Vaccinia‐Based Reporter Gene Cell‐Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors

Barna Dey1, Edward A. Berger1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.10
DOI:  10.1002/0471142735.im1210s54
Online Posting Date:  May, 2003
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Abstract

This unit describes quantitation of functional interactions between HIV envelope glcoprotein and target cell receptors, using assay of cell fusion‐dependent reporter gene activation. The method is particularly useful since it isolates the fusion reaction from the rest of the HIV replication cycle, and obviates the need for infectious HIV particles.Reporter Gene Cell Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors

     
 
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Table of Contents

  • Basic Protocol 1: Fusion Assay Using Plasmids to Express ENV and Receptors
  • Alternate Protocol 1: Fusion Assay Using Recombinant Viruses for All Components
  • Alternate Protocol 2: SCD4‐Activated Fusion Assay
  • Support Protocol 1: Colorimetric Measurement of Fusion‐Dependent Reporter Gene Activation
  • Support Protocol 2: In Situ Staining to Measure Activation
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fusion Assay Using Plasmids to Express ENV and Receptors

  Materials
  • Cells (see )
  • recipeEMEM‐2.5 and ‐10 (see recipe)
  • Plasmids with the following elements (Table 12.10.2):
    • Env gene under vaccinia‐promoter control
    • CD4 under vaccinia‐promoter control
    • Coreceptor (e.g. CCR5, CXCR4) under vaccinia‐promoter control
    • T7 RNA polymerase under vaccinia‐promoter control
    • Reporter gene (E. coli LacZ) under T7‐promoter control
    • Negative control Env (Unc)
  • Transfection buffer: 20 mM HEPES, pH 7.4 (store up to several weeks at 4°C)
  • DOTAP transfection reagent (1,2‐dioleoyl‐3‐trimethylammonium propane; Boehringer Mannheim)
  • 0.25% (w/v) trypsin/0.02% (w/v) EDTA in HEPES buffer without calcium and magnesium (Quality Biochemicals)
  • Purified or crude vaccinia virus, wild type and/or recombinant
  • 25‐cm2 tissue culture flasks
  • 12 × 75–mm polystyrene tubes
  • 50‐ml conical polypropylene tubes
  • Cup sonicator
  • Humidified 31°C, 5% CO 2 tissue culture incubator
  • 96‐well flat‐bottom tissue culture plates (Costar)
  • Additional reagents and equipment for counting cells ( appendix 3A) and measuring fusion‐dependent reporter gene activation (see Support Protocols protocol 41 and protocol 52)
    Table 2.0.2   MaterialsVaccinia recombinants used in HIV Env‐mediated cell fusion assays

    Expressed protein Vaccinia recombinant Promoter Strength a Replication stage b Comment Reference
    HIV Envs
    LAV vCB‐41 Vaccinia synthetic High Early and late CXCR4 using Broder and Berger ( )
    Ba‐L vCB‐43 Vaccinia synthetic High Early and late CCR5 using Broder and Berger ( )
    89.6 MVA/89.6 Vaccinia modified H5 High Early and late CCR5 and CXCR4 using Belyakov et al. ( )
    Unc vCB‐16 Vaccinia synthetic High Early and late Uncleaveable, nonfusogenic control Broder and Berger ( )
    Receptors
    CD4 vCB‐3 Vaccinia synthetic High Early and late Primary receptor Broder et. al. ( )
    CXCR4 vCBYF1‐fusin Vaccinia synthetic High Early and late Coreceptor Feng et. al. ( )
    CCR5 vvCCR5‐1107 Vaccinia 7.5 K Moderate Early and late Coreceptor Xiao et. al. ( )
    Reporter gene activation read out
    T7 RNA vTF7‐3 Vaccinia 7.5 K Moderate Early and late Fuerst et. al. ( )
    Polymerase vP11T7gene1 Vaccinia 11K Moderate Early and late Alexander et. al. ( )
    β‐gal vCB21R‐LacZ T7 High Late LacZ gene linked to T7 promoter Alkhatib et. al. ( )

     aStrength refers to expression level.
     bStage of vaccinia virus life cycle during which the promoter is active.

Alternate Protocol 1: Fusion Assay Using Recombinant Viruses for All Components

  • sCD4, purified (NIH AIDS Research and Reference Reagent Program)
NOTE: Typically, four‐domain sCD4 (i.e., amino acids 1 to 369) is used, but other forms (e.g., two domain) are also acceptable.

