HIV‐1 Interactions with Cells: From Viral Binding to Cell‐Cell Transmission

Alicia M. Janas1, Li Wu1

1 Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.5
DOI:  10.1002/0471143030.cb2605s43
Online Posting Date:  June, 2009
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Abstract

Characterization of HIV‐1 interactions with host cells is critical for cell biology studies of HIV‐1. This unit describes a set of methods and protocols to perform quantitative assays of HIV‐1 binding, internalization, infection, and cell‐cell transmission. The protocols include: (1) generating infectious single‐cycle or replication‐competent HIV‐1 stocks, (2) an HIV‐1 binding and internalization assay, (3) HIV‐1 infection of target cells and quantification of viral infection, and (4) HIV‐1 cell‐cell transmission assays. These functional assays provide useful tools to quantitatively study HIV‐1 infection and viral transmission. Curr. Protoc. Cell Biol. 43:26.5.1‐26.5.20. © 2009 by John Wiley & Sons, Inc.

Keywords: human immunodeficiency virus type 1 (HIV‐1); binding; internalization; infection; viral transmission; interactions

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Generating Infectious Single‐Cycle or Replication‐Competent HIV‐1 Stocks
  • Basic Protocol 2: HIV‐1 Binding and Internalization Assays
  • Basic Protocol 3: HIV‐1 Infection of Target Cells and Quantification of Viral Infection
  • Alternate Protocol 1: Viral Infection and Quantification Using Reporter HIV‐1
  • Support Protocol 1: Detection of Luciferase Activity in Infected Cells
  • Support Protocol 2: GFP Detection in Infected Cells
  • Support Protocol 3: HIV‐1 p24 Detection in Supernatants of Infected Cells
  • Basic Protocol 4: HIV‐1 Cell‐Cell Transmission Assays
  • Alternate Protocol 2: HIV‐1 Enhancement Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generating Infectious Single‐Cycle or Replication‐Competent HIV‐1 Stocks

  Materials
  • 2.5 M CaCl 2
  • Tissue‐culture‐grade double distilled water
  • 2× BES‐buffered saline (BBS; see recipe)
  • Human embryonic kidney cell line HEK293T cells (a gift from Dr. V. KewalRamani, vineet@ncifcrf.gov; Wang et al., , b)
  • HEK293T cell culture medium (see recipe), 37°C
  • HIV‐1 proviral DNA pHIV‐Luc (pLai3ΔenvLuc2) env‐deleted and nef‐inactivated viral genome with a luciferase reporter insertion, and contains all other viral genes (Yamashita and Emerman, ; gifts from Michael Emerman, Fred Hutchinson Cancer Research Center; memerman@fhcrc.org)
  • HIV‐1 full‐length proviral construct pNL4‐3 and pNLAD8 (Freed et al., ; gifts from Eric Freed, National Cancer Institute, Frederick, Maryland)
  • Expression plasmids: R5‐tropic Env of HIV‐1 JRFL (pJRFL) or HIV‐1 ADA (pADA), X4‐tropic Env of HIV‐1 HXB2 (pHXB2), and VSV‐G‐expressing pVSV‐G (gifts from Vineet KewalRamani, National Cancer Institute, Frederick, Maryland; Dong et al., ; Wang et al., , b)
  • Green fluorescent protein–expressing HIV‐1 vector (pHIV‐GFP) generated from a pNL4‐3‐based HIV‐1 provirus with deletions in the env, vif, vpr, vpu, and nef genes; the GFP gene was inserted in place of the nef open reading frame (Unutmaz et al., )
  • Human CD4+ T cell line Hut/CCR5 cells (a gift from Dr. V. KewalRamani, vineet@ncifcrf.gov; Wang et al., , b)
  • 0.2‐µm sterile filters
  • Tissue culture plates (standard 10‐cm diameter dishes or 6‐well plates)
  • 37°C water bath
  • 5‐ ml round‐bottom, polystyrene tubes (Falcon cat. no. 2058)
  • 37°C, 5% CO 2 cell culture incubator designated for HIV‐1 infections
  • 15‐ml conical tubes
  • Refrigerated centrifuge
  • 2‐ml cryotubes (sterile)
  • Additional equipment and reagents for cell culture (unit 1.1)
CAUTION: Follow biosafety protocol and use 10% bleach to inactivate HIV‐1 in the plates, tubes, plastic pipets, etc.

