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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
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...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893590/ https://www.ncbi.nlm.nih.gov/pubmed/31731579 http://dx.doi.org/10.3390/v11111063 |
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author | Al-Allaf, Faisal A. Abduljaleel, Zainularifeen Taher, Mohiuddin M. Abdellatif, Ahmed A. H. Athar, Mohammad Bogari, Neda M. Al-Ahdal, Mohammed N. Al-Mohanna, Futwan Al-Hassnan, Zuhair N. Alzabeedi, Kamal H. Y. Banssir, Talib M. Bouazzaoui, Abdellatif |
author_facet | Al-Allaf, Faisal A. Abduljaleel, Zainularifeen Taher, Mohiuddin M. Abdellatif, Ahmed A. H. Athar, Mohammad Bogari, Neda M. Al-Ahdal, Mohammed N. Al-Mohanna, Futwan Al-Hassnan, Zuhair N. Alzabeedi, Kamal H. Y. Banssir, Talib M. Bouazzaoui, Abdellatif |
author_sort | Al-Allaf, Faisal A. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6893590 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-68935902019-12-23 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 Al-Allaf, Faisal A. Abduljaleel, Zainularifeen Taher, Mohiuddin M. Abdellatif, Ahmed A. H. Athar, Mohammad Bogari, Neda M. Al-Ahdal, Mohammed N. Al-Mohanna, Futwan Al-Hassnan, Zuhair N. Alzabeedi, Kamal H. Y. Banssir, Talib M. Bouazzaoui, Abdellatif Viruses Article 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. MDPI 2019-11-15 /pmc/articles/PMC6893590/ /pubmed/31731579 http://dx.doi.org/10.3390/v11111063 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Al-Allaf, Faisal A. Abduljaleel, Zainularifeen Taher, Mohiuddin M. Abdellatif, Ahmed A. H. Athar, Mohammad Bogari, Neda M. Al-Ahdal, Mohammed N. Al-Mohanna, Futwan Al-Hassnan, Zuhair N. Alzabeedi, Kamal H. Y. Banssir, Talib M. Bouazzaoui, Abdellatif 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 |
title | 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 |
title_full | 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 |
title_fullStr | 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 |
title_full_unstemmed | 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 |
title_short | 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 |
title_sort | 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 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893590/ https://www.ncbi.nlm.nih.gov/pubmed/31731579 http://dx.doi.org/10.3390/v11111063 |
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