Alternate Protocol 2: SCD4‐Activated Fusion Assay

  • Cell mixtures following fusion reactions (see protocol 1 or Alternate Protocols protocol 21 and protocol 32)
  • 10% (v/v) NP‐40
  • recipeEMEM‐2.5 (see recipe)/0.5% (v/v) NP‐40
  • 1000 U/ml β‐galactosidase standard stock solution (Boehringer Mannheim; optional): store 50‐µl aliquots at −20°C
  • recipe10× CPRG (see recipe)
  • Count‐up timer
  • Microplate absorbance reader equipped with 570‐nm filter

Support Protocol 1: Colorimetric Measurement of Fusion‐Dependent Reporter Gene Activation

  Materials
  • recipeStaining buffer (see recipe)
  • recipe40 mg/ml Xgal (see recipe)
  • Cell fusion (see protocol 1 or Alternate Protocols protocol 21 and protocol 32)
  • recipe10× fixative solution (see recipe)
  • Additional reagents and equipment for microscopy (unit 21.1)
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Figures

Videos

Literature Cited

Literature Cited
   Alexander, W.A., Moss, B., and Fuerst, T.R. 1992. Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor. J. Virol. 66:2934‐2942.
   Alkhatib, G., Broder, C.C., and Berger, E.A. 1996a. Cell type‐specific fusion cofactors determine human immunodeficiency virus type 1 tropism for T‐cell lines versus primary macrophages. J. Virol. 70:5487‐5494.
   Alkhatib, G., Combadiere, C., Broder, C.C., Feng, Y., Kennedy, P.E., Murphy, P.M., and Berger, E.A. 1996b. CC CKR5: A RANTES, MIP‐1α, MIP‐1β receptor as a fusion cofactor for macrophage‐tropic HIV‐1. Science 272:1955‐1958.
   Alkhatib, G., Liao, F., Berger, E.A., Farber, J.M., and Peden, K.W.C. 1997. A new SIV co‐receptor, STRL33. Nature 388:238.
   AstierGin, T., Portail, J.P., LondosGagliardi, D., Moynet, D., Blanchard, S., Dalibart, R., Pouliquen, J.F., GeorgesCourbot, M.C., Hajjar, C., SainteFoie, S., and Guillemain, B. 1997. Neutralizing activity and antibody reactivity toward immunogenic regions of the human T cell leukemia virus type I surface glycoprotein in sera of infected patients with different clinical states. J. Infect. Dis. 175:716‐719.
   Bagai, S. and Lamb, R.A. 1995. Quantitative measurement of paramyxovirus fusion‐differences in requirements of glycoproteins between simian‐virus‐5 and human parainfluenza‐virus‐3 or Newcastle‐disease virus. J. Virol. 69:6712‐6719.
   Belyakov, I.M., Wyatt, L.S., Ahlers, J.D., Earl, P., Pendleton, C.D., Kelsall, B.L., Strober, W., Moss, B., and Berzofsky, J.A. 1998. Induction of mucosal CTL response by intrarectal immunization with a replication‐deficient recombinant vaccinia virus expressing HIV 89.6 envelope protein. J. Virol. 72:8264‐8272.
   Berger, E.A., Murphy, P.M., and Farber, J.M. 1999. Chemokine receptors as HIV‐1 coreceptors: Roles in viral entry, tropism, and disease. Annu. Rev. Immunol. 17:657‐700.
   Broder, C.C. and Berger, E.A. 1995. Fusogenic selectivity of the envelope glycoprotein is a major determinant of human immunodeficiency virus type 1 tropism for CD4+ T‐cell lines vs. primary macrophages. Proc. Natl. Acad. Sci. U.S.A. 92:9004‐9008.
   Broder, C.C., Dimitrov, D.S., Blumenthal, R., and Berger, E.A. 1993. The block to HIV‐1 envelope glycoprotein‐mediated membrane fusion in animal cells expressing human CD4 can be overcome by a human cell component(s). Virol. 193:483‐491.
   Broder, C.C., Nussbaum, O., Gutheil, W.G., Bachovchin, W.W., and Berger, E.A. 1994a. CD26 antigen and HIV fusion? Science 264:1156‐1159.
   Broder, C.C., Kennedy, P.E., Michaels, F., and Berger, E.A. 1994b. Expression of foreign genes in cultured human primary macrophages using recombinant vaccinia virus vectors. Gene 142:167‐174.
   Cleverley, D.Z. and Lenard, J. 1998. The transmembrane domain in viral fusion: Essential role for a conserved glycine residue in vesicular stomatitis virus G protein. Proc. Natl. Acad. Sci. U.S.A. 95:3425‐3430.
   Combadiere, C., Salzwedel, K., Smith, E.D., Tiffany, H.L., Berger, E.A., and Murphy, P.M. 1998. Identification of CX3CR1: A chemotactic receptor for the human CX3C chemokine fractalkine, and a fusion coreceptor for HIV‐1. J. Biol. Chem. 273:23799‐23804.
