Basic Residues Are Critical to the Activity of Peptide Inhibitors of Human T Cell Leukemia Virus Type 1 Entry

Abstract

A synthetic peptide based on the leash and alpha-helical region (LHR) of human T cell leukemia virus type 1 envelope is a potent inhibitor of viral entry into cells. The inhibitory peptide targets a triple-stranded coiled-coil motif of the fusion-active transmembrane glycoprotein and in a trans-dominant negative manner blocks resolution to the trimer-of-hairpins form. The LHR-mimetic is, therefore, functionally analogous to the C34/T20-type inhibitors of human immunodeficiency virus. Previous attempts to shorten the bioactive peptide produced peptides with severely attenuated activity. We now demonstrate that truncated peptides often suffer from poor solubility and impaired coiled coil binding properties, and we identify features that are critical to peptide function. In particular, the alpha-helical region of the LHR-mimetic is necessary but not sufficient for inhibitory activity. Moreover, two basic residues are crucial for coiled-coil binding and efficient inhibition of membrane fusion. By retaining these basic residues and a region of main chain peptide contacts with the coiled coil, a core LHR-mimetic was obtained that retains both the inhibitory properties and solubility profile of the parental peptide. Variants of the core peptide inhibit both membrane fusion and infection of cells by free viral particles, but unexpectedly, infection by virions was more susceptible to inhibition by low activity inhibitors than syncytium formation. The core inhibitor provides a valuable lead in the search for smaller more bio-available peptides and peptido-mimetics that possess anti-viral activity. Such molecules may be attractive candidates for therapeutic intervention in human T cell leukemia virus type 1 infections.

Cellular Factors Involved in HTLV-1 Entry and Pathogenicit

“…The dissociation of TM from the SU-TM complex results in the projection of FD into the cell membrane. This dissociation involves the formation of a six-helix coiled-coil bundle that induces membrane fusion (Maerz et al, 2000; Marsh and Helenius, 2006; Mirsaliotis et al, 2007; Lamb et al, 2009). …”

“…Four domains conserved in retroviral TM glycoproteins are also present in HTLV-1 TM (Kobe et al, 1999): (1) an amino-terminal hydrophobic domain with characteristics of a FD (Kim et al, 2000); (2) a leucine zipper-like region (LZR) with an amphipathic α-helical structure capable of self-association as a coiled-coil (Maerz et al, 2000; Pinon et al, 2003b; Mirsaliotis et al, 2007; Lamb et al, 2009); (3) a domain containing the conserved motif, which forms an intramolecular disulfide link; and (4) a FD linked to the coiled-coil core through a conserved sequence rich in glycine [glycine-rich segment (GRS), M(326) to S(337); Wilson et al, 2005; Figure 3]. …”

Molecular Aspects of HTLV-1 Entry: Functional Domains of the HTLV-1 Surface Subunit (SU) and Their Relationships to the Entry Receptors

“…The Ser residue at position 101 also controls SU/TM association [40] (Figure 3). Details of the mechanism of HTLV-1 gp41-mediated fusion have been described elsewhere [41,42]. …”

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

“…Syncytium interference assays were performed by standard methods (28,29,43,44). HeLa cells (2 3 10 5 ) transfected with the envelope expression vector pHTE-1 were added to untransfected HeLa target cells (8 3 10 5 ).…”

“…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. 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.…”