Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surfaceexposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Å resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1͞simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Hepatitis C virus (HCV) glycoproteins E1 and E2 are believed to be retained in the endoplasmic reticulum (ER) or cis-Golgi compartment via retention signals located in their transmembrane domains. Here we describe the detection of E1 and E2 at the surface of transiently transfected HEK 293T and Huh7 cells. Surface-localized E1E2 heterodimers presented exclusively as non-covalently associated complexes. Surface-expressed E2 contained trans-Golgi modi¢ed complex/hybrid type carbohydrate and migrated di¡usely between 70 and 90 kDa while intracellular E1 and E2 existed as high mannose 35 kDa and 70 kDa precursors, respectively. In addition, surface-localized E1E2 heterodimers were incorporated into E1E2-pseudotyped HIV-1 particles that were competent for entry into Huh7 cells. These studies suggest that functional HCV glycoproteins are not retained exclusively in the ER and transit through the secretory pathway.

CD4, a member of the immunoglobulin superfamily of receptors that mediates cell-cell interactions in the immune system, is the primary receptor for HIV-1. The extracellular portion of CD4 is a concatenation of four immunoglobulin-like domains, D1 to D4. The D1, D2 and D4 domains each contain a disulfide bond. We show here that the D2 disulfide bond is redox-active. The redox state of the thiols (disulfide versus dithiol) appeared to be regulated by thioredoxin, which is secreted by CD4(+) T cells. Locking the CD4 and the thioredoxin active-site dithiols in the reduced state with a hydrophilic trivalent arsenical blocked entry of HIV-1 into susceptible cells. These findings indicate that redox changes in CD4 D2 are important for HIV-1 entry and represent a new target for HIV-1 entry inhibitors.

The binding of hepatitis C virus glycoprotein E2 to the large extracellular loop (LEL) of CD81 has been shown to modulate human T-cell and NK cell activity in vitro. Using random mutagenesis of a chimera of maltose-binding protein and LEL residues 113 to 201, we have determined that the E2-binding site on CD81 comprises residues Ile 182 , Phe 186 , Asn 184 , and Leu 162 . These findings reveal an E2-binding surface of approximately 806 Å 2 and potential target sites for the development of small-molecule inhibitors of E2 binding.CD81 is a member of the tetraspanin membrane protein superfamily, characterized by the presence of four transmembrane domains, three intracellular loops, and two extracellular domains, which in the case of CD81 are referred to as the small extracellular loop and the large extracellular loop (LEL). Tetraspanins appear to have an essential function in organizing signaling molecules at the cell surface by associating with other tetraspanins, lineage-specific proteins, integrins, major histocompatibility complex molecules, and signaling proteins in a cell type-dependent manner (for a review, see reference 8). The recent elucidation of the CD81 LEL crystal structure revealed that it is a homodimer, with each monomer being composed of five ␣-helices (A to E) arranged in a head subdomain (consisting of the last two turns of the A helix, the B, C, and D helices, and their interconnecting loops) atop a stalk subdomain (comprising antiparallel A and E helices) (6). Four cysteine residues, conserved in all tetraspanin sequences, participate in the formation of two disulfide bonds, Cys and Cys 157 -Cys 175 , which stabilize the head subdomain. Hepatitis C virus (HCV) encodes a ϳ3,300-amino-acid polyprotein from which the E1 (polyprotein residues 191 to 383) and E2 (residues 384 to 746) glycoproteins are cleaved cotranslationally. The mature forms of E1 and E2 are noncovalently associated, and each contains an N-terminal ectodomain and a C-terminal transmembrane domain. Recently, it was shown that recombinant soluble E2, E1-E2 complex, and HCV-like particles (12, 14) as well as HCV particles from infectious plasma (14) bind to the CD81 LEL with nanomole-level affinity (13,14). Furthermore, the LEL can inhibit the binding of E2 to liver sections (13, 14) and of HCV-like particles to MOLT-4 T cells (21). The available evidence suggests that CD81 is unlikely to play a role in HCV entry (1,4,13,16,17,21,22). However, E2-CD81 ligation was recently found to induce in naive and antigen-experienced T cells in vitro a costimulatory signal leading to the production of the proinflammatory cytokine gamma interferon (20). As HCV-associated liver damage is primarily due to a massive infiltration by activated proinflammatory lymphocytes (for a review, see reference 19), these findings raise the possibility that the CD81-E2 interaction plays a role in T-cell-mediated liver inflammation and pathology.To further characterize the E2-binding residues on the LEL of CD81, we produced a protein chimera consisting of maltos...

The hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer that mediates CD81 receptor binding and viral entry. In this study, we used site-directed mutagenesis to examine the functional role of a conserved G 436 WLAGLFY motif of E2. The mutants could be placed into two groups based on the ability of mature virion-incorporated E1E2 to bind the large extracellular loop (LEL) of CD81 versus the ability to mediate cellular entry of pseudotyped retroviral particles. Group 1 comprised E2 mutants where LEL binding ability largely correlated with viral entry ability, with conservative and nonconservative substitutions (W437 L/A, L438A, L441V/F, and F442A) inhibiting both functions. These data suggest that Trp-437, Leu-438, Leu-441, and Phe-442 directly interact with the LEL. Group 2 comprised E2 glycoproteins with more conservative substitutions that lacked LEL binding but retained between 20% and 60% of wild-type viral entry competence. The viral entry competence displayed by group 2 mutants was explained by residual binding by the E2 receptor binding domain to cellular full-length CD81. A subset of mutants maintained LEL binding ability in the context of intracellular E1E2 forms, but this function was largely lost in virion-incorporated glycoproteins. These data suggest that the CD81 binding site undergoes a conformational transition during glycoprotein maturation through the secretory pathway. The G436P mutant was an outlier, retaining near-wild-type levels of CD81 binding but lacking significant viral entry ability. These findings indicate that the G 436 WLA GLFY motif of E2 functions in CD81 binding and in pre-or post-CD81-dependent stages of viral entry.Hepatitis C virus (HCV) is a member of the Flaviviridae family of small, enveloped plus-strand RNA viruses that has infected over 3% of the global human population, causing significant morbidity and mortality. Hepatitis C virus encodes two type I transmembrane glycoproteins, E1 and E2, which are cleaved from the viral polyprotein precursor by signal peptidases in the endoplasmic reticulum (ER). E1 and E2 form heterogeneous mixtures of covalently and noncovalently associated heterodimers that are largely retained in the ER via retention sequences located in their transmembrane domains (5,6,14). However, a small proportion of E1E2 heterodimer escapes the ER and matures through the secretory pathway (11). Retroviruses such as human immunodeficiency virus type 1 (HIV-1) can be pseudotyped with cell surface-expressed E1E2 (E1E2-pseudotyped particles [E1E2-pps]). E1E2-pps contain noncovalently associated E1E2 heterodimers and are capable of infecting primary human hepatocytes and various human liver cell lines including Huh7 cells (2,11,23). Viral entry of E1E2-pps and cell culture-grown HCV occurs via receptor-mediated endocytosis, the E1E2 glycoproteins mediating low-pH-dependent fusion (1, 2, 23, 24, 40).The E2 glycoprotein mediates binding to cellular receptors, including the tetraspanin CD81 (34) and the high-density lipoprotein receptor scavenger recep...