Fluorescently Labeled Human Papillomavirus Pseudovirions for Use in Virus Entry Experiments

Pilar Samperio Ventayol1, Mario Schelhaas1

1 Emmy‐Noether Group, Virus Endocytosis’, Institutes of Molecular Virology and Medical Biochemistry, ZMBE, University of Münster, Münster
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14B.4
DOI:  10.1002/9780471729259.mc14b04s37
Online Posting Date:  May, 2015
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Abstract

Human papillomaviruses (HPV) infect skin or mucosal epidermis. The simplistic capsid consists of a major capsid protein L1, a minor capsid protein L2, and a double‐stranded circular DNA of about 8kB in size. The development of HPV‐based vectors [i.e., pseudovirions (PsV)] as tools to study the initial infection has facilitated our understanding of HPV entry. The covalent coupling of fluorescent molecules to these PsV allows following the viruses en route to the nucleus, i.e., the site of replication. In the first section, we describe a facile method to covalently label HPV PsV that retain their infectivity. In this method, fluorophores coupled to a reactive succinimidyl ester are covalently attached to amine residues in the virion in a one‐step chemical reaction. In the second section of this unit, several assays are outlined that use the fluorescently labeled virions for entry studies in live and fixed cells. © 2015 by John Wiley & Sons, Inc.

Keywords: HPV; papillomavirus; virus entry; labeling; microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Fluorescence Labeling of HPV PsV
  • Alternate Protocol 1: Labeling of HPV with Higher Degrees of Labeling
  • Support Protocol 1: Determining a Visible Presence of Free Dye
  • Support Protocol 2: Testing for the Monodispersity of Viruses in Solution
  • Support Protocol 3: Quantification of Labeled HPV
  • Support Protocol 4: Assessing the Degree of Labeling (Optional)
  • Support Protocol 5: Infectivity Test of Labeled HPV
  • Basic Protocol 2: Internalization Assay by Gain of pH Sensitive Fluorescence
  • Basic Protocol 3: Internalization Assay After Quenching of Extracellular Fluorescence with Trypan Blue
  • Alternate Protocol 2: TCEP Internalization Assay
  • Basic Protocol 4: Fluorescence Microscopy of HPV Particles
  • Alternate Protocol 3: HPV Colocalization Analysis with Cellular Markers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorescence Labeling of HPV PsV

  Materials
  • HPV PsV (purified over an Optiprep gradient according to Buck and Thompson, )
  • Virion buffer: modified PBS/0.8 NaCl, pH 7.4 (see recipe)
  • Succinimidyl ester‐conjugated fluorophores dissolved in DMSO, (e.g., Alexa Fluor dyes from Invitrogen; see Table 14.4.1) (see recipe)
  • Low binding products: 1.5‐ml tubes, tips, etc. (e.g., MAXYMum Recovery from Axygen)
  • Vortex mixer
  • Aluminum foil
  • Head‐over‐head mixer (e.g., adjustable mixing angle rotator from Stuart)
  • Size‐exclusion column (e.g., illustra NAP‐5 columns from GE Healthcare: prepacked with Sephadex G‐25 DNA Grade, 0.5‐ml size)
Table 4.0.1   MaterialsFluorescence Characteristics of Selected Succinimidyl Ester Conjugates

Dye Molecular Weight λ max a Em a pK a dilution/conjugation Source
Alexa Fluor488 643 495 519 Life Technologies
Alexa Fluor594 820 590 617 Life Technologies
Alexa Fluor647 ≈1300 650 665 Life Technologies
pHrodo ≈650 566 590 ≈7.3/6.5 Life Technologies

 aFluorescence absorbance and emission maxima, in nm, conjugated to an IgG antibody.
NOTE: The protocol is performed at room temperature (RT).NOTE: Always use low‐binding products for pipetting and reaction tubes.

