Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins

Abstract

Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways. T he specificity of retroviral vectors is initially conferred by the envelope (Env) protein present on the surface of virus particles. This Env protein is responsible for binding to a host cell receptor, thereby initiating virus entry. In gammaretroviruses, the Env protein is composed of a transmembrane protein (TM) and a surface protein (SU). For murine leukemia virus (MLV) and feline leukemia virus (FeLV) SUs, primary receptor binding is localized to the N-terminal half of SU (1, 2, 12). Critical residues for specificity are encoded within the N-terminal variable regions, VRA and VRB. Secondary receptor binding sites that promote viral infection have also been identified within the SU C terminus (3-5).FeLV Envs are classified as A, B, and C, originally based on interference assays (6, 7). The receptors for these three major subgroups have been identified as THTR1 (8), PiT1 (9), and FLVCR1 (10, 11), respectively, molecularly confirming the distinct receptor usage for each subgroup. For FeLV-A, 19 residues in the VRA can be replaced with 16 residues of FeLV-C VRA to sufficiently alter the binding specificity, thus switching receptor usage (12). In FeLV-A, the VRA and VRB each include a pair of cysteines, which have the potential to form intramolecular disulfide bonds (5). The cysteine contents of MLV and FeLV-B SUs are more complex; ecotropic MLV ...

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model

“…Cultures were maintained for at least 14 days prior to analysis. [88]. Viral supernatant was collected, filtered through a 0.45 μm syringe and viral particles were quantified using ELISA against the MLV p30 [86].…”

Direct mass spectrometric characterization of disulfide linkages

“…However, the reduction of disulfide linkages does not always lead to a shift of retention time; thus, the potential location of disulfide linkages could not be confirmed by CID MS 2 product spectra without the presence of a new peak in the reduced peptide mapping. For example, in the case of internally disulfide-linked peptides, the disulfide-linked and disulfide-dissociated peptides elute closely, 44 and the mass shift in MS 1 peak of a possible “disulfide-linked” peptide can be misinterpreted as the isotopic distribution. Therefore, it is hard to conclude whether there is an internal disulfide linkage based on the LC traces and CID MS 2 data.…”