Intersubunit Disulfide Isomerization Controls Membrane Fusion of Human T-Cell Leukemia Virus Env

Abstract

Human T-cell leukemia virus (HTLV-1) Env carries a typical disulfide isomerization motif, C 225 XXC, in the C-terminal domain SU. Here we have tested whether this motif is used for isomerization of the intersubunit disulfide of Env and whether this rearrangement is required for membrane fusion. We introduced the C225A and C228A mutations into Env and found that the former but not the latter mutant matured into covalently linked SU-TM complexes in transfected cells. Next, we constructed a secreted Env ectodomain and showed that it underwent incubation-dependent intersubunit disulfide isomerization on target cells. However, the rearrangement was blocked by the C225A mutation, suggesting that C 225 carried the isomerization-active thiol. Still, it was possible to reduce the intersubunit disulfide of the native C225A ectodomain mutant with dithiothreitol (DTT). The importance of the CXXC-mediated disulfide isomerization for infection was studied using murine leukemia virus vectors pseudotyped with wild-type or C225A HTLV-1 Env. We found that the mutant Env blocked infection, but this could be rescued with DTT. The fusion activity was tested in a fusion-from-within assay using a coculture of rat XC target and transfected BHK-21 effector cells. We found that the mutation blocked polykaryon formation, but this could be reversed with DTT. Similar DTT-reversible inhibition of infection and fusion was observed when a membrane-impermeable alkylator was present during the infection/fusion incubation. We conclude that the fusion activity of HTLV-1 Env is controlled by an SU CXXC-mediated isomerization of the intersubunit disulfide. Thus, this extends the applicability of the isomerization model from gammaretroviruses to deltaretroviruses.Human T-cell leukemia virus 1 (HTLV-1), which belongs to the group of deltaretroviruses, matures by budding at the plasma membrane of an infected cell and enters into a new one by virus-cell membrane fusion (16,17,32,35). The membrane fusion protein or envelope protein (Env) of HTLV-1 is a trimer of a gp46-gp21 subunit pair (9,15,43). The fusion activity is carried by the transmembrane (TM) gp21 and the host cell receptor binding activity by the peripheral (surface [SU]) gp46. The two subunits are made as a precursor, gp62, in the rough endoplasmic reticulum of the infected cell (37). The gp62 becomes cleaved into the subunits when it passes through the Golgi complex on its way to budding sites at the cell surface (32). The cell receptor of HTLV-1 is the glucose transporter protein I, which gives the virus a very wide host range (31). The neurophilin-1 and heparan sulfate proteoglycans have also been shown to serve as components of a receptor complex and an attachment factor, respectively (13, 39). We are interested in the way by which the HTLV-1 Env is activated for membrane fusion.The HTLV-1 SU has been shown to have a modular organization (20). The first 160 residues are folded as a receptor binding N-terminal domain (RBD), which is linked by a 35-residue-long Pro-rich region (PRR) to a C...

Betaretroviral Envelope Subunits Are Noncovalently Associated and Restricted to the Mammalian Class

“…Retroviruses have evolved various mechanisms of SU-TM bonding and dissociation that accompany the fusion and entry steps (2,(6)(7)(8)(9)(10)(11). Among retroviruses with covalently bonded Env subunits, the interaction of SU and TM has been most extensively studied in murine leukemia virus (MLV), a gammaretrovirus.…”

Antibodies to the Envelope Glycoprotein of Human T Cell Leukemia Virus Type 1 Robustly Activate Cell-Mediated Cytotoxic Responses and Directly Neutralize Viral Infectivity at Multiple Steps of the Entry Process

“…Alkylation studies coupled with elegant cryo-electron microscopy (23,24,41,53) demonstrate that, upon receptor binding, the envelope protein of MLV undergoes a radical change in conformation. Receptor binding induces an isomerization and disruption of the SU-TM intersubunit thiol, which triggers the reorganization of the SU subunits to form an open ring-shaped structure.…”

“…Moreover, transfer of the RBD into a heterologous viral envelope confers upon the recipient virus the cellular tropism that is typical of HTLV-1 (32,37). By contrast, the C-terminal domain (CTD) is less well characterized, but accumulating evidence suggests that the CTD not only provides an important structural function in the envelope trimer, but is also involved in recognition of additional entry factors or coreceptors (38,39), in determining the differing tropisms of HTLV-1 and HTLV-2 (40), and in transducing the envelope activation signal upon receptor binding (23,24,41). To date, the contribution of these domains to recognition by Ab and to the sensitivity of HTLV-1 to Ab-mediated neutralization is incompletely understood.…”

“…The surface glycoprotein (SU) component of envelope mediates viral infection of cells by binding to the cellular entry factors heparan sulfate proteoglycans (19,20), glucose transporter 1 (Glut-1) (21), and neuropilin 1 (22). Subsequently, through a cascade of conformational changes in envelope that includes isomerization of a SU-transmembrane glycoprotein (TM) intersubunit disulfide (23,24), envelope catalyzes the fusion of the viral and cell membranes allowing entry of the virus into the cell. Neutralizing Abs (10)(11)(12)14) and synthetic inhibitory peptides block envelope-mediated entry (25)(26)(27)(28), suggesting that interfering with envelope function can prevent dissemination of viral infection.…”

Last 20 aa of the West Nile virus NS1′ protein are responsible for its retention in cells and the formation of unique heat-stable dimers

“…It has previously been suggested (M. Lobigs, personal communication) that NS1 may itself catalyse disulfide bond exchange. Viral proteins have been identified previously to contain the disulfide isomerization motif, CXXC (Li et al, 2008). NS1 contains a CXXC motif that is conserved for dengue virus, yellow fever virus and the JEV serogroup.…”