Alternate Protocol 1: Labeling of HPV with Higher Degrees of Labeling

  Materials
  • Adherent cells (e.g., HeLa cells)
  • Cultivation medium (depending on the cell type; for HeLa we use DMEM including 10% FBS)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free, pH 7.4 (see recipe)
  • Labeled HPV fractions
  • Glass‐bottom dishes, or coverslips and corresponding microscope coverslip chamber (e.g., from Life Technologies)
  • Protein low‐binding products: 1.5 ml‐tubes, tips, etc. (e.g., MAXYMum Recovery from Axygen)

Support Protocol 1: Determining a Visible Presence of Free Dye

  Materials
  • Phosphate‐buffered saline (PBS; calcium‐ and magnesium‐free), pH 7.4 (see recipe)
  • Labeled HPV
  • Glass‐bottom dishes, or coverslips and corresponding microscope coverslip chamber (e.g., from Life Technologies)
  • Software for fluorescence signal analysis, for example ImageJ (free open‐source; http://hrsb.info.nih.gov/ij; see Table 14.4.2)
Table 4.0.2   MaterialsImage‐Processing Software

Software Principal functions Used in protocol Is it open source? Source Link
ImageJ (Fiji) Wide array of image analyses and processing algorithms (without automation for multiple samples) protocol 11 and protocol 4 Yes National Institutes of Health (NIH), US http://hrsb.info.nih.gov/ij(fiji.sc/Fiji)
Matlab Allows programming of custom algorithms for image analysis protocol 8 No MathWorks http://www.mathworks.com
Cell Profiler Image segmentation and quantitative analysis; can be automated Basic Protocols protocol 82 and protocol 93; protocol 10 Yes Broad Institute Imaging Platform http://www.cellprofiler.org
Imaris 3‐D image analysis. Modules for automated analysis protocol 11 and protocol 12 No Bitplane http://www.bitplane.com/imaris
BioimageXD Package for analyzing, processing ,and visualizing multi‐dimensional microscopy images protocol 12 Yes Universities of Jyväskylä and Turku in Finland, Max Planck Institute CBG in Dresden, Germany and collaborators worldwide http://www.bioimagexd.net
Metamorph Acquisition, processing, and analysis packages No Molecular Devices http://www.moleculardevices.com/systems/metamorph‐research‐imaging
Volocity 3‐D image analysis No Perkin Elmer http://www.perkinelmer.com/pages/020/cellularimaging/products/volocity.xhtml

Support Protocol 2: Testing for the Monodispersity of Viruses in Solution

  Materials
  • Labeled HPV
  • 2 mg/ml bovine serum albumin (BSA) in PBS
  • Input HPV prior to labeling with known concentration
  • 5 × SDS loading buffer containing 20 mM DTT (see recipe)
  • De‐ionized water: ddH 2O
  • 10% acrylamide gel
  • Coomassie Brilliant Blue staining solution (see recipe)
  • Destaining solution: 10% (v/v) acetic acid
  • Low‐binding products: 1.5‐ml tubes, tips, etc. (e.g., MAXYMum Recovery from Axygen)

Support Protocol 3: Quantification of Labeled HPV

  Materials
  • Labeled and unlabeled HPV
  • Phosphate‐buffered saline (PBS; calcium‐ and magnesium‐free), pH 7.4 (see recipe)
  • Dye used for virus labeling
  • Low‐binding products: 1.5‐ml tubes, tips, etc. (e.g., MAXYMum Recovery from Axygen)
  • Fluorospectophotometer
  • Quartz cuvette

Support Protocol 4: Assessing the Degree of Labeling (Optional)

  Materials
  • Cells (e.g., HeLa Kyoto cells)
  • Cultivation medium (depending on the cell type; for HeLa DMEM including 10% FBS)
  • HPV labeled with a fluorophore that fluoresces only at low pH (e.g., pHrhodo from Life Technologies)
  • 10 mM HEPES, pH 7.4
  • Phosphate‐buffered saline (PBS; calcium‐ and magnesium‐free), pH 7.4 (see recipe)
  • Imaging medium (depending on the cell type; for HeLa see recipe)
  • Low‐binding products: 1.5‐ml tubes, tips, etc. (e.g., MAXYMum Recovery from Axygen).
  • Glass‐bottom dishes, or coverslips and corresponding microscope coverslip chamber (e.g., from Life Technologies)
  • Software for fluorescence signal measurement (e.g., Cell Profiler; free open‐source: cellprofiler.org)