   Dalgleish, A.G., Beverley, P.C., Clapham, P.R., Crawford, D.H., Greaves, M.F., and Weiss, R.A. 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312:763‐767.
   Dey, B., Lerner, D.L., Lusso, P., Boyd, M.R., Elder, J.H., and Berger, E.A. 2000. Multiple antiviral activities of cyanovirin‐N: Blocking of human immunodeficiency virus type 1 gp120 interaction with CD4 and coreceptor and inhibition of diverse enveloped viruses. J. Virol. 74:4562‐4569.
   Dimitrov, D.S., Golding, H., and Blumenthal, R. 1991. Initial stages of HIV‐1 envelope glycoprotein‐mediated cell fusion monitored by a new assay based on redistribution of fluorescent dyes. AIDS Res. Hum. Retrovir. 7:799‐805.
   Doranz, B.J., Rucker, J., Yi, Y.J., Smyth, R.J., Samson, M., Peiper, S.C., Parmentier, M., Collman, R.G., and Doms, R.W. 1996. A dual‐tropic primary HIV‐1 isolate that uses fusin and the beta‐chemokine receptors CKR‐5, CKR‐3, and CKR‐2b as fusion cofactors. Cell 85:1149‐1158.
   Earl, P.L., Moss, B., Wyatt, L.S., and Caroll, M.W. 1998. Generation of Recombinant Vaccinia Viruses. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.17.1‐16.17.9. John Wiley & Sons, Hoboken, N.J.
   Feng, Y., Broder, C.C., Kennedy, P.E., and Berger, E.A. 1996. HIV‐1 entry cofactor: Functional cDNA cloning of a seven‐ transmembrane, G protein‐coupled receptor. Science 272:872‐877.
   Fuerst, T.R., Niles, E.G., Studier, F.W., and Moss, B. 1986. Eukaryotic transient‐expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83:8122‐8126.
   Gallagher, T.M. 1996. Murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein. J. Virol. 70:4683‐4690.
   Helseth, E., Kowalski, M., Gabuzda, D., Olshevsky, U., Haseltine, W., and Sodroski, J. 1990. Rapid complementation assays measuring replicative potential of human immunodeficiency virus type 1 envelope glycoprotein mutants. J. Virol. 64:2416‐2420.
   Hernandez, L.D. and White, J.M. 1998. Mutational analysis of the candidate internal fusion peptide of the avian leukosis and sarcoma virus subgroup A envelope glycoprotein. J. Virol. 72:3259‐3267.
   Kimpton, J. and Emerman, M. 1992. Detection of replication‐competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated β‐galactosidase gene. J. Virol. 66:2232‐2239.
   Konopka, K., Davis, B.R., Larsen, C.E., Alford, D.R., Debs, R.J., and Duzgunes, N. 1990. Liposomes modulate human‐immunodeficiency‐virus infectivity. J. Gen. Virol. 71:2899‐2907.
   Li, Q.X., Camerini, D., Xie, Y.M., Greenwald, M., Kuritzkes, D.R., and Chen, I.S.Y. 1996. Syncytium formation by recombinant HTLV‐II envelope glycoprotein. Virology 218:279‐284.
   Liao, F., Alkhatib, G., Peden, K.W.C., Sharma, G., Berger, E.A., and Farber, J.M. 1997. STRL33, a novel chemokine receptor‐like protein, functions as a fusion cofactor for both macrophage‐tropic and T cell line‐tropic HIV‐1. J. Exp. Med. 185:2015‐2023.
   Lifson, J.D., Feinberg, M.B., Reyes, G.R., Rabin, L., Banapour, B., Chakrabarti, S., Moss, B., Wong‐Staal, F., Steimer, K.S., and Engleman, E.G. 1986. Induction of CD4‐dependent cell fusion by the HTLV‐III/LAV envelope glycoprotein. Nature 323:725‐728.
   Litwin, V., Nagashima, K.A., Ryder, A.M., Chang, C.H., Carver, J.M., Olson, W.C., Alizon, M., Hasel, K.W., Maddon, P.J., and Allaway, G.P. 1996. Human immunodeficiency virus type 1 membrane fusion mediated by a laboratory‐adapted strain and a primary isolate analyzed by resonance energy transfer. J. Virol. 70:6437‐6441.
   Lyall, J.W., Solanky, N., and Tiley, L.S. 2000. Restricted species tropism of maedi‐visna virus strain EV‐1 is not due to limited receptor distribution. J. Gen. Virol. 81:2919‐2927.
   Moss, B., Earl, P.L., Cooper, N., Wyatt, L.S., Carroll, M.W., and Elroy‐Stein, O. 1998. Expression of protein in mammalian cells using vaccinia viral vectors. In Current Protocols in Molecular Biology, Vol.2, Chapter 16, Suppl. 43. (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.15.1‐16.19.11. John Wiley & Sons, Hoboken, N.J.
