Truncation of the Cytoplasmic Domain Induces Exposure of Conserved Regions in the Ectodomain of Human Immunodeficiency Virus Type 1 Envelope Protein

Abstract

We have described a CD4-independent variant of HXBc2, termed 8x, that binds directly to CXCR4 and mediates CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4-C4 domains together with a single nucleotide deletion in the transmembrane domain which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of 27 amino acids. We demonstrate here that while introduction of the 8x FS mutation into heterologous R5, X4, or R5X4 Env proteins did not impart CD4 independence, it did affect the conformation of the gp120 surface subunit, exposing highly conserved domains involved in both coreceptor and CD4 binding. In addition, antigenic changes in the gp41 ectodomain were also observed, consistent with the idea that the effects of cytoplasmic domain truncation must in some way be transmitted to the external gp120 subunit. Truncation of gp41 also resulted in the marked neutralization sensitivity of all Env proteins tested to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may prove useful for vaccine development.The human immunodeficiency virus (HIV) envelope protein is a trimeric type I integral membrane protein in which each monomer consists of a heavily glycosylated surface subunit (gp120) noncovalently associated with a transmembrane (TM) domain subunit (gp41) (reviewed 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 in gp120 that is important for coreceptor binding (33,46,56,60). Binding to a coreceptor, most often the CCR5 or CXCR4 chemokine receptor (reviewed 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 (reviewed in reference 4), the membrane-spanning gp41 subunit plays a critical role in the actual membrane fusion process (reviewed in references 9 and 62). The gp41 subunit contains at its N terminus a hydrophobic fusion peptide that is thought to insert into the membrane of the cell, thus linking the cellular membrane with that of the virus. A peculiar feature of gp41 is its unusually long cytoplasmic domain, ...

Photoinduced Reactivity of the HIV-1 Envelope Glycoprotein with a Membrane-Embedded Probe Reveals Insertion of Portions of the HIV-1 Gp41 Cytoplasmic Tail into the Viral Membrane

“…During the circulation of the virus, the length of the tail has been correlated to a differential exposure of the virus to neutralizing antibodies (41). At the stage of entry, the cytoplasmic tail has also been implicated in regulating the kinetics of fusion and in the ability of the Env to promote syncytia (42,43).…”

Surface Exposure of the HIV-1 Env Cytoplasmic Tail LLP2 Domain during the Membrane Fusion Process

“…Thus, these domains may possibly interact with biological membranes, taking part in fusion reactivity (6, 16, 36 -40). (iii) Accumulated data indicate that gp41 CT LLP regions, which can interact with phospholipid membranes, may be involved in modulating cell-cell fusion by certain as-yet-undefined pathways (10,15,(17)(18)(19)(20)(21). It has been widely recognized that Gag matrix proteins mediate a mechanism of inside-out regulation of HIV-1 Env fusogenicity (18,41,42).…”

“…Recent studies have shown that the gp41 CTs of HIV and simian immunodeficiency virus correlate with their infectivity and Env-mediated cell-cell fusion (10,15). The mutations or truncations of gp41 CT can affect HIV-1 Env-mediated cell-cell fusion, presumably because of modulation of the fusogenicity of Env (17,18). Kalia et al (15) discovered that one Env having a mutation in the gp41 CT LLP2 region (MX2) exhibited a decrease of about 90% in fusogenicity compared with the wildtype Env and that the mutation did not correspond to apparent defects in the levels of cell surface Env expression.…”

Maturation cleavage within the ectodomain of Lassa virus glycoprotein relies on stabilization by the cytoplasmic tail

“…Many examples in the literature describe the cytoplasmic domain as important for the function of the ectodomains of glycoproteins in a variety of virus families [16][17][18][19][20]. Studies examining conformation dependent binding of monoclonal antibodies showed that alterations in the cytoplasmic domain led to structural changes in the ectodomain [21,22]. Moreover, our data highlight that the cytoplasmic domain is an important factor for a correct quaternary structure, which subsequently is required for proper proteolytic processing and function of the viral glycoprotein.…”