BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of Cell Entry

Takahito Moriyama1, Andrey Sorokin2

1 Tokyo Women's Medical University, Tokyo, Japan, 2 Medical College of Wisconsin, Milwaukee, Wisconsin
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.2
DOI:  10.1002/0471143030.cb2602s42
Online Posting Date:  March, 2009
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Abstract

BK virus (BKV) can cause BKV nephritis in renal transplant patients and has become a significant reason for graft loss in this decade. BKV is latent in the urogenital tract and most likely is transported with the donor kidney to recipients. BKV replication occurs in the nucleus of human renal proximal tubular cells (HRPTEC) and daughter viruses are delivered to other cells to spread infection. A few in vitro studies have been reported about the mechanism and kinetics of BKV infection. However, there are still a lot of unknown factors regarding BKV infection. This unit describes the handling of BKV, BKV propagation, determination of titer and ability to infect cells, as well as purification and labeling of BKV in order to analyze BKV cell entry. Curr. Protoc. Cell Biol. 42:26.2.1‐26.2.13. © 2009 by John Wiley & Sons, Inc.

Keywords: BK virus; human renal proximal tubular epithelial cells; infection; propagation; quantitation; purification; labeling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Viral Propagation of BKV
  • Support Protocol 1: Viral Titration Using the Fluorescent Focus Assay
  • Support Protocol 2: Purification of BKV
  • Support Protocol 3: Labeling Purified Virus
  • Basic Protocol 2: BKV Infection of Human Renal Proximal Tubular Epithelial Cells (HRPTEC)
  • Basic Protocol 3: Analysis of BKV Entry and Intracellular Trafficking Pathway in HRPTEC Using Confocal Microscope and MetaVue Imaging System
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Viral Propagation of BKV

  Materials
  • HRPTEC (Cambrex Bio Science Inc.), passage 6
  • REBM containing 5% FBS (see recipe)
  • BKV stock (ATCC no. VR‐837), titered by fluorescent focus assay ( protocol 2)
  • 2.5% (w/v) deoxycholic acid
  • 75‐cm2 tissue culture flasks
  • Refrigerated centrifuge
  • Cell scrapers
  • Sonicator (e.g., Sonic Dismembrator 550; Fisher Scientific), with microtip

Support Protocol 1: Viral Titration Using the Fluorescent Focus Assay

  Materials
  • 70% ethanol
  • HRPTEC (Cambrex Bio Science Inc.), passage 6
  • REBM containing 0.5% FBS (see recipe)
  • BKV stock (ATCC no. VR‐837) for titration ( protocol 1)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 100% methanol
  • Tris‐buffered saline with 0.1% (v/v) Tween (TTBS; see recipe)
  • TTBS (see recipe) containing 3% (v/v) FBS
  • TTBS (see recipe) containing 1% (v/v) FBS
  • Primary antibody: antibody (mouse PA 416) against SV40 T‐antigen, recognizing BKV T‐Ag (Calbiochem)
  • Secondary antibody: goat antibody against mouse Ig, conjugated to Alexa Fluor 350 (detect blue color as nuclei in BKV‐infected cells), Alexa Fluor 488 (green color), and Alexa Fluor 680 (red color), all from Invitrogen
  • Mounting medium: SlowFade Antifade Kit (Molecular Probes) or 70% (v/v) glycerol/30% (v/v) PBS (see appendix 2A for PBS; store up to 1 year at 4°C)
  • Clear nail polish
  • 13‐mm‐diameter glass coverslips
  • 24‐well tissue culture plates
  • Fluorescence microscope (unit 4.2) with FITC filters (which are a match for Alex Fluor 488)

Support Protocol 2: Purification of BKV

  Materials
  • Virus‐containing supernatant ( protocol 1)
  • 20% (w/v) sucrose in reassociation buffer (see recipe for buffer)
  • Buffer A (see recipe)
  • 1.40 g/ml and 1.20 g/ml cesium chloride (CsCl) solutions in distilled water
  • Slide‐A‐Lyzer dialysis cassette (MWCO 10,000, Pierce)
  • Ultracentrifuge
  • 10‐ml and 20‐ml ultracentrifuge tubes
  • Sonicator (e.g., Sonic Dismembrator 550; Fisher Scientific), with microtip
  • 5‐ml syringe
  • 18‐G needle

Support Protocol 3: Labeling Purified Virus

  Materials
  • Alexa Fluor 488 Microscale Protein Labeling Kit (Invitrogen) containing:
    • Alexa Fluor 488 tetrafluorophenyl (TFP) ester (Component A), 3 vials
    • Sodium bicarbonate (Component B), 84 mg
    • Reaction tubes (Component C), 3 tubes
    • Spin filters (Component D) Nanosep MF 0.2‐µm centrifugation devices, 3 tubes
    • Purification resin (Component E) Bio‐Gel P‐6 fine resin suspended in PBS, 3 ml
  • Purified BKV ( protocol 3)

Basic Protocol 2: BKV Infection of Human Renal Proximal Tubular Epithelial Cells (HRPTEC)

  Materials
  • HRPTEC (Cambrex Bio Science Inc.), no later than passage 6
  • REBM containing 0.5% FBS (see recipe)
  • BKV stocks, titrated ( protocol 2), purified ( protocol 3), and labeled ( protocol 4, for observing virus particles)
  • 60‐mm tissue culture dishes or 24‐well tissue culture plates with glass coverslip in each well (see protocol 2, step 1)
  • Cell scrapers
  • Sonicator (e.g., Sonic Dismembrator 550; Fisher Scientific) with microtip

