Molecular Dynamics Simulation Reveals Exposed Residues in the Ligand-Binding Domain of the Low-Density Lipoprotein Receptor that Interacts with Vesicular Stomatitis Virus-G Envelope

Abstract

Familial hypercholesterolemia (FH) is an autosomal dominant disease most often caused by mutations in the low-density lipoprotein receptor (LDLR) gene, which consists of 18 exons spanning 45 kb and codes for a precursor protein of 860 amino acids. Mutations in the LDLR gene lead to a reduced hepatic clearance of LDL as well as a high risk of coronary artery disease (CAD) and sudden cardiac death (SCD). Recently, LDLR transgenes have generated interest as potential therapeutic agents. However, LDLR packaging using a lentiviral vector (LVV) system pseudotyped with a vesicular stomatitis virus (VSV)-G envelope is not efficient. In this study, we modified the LVV system to improve transduction efficiency and investigated the LDLR regions responsible for transduction inhibition. Transduction efficiency of 293T cells with a 5′-LDLReGFP-3′ fusion construct was only 1.55% compared to 42.32% for the eGFP construct. Moreover, co-expression of LDLR affected eGFP packaging. To determine the specific region of the LDLR protein responsible for packaging inhibition, we designed constructs with mutations or sequential deletions at the 3′ and 5′ ends of LDLR cDNA. All constructs except one without the ligand-binding domain (LBD) (pWoLBD–eGFP) resulted in low transduction efficiency, despite successful packaging of viral RNA in the VSV envelope, as confirmed through RT-PCR. When we evaluated a direct interaction between LDLR and the VSV envelope glycoprotein using MD simulation and protein–protein interactions, we uncovered Val119, Thr120, Thr67, and Thr118 as exposed residues in the LDLR receptor that interact with the VSV protein. Together, our results suggest that the LBD of LDLR interacts with the VSV-G protein during viral packaging, which significantly reduces transduction efficiency.

Compound A Increases Cell Infiltration in Target Organs of Acute Graft-versus-Host Disease (aGVHD) in a Mouse Model

“…First strand cDNA was synthesized using 1 µg of total RNA (DNase-treated) and the Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C until use. All real-time PCRs were performed according to a standard protocol as described previously [ 39 , 56 ]. Quantitative real-time PCR was performed using the ABI PRISM ® 7900HT sequence detection system from Applied Biosystems (Foster City, CA, USA).…”

Strategies for Vaccination: Conventional Vaccine Approaches Versus New-Generation Strategies in Combination with Adjuvants

“…However, critical complications, such as leukemias in patients with SCID-X1 and myelodysplastic-like syndromes in patients with CGD [ 47 ], highlighted the limitations of the first generation of retroviral vectors. Subsequently, modified vectors with modest architectural changes and a more favorable profile than the first-generation retroviral vectors were developed [ 48 , 49 , 50 , 51 , 52 , 53 ]. These vectors are characterized by the deletion of the long terminal repeat (LTR) promoter region, which results in the self-inactivation (SIN) of the vector and good biosafety ( Figure 1 ).…”