Support Protocol 5: Infectivity Test of Labeled HPV

  Materials
  • Cells of choice (e.g., HeLa cells)
  • Cultivation medium (depending on the cell type; for HeLa, we use DMEM including 10% FBS)
  • Labeled HPV (freshly thawed): we recommend AF594‐HPV
  • Phosphate‐buffered saline (PBS; calcium‐ and magnesium‐free), pH 7.4 (see recipe)
  • Imaging medium (depending on cell type; for HeLa cells, see recipe)
  • 0.4% (w/v) Trypan Blue (see recipe)
  • Low‐binding products: 1.5‐ml tubes, tips, etc. (e.g., MAXYMum Recovery from Axygen)
  • Glass‐bottom dishes, or coverslips and corresponding microscope coverslip chamber (e.g., from Life Technologies)
  • 37°C and 5% CO 2 incubator
  • Software for fluorescence signal measurement (e.g., Cell Profiler: free open‐source; cellprofiler.org)
NOTE: Live imaging of all samples will be performed 12 hr p.i., before and after addition of Trypan Blue. Therefore, the number of samples equals the number of images that can be taken with a normal microscope. If your setup is automated, you may be able to save multiple positions on your sample and thus automatically take images from the same positions prior to and after Trypan Blue addition. As control, include a sample where virus was bound to cells (e.g., for 1 hr), and where all virus signal should be quenched. Prepare this sample 1 hr prior to imaging. In addition, prepare an uninfected sample.

Basic Protocol 2: Internalization Assay by Gain of pH Sensitive Fluorescence

  Additional materials (also see protocol 9)
  • Cells of choice (e.g., HeLa Kyoto H2BmCherry cells as they exhibit nuclear fluorescence; see Steigemann et al., )
  • AF647‐labeled HPV (freshly thawed)
  • 4% paraformaldehyde (PFA) in PBS; prepared freshly before use
  • TCEP dilution (see recipe); prepared freshly before use

Basic Protocol 3: Internalization Assay After Quenching of Extracellular Fluorescence with Trypan Blue

  Materials
  • Cells of choice (e.g., HeLa cells)
  • Cultivation medium (depending on the cell type; for HeLa cells, we use DMEM including 10%FBS)
  • Imaging medium (depending on cell type; for HeLa cells, see recipe)
  • Labeled HPV (freshly thawed)
  • Low‐binding products: 1.5‐ml tubes, tips, etc. (e.g., MAXYMum Recovery from Axygen)
  • Glass‐bottom dishes, or coverslips and corresponding microscope coverslip chamber (e.g., from Life Technologies)
  • Total internal reflection (TIRF) microscope
  • Software for fluorescence signal measurement (e.g., the MOSAIC tracking plugin of FIJI; free open‐source; http://mosaic.mpi‐cbg.de/?q=downloads/imageJ)

Alternate Protocol 2: TCEP Internalization Assay

  Additional Materials (also see protocol 11)
  • Cultivation medium
  • 4% paraformaldehyde (PFA) in PBS, prepared freshly before use
  • Permeabilization solution: 0.1% Triton X‐100 in PBS (see recipe)
  • Blocking solution: 3% (w/v) BSA in PBS
  • LAMP1 antibody (ab; e.g., from Santa Cruz)
  • Fluorescence dye conjugated secondary antibody (e.g., Alexa AlexaFluor‐conjugated secondary Ab from Life Technologies)
  • Phosphate‐buffered saline (PBS, calcium‐ and magnesium‐free), pH 7.4 (see recipe)
  • Mounting medium (e.g., CitiFluor AF1)
  • Nail polish
  • Humidified chamber
  • Parafilm
  • Confocal laser scanning microscope
  • Software for fluorescence colocalization analysis (e.g., Imaris, from Bitplane)
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Figures

Videos

Literature Cited

Literature Cited
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