Basic Protocol 2: HIV‐1 Binding and Internalization Assays

  Materials
  • Raji cells, Raji/DC‐SIGN cells or another appropriate cell type (NIH AIDS Research and Reference Reagent Program, Raji cells cat. no. 9944, Raji/DC‐SIGN cells cat. no. 9945; http://www.aids.reagent.org/index.cfm)
  • Sterile PBS without Ca2+ and Mg2+ (CMF‐PBS; appendix 2A), cold
  • Cell culture media (see recipes)
  • AT‐2–inactivated R5‐tropic HIV (Bal/Supt1‐CCR5 cl30; from Jeffery Lifson, AIDS Vaccine Program, SAIC‐Fredrick)
  • RPMI‐1640 medium with 10% (v/v) fetal bovine serum (FBS)
  • 0.25% (w/v) trypsin without EDTA
  • 1× cell lysis buffer (see recipe)
  • 10% (v/v) Triton X‐100
  • HIV‐1 p24 ELISA kit (anti‐p24‐coated plates from the AIDS Vaccine Program, SAIC, Frederick, Maryland) or p24 ELISA kit (PerkinElmer)
  • 1.5‐ml screw‐cap tubes and microcentrifuge tubes
  • Refrigerated microcentrifuge (e.g., rotor model F241.5P in Beckman Coulter microcentrifuge 22R centrifuge)
  • 37°C water bath or incubator
  • Additional equipment and reagents for cell culture (unit 1.1)

Basic Protocol 3: HIV‐1 Infection of Target Cells and Quantification of Viral Infection

  Materials
  • HIV indicator cell line: GHOST/X4/R5 cells (available from NIH AIDS Research and Reference Reagent Program cat. no. 3942 or from Dr. Vineet KewalRamani, National Cancer Institute, Frederick, Maryland)
  • Cell culture medium (see recipe)
  • Infectious HIV‐1 stocks
  • Sterile PBS without Ca2+ and Mg2+ (CMF‐PBS; appendix 2A)
  • Sterile 1 mM EDTA
  • 0.05% (w/v) trypsin with 0.2 g/liter EDTA, optional
  • 4% (w/v) paraformaldehyde
  • CMF‐PBS/2% (v/v) FBS
  • 12‐well tissue culture plate
  • 37°C incubator
  • Centrifuge with plate rotor
  • Flow cytometer
  • Additional reagents and equipment for cell culture (unit 1.1)
CAUTION: Follow the biosafety protocol and use 10% bleach to inactivate HIV‐1 in the plates, tubes, plastic pipets, etc.

Alternate Protocol 1: Viral Infection and Quantification Using Reporter HIV‐1

  Materials
  • HIV‐1 stocks with known viral infectivity (see protocol 3)
  • Hut/CCR5 cells (available from Dr. Vineet KewalRamani, vineet@ncifcrf.gov; Wang et al., , b)
  • Cell culture medium (see recipes)
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • 24‐well culture plates
  • 37°C incubator
  • Additional equipment and reagents for cell culture (unit 1.1)

Support Protocol 1: Detection of Luciferase Activity in Infected Cells

  Materials
  • Cell samples
  • PBS ( appendix 2A)
  • 5× passive lysis buffer (Promega)
  • Luciferase assay kit (Promega)
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • Microcentrifuge (e.g., rotor model F241.5P in Beckman Coulter microcentrifuge, 22R centrifuge)
  • Platform shaker
  • 96‐well black plate (Thermo)
  • Chemluminescence microplate reader (Wallac 1420 VICTOR2 Multilabel Plate Reader)

Support Protocol 2: GFP Detection in Infected Cells

  Materials
  • Infected cells
  • 4% (w/v) paraformaldehyde
  • Sterile PBS without Ca2+ and Mg 2+ (CMF‐PBS; appendix 2A)
  • CMF‐PBS/2% (v/v) FBS
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • Microcentrifuge (e.g., rotor model F241.5P in Beckman Coulter microcentrifuge, 22R centrifuge)
  • Flow cytometer

Support Protocol 3: HIV‐1 p24 Detection in Supernatants of Infected Cells

  Materials
  • Infected cells
  • 10% (w/v) Triton X‐100
  • HIV‐1 p24 ELISA kit (anti‐p24‐coated plates from the AIDS Vaccine Program, SAIC, Frederick, Maryland) or p24 ELISA kit (PerkinElmer)
  • Refrigerated centrifuge
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • 37°C incubator

Basic Protocol 4: HIV‐1 Cell‐Cell Transmission Assays

  Materials
  • Raji/DC‐SIGN cells
  • Sterile PBS without Ca2+ and Mg 2+ (CMF‐PBS; appendix 2A)
  • Inhibitors: anti‐DC‐SIGN or mannan, optional
  • Single‐cycle luciferase reporter HIV‐1 stocks (see protocol 3)
  • Cell culture medium (see recipe)
  • Hut/CCR5 target cells
  • Polybrene
  • RPMI‐1640 with 10% FBS
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • Refrigerated microcentrifuge (e.g., rotor model F241.5P in Beckman Coulter microcentrifuge, 22R centrifuge)
  • 37°C incubator
  • 24‐well tissue culture plates
  • Additional reagents and equipment for cell culture (unit 1.1)
CAUTION: Follow the biosafety protocol and use 10% bleach to inactivate HIV‐1 in the plates, tubes, plastic pipets, etc.
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Figures

Videos

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Internet Resources
  http://www.niaid.nih.gov/DAIDs/pdatguide/vmol.htm
  The Virology Manual for HIV Laboratories is freely available to download from is Website.
  https://www.aidsreagent.org/Index.cfm
  The Website of the NIH AIDS Research and Reference Reagent Program.
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