   Nussbaum, O., Broder, C.C., and Berger, E.A. 1994. Fusogenic mechanisms of enveloped‐virus glycoproteins analyzed by a novel recombinant vaccinia virus‐based assay quantitating cell fusion‐dependent reporter gene activation. J. Virol. 68:5411‐5422.
   Nussbaum, O., Broder, C.C., Moss, B., Stern, L.B.L., Rozenblatt, S., and Berger, E.A. 1995. Functional and structural interactions between measles‐virus hemagglutinin and CD46. J. Virol. 69:3341‐3349.
   Page, K.A., Landau, N.R., and Littman, D.R. 1990. Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity. J. Virol. 64:5270‐5276.
   Pastey, M.K. and Samal, S.K. 1997. Analysis of bovine respiratory syncytial virus envelope glycoproteins in cell fusion. J. Gen. Virol. 78:1885‐1889.
   Rich, E.A., Chen, I.S., Zack, J.A., Leonard, M.L., and O'Brien, W.A. 1992. Increased susceptibility of differentiated mononuclear phagocytes to productive infection with human immunodeficiency virus‐1 (HIV‐1). J. Clin. Invest. 89:176‐183.
   Rucker, J., Edinger, A.L., Sharron, M., Samson, M., Lee, B., Berson, J.F., Yi, Y., Margulies, B., Collman, R.G., Doranz, B.J., Parmentier, M., and Doms, R.W. 1997. Utilization of chemokine receptors, orphan receptors, and herpesvirus‐encoded receptors by diverse human and simian immunodeficiency viruses. J. Virol. 71:8999‐9007.
   Salzwedel, K. and Berger, E.A. 2000. Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV‐1: Functional complementation of specific defects in gp120 and gp41. Proc. Natl. Acad. Sci. U.S.A. 97:12794‐12799.
   Salzwedel, K., Smith, E.D., Dey, B., and Berger, E.A. 2000. Sequential CD4‐coreceptor interactions in human immunodeficiency virus type 1 Env function: Soluble CD4 activates Env for coreceptor‐dependent fusion and reveals blocking activities of antibodies against cryptic conserved epitopes on gp120. J. Virol. 74:326‐333.
   Santoro, F., Kennedy, P.E., Locatelli, G., Malnati, M.S., Berger, E.A., and Lusso, P. 1999. CD46 is a cellular receptor for human herpesvirus 6. Cell 99:817‐827.
   Takikawa, S., Ishii, K., Aizaki, H., Suzuki, T., Asakura, H., Matsuura, Y., and Miyamura, T. 2000. Cell fusion activity of hepatitis C virus envelope proteins. J. Virol. 74:5066‐5074.
   Tanabayashi, K. and Compans, R.W. 1996. Functional interaction of paramyxovirus glycoproteins: Identification of a domain in Sendai virus HN which promotes cell fusion. J. Virol. 70:6112‐6118.
   Willett, B.J., Picard, L., Hosie, M.J., Turner, J.D., Adema, K., and Clapham, P.R. 1997. Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses. J. Virol. 71:6407‐6415.
   Wyatt, R. and Sodroski, J. 1998. The HIV‐1 envelope glycoproteins: Fusogens, antigens, and immunogens. Science 280:1884‐1888.
   Xiao, X.D., Wu, L., Stantchev, T.S., Feng, Y.‐R., Ugolini, S., Chen, H., Shen, Z., Riley, J.L., Broder, C.C., Sattentau, Q.J., and Dimitrov, D.S. 1999. Constitutive cell surface association between CD4 and CCR5. Proc. Natl. Acad. Sci. U.S.A. 96:7496‐7501.
   Yang, C.L. and Compans, R.W. 1997. Analysis of the murine leukemia virus R peptide: Delineation of the molecular determinants which are important for its fusion inhibition activity. J. Virol. 71:8490‐8496.
   Yao, Q.H. and Compans, R.W. 1995. Differences in the role of the cytoplasmic domain of human parainfluenza virus fusion proteins. J. Virol. 69:7045‐7053.
   Zeira, M., Tosi, P.F., Mouneimne, Y., Lazarte, J., Sneed, L., Volsky, D.J., and Nicolau, C. 1991. Full‐length CD4 electroinserted in the erythrocyte membrane as a long‐lived inhibitor of infection by human immunodeficiency virus. Proc. Natl. Acad. Sci. U.S.A. 88:4409‐4413.
Key References
   Nussbaum et al., 1994. See above.
  Details on the principle of standard fusion assay.
   Salzwedel et al., 2000. See above.
  Details on the method and application of sCD4‐activated fusion assay.
   Moss et al., 1998. See above.
  For detailed description of the recombinant vaccinia virus technology.
Internet Resource
  http://www.aidsreagent.org
  Web site for NIH AIDS Research and Reference Reagent Program
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