Basic Protocol 3: Analysis of BKV Entry and Intracellular Trafficking Pathway in HRPTEC Using Confocal Microscope and MetaVue Imaging System

  Materials
  • HRPTEC (Cambrex Bio Science Inc.), no later than passage 6
  • REBM containing 0.5% FBS (see recipe)
  • Purified and labeled BKV ( protocol 4)
  • Primary antibody against target organelle
  • Secondary antibody with a fluorophore different from that of the virus
  • 24‐well tissue culture plates with glass coverslip in each well
  • Confocal microscope (unit 4.5)
  • MetaVue Imaging System (Molecular Devices) or other image‐analysis program
  • Additional reagents and equipment for indirect immunofluorescence assay ( protocol 2)
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Figures

  •   FigureFigure 26.2.1 Detection of HRPTEC BKV infection by immunofluorescence. HRPTEC are either stained with anti‐T‐Ag antibodies (A) or stained with 4′,6‐diamidino‐2‐phenylindole dilactate (DAPI) (B). HRPTEC were incubated with BKV (MOI, 0.5 FFU/cell). After 72 hr, fresh medium was added and the cells were incubated for another 48 hr. After incubation, cells were fixed and blocked. Next, cells were incubated with primary antibody [5 µl of PA 416 (Calbiochem) in 1 ml of TTBS with 1% FBS] and secondary antibody [1:200 diluted Alexa Fluor 488 goat anti‐mouse IgG (H+L); (Molecular Probes) in TTBS with 1 % FBS]. To stain nuclei, cells were incubated with 300 nM DAPI (Molecular Probes) for 5 min and washed three times with PBS. Cells were observed by fluorescent microscopy (Nikon Eclipse E600) with 20× objective lens, and images were captured by SPOT version 4.0.9 (Diagnostic Instruments).
  •   FigureFigure 26.2.2 Localization of labeled BKV particles in HRPTEC. (A) Fluorescence of Alexa Fluor 488–labeled BKV particles in HRPTEC. (B) Staining for endoplasmic reticulum (ER) in HRPTEC. (C) Co‐localization of purified and labeled BKV with ER marker. HRPTEC were incubated with purified and labeled BKV for 6 hr. After incubation, cells were fixed and blocked. Then cells were incubated with primary antibody [1:100 dilution of PDI (Abcam) as ER marker against TTBS with 1% FBS] and secondary antibody [1:200 diluted Alexa Fluor 680 goat anti‐mouse IgG (H+L) (Molecular Probes) in TTBS with 1% FBS]. Cells were analyzed by confocal microscope (Leica TCS SP5) with 63× objective lens and images were captured by Leica application suite (advanced fluorescence). Bar = 10 µm.

Videos

Literature Cited

   Abend, J.R., Low, J.A., and Imperiale, M.J. 2007. Inhibitory effect of gamma interferon on BK virus gene expression and replication. J. Virol. 81:272‐279.
   Ahsan, N. and Shah, K.V. 2006. Polyomaviruses and human diseases. Adv. Exp. Med. Biol. 577:1‐18.
   Eash, S. and Atwood, W.J. 2005. Involvement of cytoskeletal components in BK virus infectious entry. J. Virol. 79:11734‐11741.
   Eash, S., Querbes, W., and Atwood, W.J. 2004. Infection of Vero cells by BK virus is dependent on caveolae. J. Virol. 78:11583‐11590.
   Gardner, S.D., Field, A.M., Coleman, D.V., and Hulme, B. 1971. New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet. I:1253‐1257.
   Liu, C.K. and Atwood, W.J. 2001. Propagation assay of the JC virus. Methods Mol. Biol. 165:9‐17.
   Lonardo, A.D., Buttinelli, G., Amato, C., Novello, F., Ridolfi, B., and Fiore, L. 2002. Rapid methods for identification of poliovirus isolates and determination of polio neutralizing antibody titers in human sera. J. Virol. Methods 101:189‐196.
   Low, J., Humes, H.D., Szczypka, M., and Imperiale, M. 2004. BKV and SV40 infection of human kidney tubular epithelial cells in vitro. Virology 323:182‐188.
   Moriyama, T. and Sorokin, A. 2008. Intracellular trafficking pathway of BK virus in human renal proximal tubular epithelial cells. Virology 371:336‐349.
   Moriyama, T., Marquez, J.P., Wakatsuki, T., and Sorokin, A. 2007. Caveolae endocytosis is critical for BK virus infection of human renal proximal tubular epithelial cells. J. Virol. 81:8552‐8562.
   Nickeleit, V., Singh, H.K., and Mihatsh, M.J. 2003. Polyomavirus nephropathy: Morphology, pathophysiology, and clinical management. Curr. Opin. Nephrol Hypertens. 12:599‐605.
   Payne, A.F., Binduga‐Gajewska, I., Kauffman, E.B., and Kramer, L.D. 2006. Quantitation of flaviviruses by fluorescent focus assay. J. Virol. Methods 134:183‐189.
   Sawa, H. and Komagome, R. 2005. The JC virus‐like particle overlay assay. Methods Mol. Biol. 292:175‐186.
Internet Resources
  http://probes.invitrogen.com/media/publications/150.pdf
  Web sites showing product descriptions of Alexa Fluor labeling kits.
  http://probes.invitrogen.com/handbook/sections/0102.html
  http://probes.invitrogen.com/handbook/sections/0103.html
  http://probes.invitrogen.com/media/pis/mp30006.pdf
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