Amara, F
Amara, F. of cytoplasmic domain truncation must in a few true way be transmitted towards the external gp120 subunit. Truncation of gp41 also led to the proclaimed neutralization sensitivity of most Env proteins examined to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 Necrostatin-1 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain name of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain name is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may show useful for vaccine development. The human immunodeficiency computer virus (HIV) envelope protein is a trimeric type I integral membrane protein in which each monomer consists of a greatly glycosylated surface subunit (gp120) noncovalently associated with a transmembrane (TM) domain name subunit (gp41) (examined in reference 62). The gp120 subunit contains highly conserved domains involved in CD4 and coreceptor binding (46). However, parts of these domains, particularly the bridging sheet, are poorly immunogenic, due in part to shielding by N-linked carbohydrate structures, the V3 loop, and the V1-V2 region (61). For its membrane fusion potential to be realized, Env must first bind CD4, which induces the exposure or formation of a highly conserved domain name in gp120 that is important for coreceptor binding (33, 46, 56, 60). Binding to a coreceptor, most often the CCR5 or CXCR4 chemokine receptor (examined in reference 13), triggers the final conformational changes in Env that ultimately result in fusion between the viral and cellular membranes. While the gp120 subunit mediates binding to cell surface receptors as well as attachment factors, such as DC-SIGN (examined in reference 4), the membrane-spanning gp41 subunit plays a critical role in the actual membrane fusion process (examined in recommendations 9 and 62). The gp41 subunit contains at its N terminus a hydrophobic fusion peptide that is thought to place into the membrane of the cell, thus linking the cellular membrane with Necrostatin-1 that of the computer virus. A peculiar feature of gp41 is usually its unusually long cytoplasmic domain name, typically about 150 amino acids. Truncation of the cytoplasmic domain name in vitro has been associated with enhanced fusion activity but with reduced viral infectivity (19, 35, 36, 40). In vitro passage Necrostatin-1 of SIVmac in certain human cell types has been shown to select for variants with truncated cytoplasmic domains, although the nature of the producing growth advantage remains unclear (27). Interestingly, these truncated cytoplasmic domains rapidly revert in infected animals, suggesting that a long cytoplasmic domain name confers an advantage to the computer virus in vivo (24, 27). We have described a CD4-impartial variant of HXBc2, termed 8x, which mediates CD4-impartial, CXCR4-dependent contamination (25, 31) as a consequence of specific mutations in the V3 and V4-C4 domains of gp120 coupled with a frameshift (FS) mutation at position 706 which results in a truncated cytoplasmic domain name of 27 amino acids (18). The CD4-impartial phenotype is usually correlated with increased exposure of the conserved coreceptor-binding site. We found that introduction of the FS into the parental HXB2c Env protein increased exposure of the coreceptor-binding site but was not sufficient to impart CD4 independence (18). To further investigate potential functions related to the cytoplasmic domain name of the HIV type 1 (HIV-1) Env protein, we placed 8x FS in several main and laboratory-adapted R5, X4, and R5X4 HIV-1 strains. We found that truncation of the cytoplasmic domain name invariably resulted in enhanced exposure of the highly conserved bridging sheet region in gp120 that is important for CCR5 binding (46). Introduction of the FS mutation also enhanced the binding of antibodies to the CD4-binding site in gp120 and to an immunodominant epitope in the ectodomain of gp41. The FS mutation experienced little effect on the binding of antibodies to the V1-V2 region, the V3 loop, or the C5 domain name of gp120. Finally, truncation of the cytoplasmic domain name in multiple Env proteins rendered them sensitive to neutralization by HIV-positive human sera. These results indicate that truncation of the cytoplasmic domain name of HIV-1 Env directly affects the conformation of the ectodomain of the protein and provides a way to enhance the exposure of neutralization determinants in gp120 that may be useful for Dcc Env-based immunogens. MATERIALS AND METHODS Antibodies. The following reagents were obtained through the AIDS Research and Reference Reagent Program, division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health: HIV-1 gp120 monoclonal antibodies (MAbs) F105, donated by Marshall Posner (7, 41-43), 2G12, donated by Hermann Katinger (57), 48d and 17b, donated by James Robinson (34, 55), and b12 (immunoglobulin G1.