3).

shown). PMA, however, activated the minimal promoter and control luciferase reporter plasmid, pTK-Ren confirming that the compound was active. To ensure the integrity and functionality of the NCCR reporter construct, similar experiments were performed in 293 cells. At 24 h serum activated the UT strain NCCR as determined by a significant increase in luciferase activity compared to serum free conditions. In contrast, PMA was unable to directly activate the NCCR in the absence of serum (Fig. 5E). Thus, while the effects of both PMA and serum on BK virus replication depend on MAP kinase signal activation, our data suggests that the effects of PMA occur independent of the NCCR. Consistent with the different mechanisms of action, inhibition of protein kinase C with the Ro-31-8220 compound only blocked PMA-enhanced T-antigen expression but not the effect of serum (Fig. 5F). 3.3. ERK1/2 signaling increases BK virus replication via cyclin D1 The lack of direct NCCR activation by PMA suggested that the positive effects of MAP kinase signaling on BK virus replication extended beyond the transcriptional regulation of viral genes. The positive impact of cell division and ERK1/2 kinase activity implied a significant role for cell cycle activation in BK virus replication. The activation of ERK1/2 directly regulates cyclin D1 expression. Cyclin D1 and its associated cyclin-dependent kinase cdk4 phosphorylate pRb (upstream of BK virus T-antigen) to initiate entry into the S-phase of the cell cycle (Lavoie et al., 1996). Analysis of cyclin D1 levels upon PMA stimulation of BK virus infected HEL-299 cells revealed a consistent increase (∼50%) in cyclin D1 protein levels as compared to untreated and BK virus infected controls (Fig. 6A and B). Furthermore, inhibition of ERK1/2 phosphorylation using the MEK1/2 inhibitor U0126 abrogated cyclin D1 expression as measured by immunoblotting (Fig. 6A). To assess the importance of cyclin D1 in BK virus replication, experiments were conducted using a selective inhibitor for cyclin D1/cdk4 complex formation, fascaplysin (FCH). FCH dose-dependently inhibited baseline BK virus replication in serum free and 5% serum conditions, however the effect was less robust in the latter likely reflecting

the pleiotropic effects of FBS (Fig. 6C and d). The ability of PMA to enhance BK virus T-antigen expression and DNA replication, however, was completely abolished by the use of FCH in all serum conditions (Fig. 6C and D). Collectively, these results demonstrate that the activation of ERK1/2 signaling increases BK virus replication in large part via its effects on cyclin D1 expression and the cell cycle. 3.4. MAP kinase activation accelerates BK virus propagation in vitro A rapid and efficient method to propagate and purify high titers of BK virus will augment research into the biology of this pathogen. Protocols that describe BK virus propagation and preparation rely on the infection of Vero cells for up to 3 weeks to obtain sufficient titers for experimental use (Acott et al., 2006; Eash and Atwood, 2005; Jiang et al., 2009; Moriyama and Sorokin, 2009). Although the natural host for BK virus replication, HPTC cells are difficult to manipulate and culture in large quantities (Low et al., 2004). Therefore, experiments were performed to determine whether the MAP kinase signaling pathway could be exploited to accelerate BK virus propagation. HEL-299 cells cultured in 5% FBS were expanded to 25 mm × 150 mm plates and infected with BK virus (UT strain) at ∼50% confluency (MOI 1.0). The cells were incubated with PMA (80 nM) and harvested prior to a decline in cell viability (consistently observed at 6–7 days post-infection). BK virus purifications were then performed as outlined in the materials and methods. Viral propagation in HEL-299 cells was compared to a more standard BK virus amplification protocol in Vero cells maintained in culture for 3.5 weeks (Jiang et al., 2009; Moriyama and Sorokin, 2009). Following purification, viral stocks were titered using real-time PCR and fluorescent focus assay (Fig. 7A). The current standard for titering BK virus is the fluorescent focus assay (Abend et al., 2007; Low et al., 2004; Moriyama and Sorokin, 2009) which is an immunofluorescence-based technique reliant on Tantigen expression requiring up to 5 days of BK virus infection in vitro. To titrate rapidly BK virus, real-time PCR was used probing directly for the viral genome that was quantified against a stan-

