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Recombinant soluble CD4 (sCD4) activated fusion between effector cells expressing Env and target cells expressing coreceptor (CCR5 or CXCR4) but lacking CD4. sCD4-activated fusion was dose dependent, occurred comparably with two- and four-domain proteins, and demonstrated Env-coreceptor specificities parallel to those reported in conventional fusion and infectivity systems. Fusion activation occurred upon sCD4 preincubation and washing of the Env-expressing effector cells but not the coreceptor-bearing target cells, thereby demonstrating that sCD4 exerts its effects by acting on Env. These findings provide direct functional evidence for a sequential two-step model of Env-receptor interactions, whereby gp120 binds first to CD4 and becomes activated for subsequent functional interaction with coreceptor, leading to membrane fusion. We used the sCD4-activated system to explore neutralization by the anti-gp120 human monoclonal antibodies 17b and 48d. These antibodies reportedly bind conserved CD4-induced epitopes involved in coreceptor interactions but neutralize HIV-1 infection only weakly. We found that 17b and 48d had minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor but potently blocked sCD4-activated fusion with target cells expressing coreceptor alone. Both antibodies strongly inhibited sCD4-activated fusion by Envs from genetically diverse HIV-1 isolates. Thus, the sCD4-activated system reveals conserved Env-blocking epitopes that are masked in native Env and hence not readily detected by conventional systems.<\/jats:p>","DOI":"10.1128\/jvi.74.1.326-333.2000","type":"journal-article","created":{"date-parts":[[2009,4,23]],"date-time":"2009-04-23T21:08:16Z","timestamp":1240520896000},"page":"326-333","update-policy":"http:\/\/dx.doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":118,"title":["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"],"prefix":"10.1128","volume":"74","author":[{"given":"Karl","family":"Salzwedel","sequence":"first","affiliation":[{"name":"<!--label omitted: 1-->Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erica D.","family":"Smith","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Barna","family":"Dey","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Edward A.","family":"Berger","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.66.5.2934-2942.1992"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.272.5270.1955"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1097\/00002030-199301001-00006"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.2309120"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1006\/viro.1999.9779"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.72.3.2500-2504.1998"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.72.10.8264-8272.1998"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.immunol.17.1.657"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.92.19.9004"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.2144\/97236st07"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81430-0"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81313-6"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.66.6.3531-3537.1992"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1038\/381661a0"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.73.4.2752-2761.1999"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81314-8"},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1038\/381667a0"},{"key":"e_1_3_2_19_2","doi-asserted-by":"publisher","DOI":"10.1097\/00002030-199508000-00006"},{"key":"e_1_3_2_20_2","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.68.5.3015-3026.1994"},{"key":"e_1_3_2_21_2","first-page":"16.15.1","article-title":"Expression of proteins in mammalian cells using vaccinia viral vectors","volume":"2","author":"Earl P. 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