Resistance to Neutralization by Antibodies Targeting the Coiled Coil of Fusion-active Envelope Is a Common Feature of Retroviruses

Abstract

The human T-cell leukemia virus transmembrane glycoprotein (TM) is a typical class 1 membrane fusion protein and a subunit of the viral envelope glycoprotein complex. Following activation, the TM undergoes conformational transitions from a native nonfusogenic state to a fusion-active pre-hairpin intermediate that subsequently resolves to a compact trimer-of-hairpins or six-helix bundle. Disruption of these structural transitions inhibits membrane fusion and viral entry and validates TM as an anti-viral and vaccine target. To investigate the immunological properties of fusion-active TM, we have generated a panel of monoclonal antibodies that recognize the coiled-coil domain of the pre-hairpin intermediate. Antibody reactivity is highly sensitive to the conformation of the coiled coil as binding is dramatically reduced or lost on denatured antigen. Moreover, a unique group of antibodies are 100 -1000-fold more reactive with the coiled coil than the trimer-of-hairpins form of TM. The antibodies recognize virally expressed envelope, and significantly, some selectively bind to envelope only under conditions that promote membrane fusion. Most importantly, many of the antibodies potently block six-helix bundle formation in vitro. Nevertheless, viral envelope was remarkably resistant to neutralization by antibodies directed to the coiled coil. The data imply that the coiled coil of viral envelope is poorly exposed to antibody during membrane fusion. We suggest that resistance to neutralization by antibodies directed to fusion-associated structures is a common property of retroviral TM and perhaps of other viral class I fusion proteins. These observations have significant implications for vaccine design.

Charge-Surrounded Pockets and Electrostatic Interactions with Small Ions Modulate the Activity of Retroviral Fusion Proteins

“…Expression of the MBP fusion protein was carried out as previously described [14,49]. Briefly, Escherichia coli BL21(DE3) cells transformed with the MBP-BLV-hairpin vector were grown at 37uC with vigorous shaking in LB supplemented with 10 mM glucose and 100 mgml 21 ampicillin until an optical density at 600 nm of ,0.6 was reached.…”

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

“…Despite the ability of anti-TM mAbs to block binding of leash and helical region-derived peptides to the coiled coil in vitro, to recognize cell surface displayed viral envelope, and to target a region critical to the membrane fusion process, these Abs unanimously failed to inhibit either envelope-mediated membrane fusion or viral entry into cells (29,30,52). By contrast, most of the mAbs targeted to SU exhibit some level of neutralizing activity.…”

“…The neutralizing properties of the HTLV-1 SU-specific mAbs contrast strikingly with the activity of mAbs previously raised against the fusion-associated structures of TM (29,30,52). Despite the ability of anti-TM mAbs to block binding of leash and helical region-derived peptides to the coiled coil in vitro, to recognize cell surface displayed viral envelope, and to target a region critical to the membrane fusion process, these Abs unanimously failed to inhibit either envelope-mediated membrane fusion or viral entry into cells (29,30,52).…”

“…For HTLV-1, it remains to be determined whether binding of an Ab to the envelope complex is compatible with envelope function, or whether Ab must be bound to a restricted set of critical epitopes for neutralization to occur. Previously, we have provided evidence that nonfunctional forms of envelope exist on the surface of infected cells and that fusion-incompetent forms of envelope may account for the binding of nonneutralizing Ab to envelope-expressing cells (29,30). The ability of nonneutralizing or poorly neutralizing Ab to bind to virus particles may reflect that nonfunctional forms of envelope are also found on virions.…”

“…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. Although envelope-specific neutralizing Abs are frequently observed in natural infections, Abs specifically targeted to a functionally critical region of fusion-active envelope fail to antagonize viral entry into cells (29,30), emphasizing that the Biomedical Research Institute, College of Medicine Dentistry and Nursing, Ninewells Hospital, The University of Dundee, Dundee DD1 9SY, Scotland geometry of native viral envelope and the mode of interaction with Abs have a profound impact on the ability of Abs to neutralize virus infectivity. Understanding the immunogenic properties of HTLV-1 envelope is therefore a critical issue for the development of effective vaccines and immunological treatments to combat HTLV-1 infection.…”