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Fig. 7. MAP Kinase activation accelerates BK virus propagation in vitro. (A) Titering of BK virus (UT strain) crude viral lysates (CVL) and two separate purified viral preparations (Pure 1 and Pure 2). Physical titer was determined by real-time PCR using a standard curve of pure BK virus DNA and the infectious titer was derived using fluorescent focus assay. (B) The physical titer of BK virus stocks (y-axis) plotted against the infectious titer (x-axis) (r = 0.9685). (C) Physical titer (real-time PCR) of BK virus stocks propagated in HEL-299 cells in the presence of PMA (80 nM) for 1 week or Vero cells for 25 days. (D) Transmission electron microscopy image of BK virus stocks generated in HEL-299 cells in the presence of PMA (80 nM).

dard curve of pure BK virus DNA specific for the amplicon of the real-time primers. A physical titer was then generated dividing the total amount of BK virus DNA by the approximate weight of one BK virus genome (5.675 × 10−9 ng) to estimate the number of genomes/ml. The determination of physical titer by real-time PCR correlated directly with the infectious titer obtained using the fluorescent focus assay and was consistently 2.0–2.5 logs higher (Fig. 7A and B). Using both assays, BK virus titers produced in HEL-299 cells incubated in PMA for 1 week were similar to viral propagation using the standard 3.5 weeks protocol in Vero cells (Fig. 7C). The presence of intact BK virus particles produced via this method was confirmed by the infectious titers and electron microscopy (Fig. 7A and D). Thus, the effect of high serum and PMA acting through ERK1/2 increases BK virus replication and increases the production of high titer stocks of pure BK virus. Furthermore, in combination with the rapid determination of physical titer using real-time PCR, the time required to prepare BK virus for experimental purposes can be significantly reduced.

4. Discussion BK virus is becoming an increasingly problematic pathogen in immunosuppressed patients. Relatively little is known regarding the biology of the virus and the factors that allow reactivation in certain clinical settings. In this study, the activation of MAP kinase signaling substantially enhanced BK virus replication in vitro. The results shed light on the cellular conditions that promote virus replication and show that manipulation of MAP kinase signaling can be utilized to facilitate the production of high titer, pure stocks of BK virus.

Mitogens such as serum and PMA are known activators of ERK1/2 (Gause et al., 1993) and functioned to enhance BK virus replication in vitro. Several studies have assessed the relationship of intracellular signaling mechanisms on BK virus replication in relation to transcriptional activation of the BK virus non-coding regulatory region (Abend and Imperiale, 2008; Abend et al., 2007; Gorrill and Khalili, 2005; Jordan et al., 2009; Moens et al., 1994). Data in this paper show that PMA alone was incapable of stimulating the BK virus-UT NCCR in reporter assays. Consistent with this observation, PMA-enhanced the replication of other BK virus clinical isolates suggesting that MAP kinase signaling enhanced viral replication via an effect on cellular function rather than cisregulation of viral genes. The effect of MAP kinase activation may therefore be applicable to all BK virus strains. Serum, not surprisingly exhibited a more pleiotropic effect compared to PMA, activating MAP kinase signaling and the viral NCCR. Although serum positively affected BK virus replication via cell cycle activation, other ERK1/2-independent mechanisms are likely involved. A major function for BK virus large T-antigen is to facilitate transition into the S-phase of the cell cycle, initiating DNA replication and ultimately division of the host cell (Bernhoff et al., 2008, 2009; Eash et al., 2006). BK virus large T-antigen binds to hypophosphorylated members of the pRb family of proteins allowing the release of E2F transcription factors that are essential in the transition to S-phase (Harris et al., 1996, 1998). Consistent with this, a recent assessment of host gene expression revealed the upregulation of numerous cell cycle genes during BK virus infection (Abend et al., 2009). Due to its relative low expression however, BK virus large T-antigen is limited in its ability to interact with members of the pRb family indicating that additional, pro-proliferative signals may be required for efficient viral replication (Harris et al., 1996). The

