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. 2015 Dec 30;90(6):2971-80.
doi: 10.1128/JVI.02544-15.

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

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Free PMC article

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

Leonardo Valdivieso-Torres et al. J Virol. .
Free PMC article

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.

Importance: Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env-host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional pair of cysteines within the randomized region have the highest transduction efficiencies. This emphasizes the importance of considering cysteine pairs in the design of new libraries. Furthermore, our data clearly indicate that these cysteines are essential for viral infectivity by presenting essential residues to the host cell receptor. These studies facilitate the screening of Env libraries for functional entry into target cells, allowing the identification of novel gammaretroviral Envs targeting alternative host cell receptors for gene and protein delivery.

Figures

FIG 1
FIG 1
Generation of bicistronic vectors for Env library screening. (A) Schematic of the various vector backbones (left) and their corresponding viral titers (neomycin CFU [NCFU]/milliliter). The internal SV40 promoter was replaced with the M1 mutant variant (27) or the MLV SA sequence. Arrows mark the transcriptional start sites. Positions of the WPRE are indicated. ANOVA and a post hoc Dunnet test using pRVL-CP as a control were used to calculate significance. **, P < 0.05; *, P < 0.1. Error bars indicate the SEMs for at least three independent experiments. Ψ, MLV packaging signal. (B) Positions of the viral long terminal repeat (LTR), splice donor (SD), splice acceptor (SA), packaging signal (Ψ), env, and WPRE elements are indicated. The arrow marks the transcriptional start site. The position of the randomized 11 amino acids (X11) within the VRA of the FeLV-A/C Env backbone is indicated.
FIG 2
FIG 2
Comparison of the Env variable regions A and B. (A) Localization of cysteines within the VRA and VRB of three closely related gammaretroviral Envs. (B) Primary sequence of the VRA from FeLV A and C with four Env library isolates. Constrained peptide (CP) Env was isolated from an Env library screen on Caki1 cells (22). Isolates identified with the highest transduction efficiencies using the pSD-Env-SA-neo-CWPRE vector (AII, BV2, and BVI) are highlighted in dark gray. Cysteines (superscripted) are numbered based on their order within the AII, BV2, and CP isolates. Cysteines within the randomized regions of VRA are underlined.
FIG 3
FIG 3
Defining the host range of virus bearing the AII and BV2 Env. A total of 2 × 105 cells were infected with viral supernatant containing the corresponding Env proteins and packaging the lacZ gene. Viral titers are LacZ staining units/milliliter of virus. Individual cell lines are indicated, with the color key at the top. Error bars indicate the SEMs for at least three independent experiments.
FIG 4
FIG 4
AII Env mutagenesis studies identify residues critical for viral titers. (A) HEK293T cells were exposed to viral particles carrying the respective Env mutant protein and quantified for lacZ gene transfer, normalized to 200 ng of capsid. Error bars indicate the SEMs for at least three independent experiments. Asterisks indicate P values of <0.05 compared to the value for the parental AII. (B) Western blot analysis of virus-associated SU. Viral supernatant was collected and lanes were normalized to analyze 100 ng of CA. Env was detected using C11D8 mouse anti-GP70 (AbD Serotec; 1:1,000 dilution). (C) Western blot analysis of whole-cell extracts. SU was detected using C11D8 mouse anti-GP70 (AbD Serotec; 1:1,000 dilution). Positions of protein size markers are indicated on the left in panels B and C.
FIG 5
FIG 5
BV2 mutagenesis studies revealed that the pair of cysteines and residues in between are critical for viral titers. (A) Viral titers (LacZ units/200 ng of CA) were determined as for Fig. 4. (B) Western blot analysis of virus-associated SU. Viral samples were normalized to 100 ng of CA. Env was detected using C11D8 mouse anti-GP70 (AbD Serotec; 1:1,000 dilution).
FIG 6
FIG 6
CP-SU1-235 monomer has binding and interference activity and does not block AII and BV2 infection. (A) Schematic of the vector pJG expressing CP-SU1-235. The black box in the LTR indicates SIN vector. Individual components are as indicated. (B) SU binding studies on HEK293T cells. Secreted CP-SU1-235 from HEK293T (1 ml of medium, red line) and CP viral particles (blue line) produced in TELCeB6 (1 ml) was incubated with HEK293T (control green line), and binding was verified by flow cytometry as described on the methodology. (C) CP-SU1-235 interference assay, with comparison of infection of 2 × 105 HEK293T cells or HEK293T cells constitutively producing CP-SU1-235. LacZ staining units/milliliter of virus were determined for virus expressing CP, AII, or BV2 Env, as indicated. Error bars indicate the SEMs for at least three independent experiments.
FIG 7
FIG 7
Identification of CP-SU1-235 cysteine bonds using mass spectrometry. (A to D) Identification of the C2-C3 bond; (E to H) identification of the C3-C4 bond. (A and E) Analysis of products in the presence and absence of DTT); (B and F) schematic of the peptide structure corresponding to the identified products; (C and G) fragmentation pattern of the identified peptides containing a disulfide bond; (D and H) assignment of the experimental mass of peaks in panels C and G with the y and b fragment ions, defined in panels B and F. M, full peptide mass.
FIG 8
FIG 8
Summary of disulfide bonds and mutagenesis studies. Critical amino acids required for viral entry are indicated in red for each isolate. Cysteines that form intramolecular bonds are indicated via bars (solid and dashed). For AII, the linkage is based on functional substitution of a putative salt bridge. For CP, disulfide bonds were identified by mass spectrometry.

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