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ERK1/2 signaling axis has been directly implicated in S-phase transition and the onset of mitosis (Lavoie et al., 1996; Lewis et al., 1998; Liu et al., 2004; Pages et al., 1993). Signaling via ERK1/2 directly regulates the expression of cyclin D1 that control the early steps in initiating S-phase transition. Cyclin D1 expression results in the hyperphosphorylation of pRb family members and release of E2F transcription factors. The importance of these proliferative signals in the polyomavirus life cycle is exemplified by studies examining the ability of MPyV and SV40 small t-antigens to sequester the phosphatase PP2A and promote MAP kinase signaling. Furthermore, SV40 is capable of eluding G0 arrest in quiescent cells through small t-dependent induction of the cyclin D1 promoter (Rodriguez-Viciana et al., 2006; Sontag et al., 1993; Watanabe et al., 1996). Interestingly, unlike other polyomaviruses, BK virus itself did not induce ERK1/2 phosphorylation or cyclin D1 expression in vitro. Nonetheless, the inhibition of cyclin D1 activity reduced BK virus replication confirming that ERK1/2 pathway is synergistic with T-antigen likely converging at pRb to enhance viral replication. Consistent with the premise that large T-antigen effects on pRb are independent and likely occur downstream of ERK1/2 and cyclin D1, viral replication could still be detected in low serum or in the presence of PKC, MEK1/2 and cyclin D1/cdk4 inhibitors. From a clinical standpoint these observation are significant since activation of host cell signaling pathways such as ERK1/2 or the cell cycle by cellular injury, inflammation or during regeneration may be a trigger that enables BK virus reactivation. The standard for amplification of BK virus in vitro relies on propagation in Vero and requires an incubation period of 3–4 weeks to generate high titer BK virus stocks (Acott et al., 2006; Eash and Atwood, 2005; Jiang et al., 2009; Moriyama and Sorokin, 2009). The development of a protocol that results in rapid amplification of BK virus would be beneficial to the future study of BK virus biology. Although HPTC are a natural host for BK virus, they are difficult to manipulate in culture. Vero cells are more often used in BK virus protocols but are of green monkey origin. Given the speciesrestricted replication of polyomaviruses, it was hypothesized that BK virus propagation in vitro could be improved in a human cell culture model. HEL-299 cells supported the most productive BK virus replication especially in the presence of serum and PMA and were therefore used to propagate the virus. Using this system, high titers of BK virus were generated in less time than conventional protocols. Incubation of Vero cells in PMA also increased the titer of BK virus. However, the observed increase was less than that observed in HEL-299 cells. The reason for this variability is unclear, but PMAresponsiveness may be a function of the PKC isoforms present in a given cell type or the basal level of MAP kinase activation (Liu and Heckman, 1998). Currently, the fluorescent focus assay represents the standard for quantitation of infectious BK virus. This method relies on the expression of BK virus large T-antigen and is limited by interoperator variability and virus infection kinetics (Abend et al., 2007; Moriyama and Sorokin, 2009). Real-time PCR is used to detect BK virus in clinical human serum and urine specimens (Marinelli et al., 2007; McQuaig et al., 2009; Mischitelli et al., 2007). In this study, real-time PCR was employed to generate a physical titer of BK virus based on genome quantification. The physical titer determined in this manner correlated directly with the infectious titer generated by fluorescent focus assay, but approximately 2–2.5 logs lower. The difference between the physical and infectious titers is likely due to a number of factors including defective virions, unpackaged DNA and the relative efficiency of BK virus infection/inter-operator variability in the context of the fluorescent focus assay in vitro. Thus, while the use of real-time PCR to titrate BK virus may have some limitations in extrapolating to infectious titers, it is performed rapidly, objective and can be used as an adjunct to the fluorescent focus assay.

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