The aim of the study is to characterize the critical amino acid residues on the Zika virus envelope protein required for the host GRP78 protein interaction, which may be a potential therapeutic target to combat Zika virus. The Zika virus is typically spread in tropical and subtropical regions through the bite of an infected mosquito of the genus Aedes, specifically Aedes (A) aegypti and A. One of the receptors on cell membrane proteins of the host cell for the Zika virus is the GRP78 protein, which also plays an important role. roles in viral replication.
The finding of crucial amino acid residues on Zika envelope protein required for GRP78 binding will result in a better understanding of the function and regulation of the Zika virus towards host cells. The spread of the Zika virus is also a concern in Indonesia as Indonesia is a tropical country and an endemic area for dengue every year, and there is currently no vaccine available to protect against or a drug to prevent Zika virus infection. to deal with. Total protein lysate (Input) and the immunoprecipitated proteins (Output) were examined for the presence of ZIKV E, EGFP-tag-GRP78 and actin by western blot analysis.
The Zika virus is a flavivirus that is transmitted by mosquitoes, and is also transmitted through sexual contact, the placenta, and blood transfusions. The spread of the Zika virus is also a concern in Indonesia, as Indonesia is a tropical country and an endemic area for dengue fever every year. There is currently no vaccine to protect against Zika virus infection or medicine to treat it.
Understanding the function and regulation of the Zika virus in host cells will be improved by the identification of key amino acid residues on the Zika envelope protein.
INTRODUCTION
Description of Department
Product of the Host Institution/Company
PROJECT DESCRIPTION
- Project Background
- Scope of the Project
- Objectives/Aims
- Problem Formulation and Proposed Solution (Methodology)
- Quikchange site directed mutagenesis
- Transformation of mutated plasmid into E. coli DH5α
- Colony PCR screening
- Bacteria culture and plasmid preparation
- Restriction enzyme digestion
- Mammalian cell culture and cell seeding
- Mammalian cell transfection
- Co- immunoprecipitation (co-IP ) assay
- Western blot analysis
- Statistical analysis
However, we know that E protein binds to the substrate binding domain of GRP78, but its precise position and what amino acid residues it is remains unknown. The interaction domain between ZIKV E and GRP78 protein was estimated through molecular coupling and molecular dynamic simulations (Elfiky & Ibrahim., 2020). In this experiment, the amino acids at positions C308A, T309A and A311S on ZIKV E domain III served as the crucial binding residues to substrate the binding domain of the GRP78 protein.
This study aimed to characterize the critical amino acid residues on ZIKV envelope protein domain III required for host GRP78 protein interaction. The candidate amino acid residues on ZIKV E protein domain III for GRP78 interaction are shown in green, yellow, magenta and red color. A single amino acid mutation from each candidate will be introduced through QuikChange site-directed mutagenesis.
This project aims to study the interaction between ZIKV E and GRP78 protein and to identify which amino acid residues on ZIKV E protein are critical for binding to the host's GRP78 protein. Two amino acid positions at T309 and A311 on ZIKV E were selected to determine the effect of mutation on GRP78 binding. Site-directed mutagenesis is usually substituted with alanine because it is the smallest amino acid and inert and contains a methyl group as a side chain.
To detect the effects of mutation, substituting amino acids should use different amino acid properties. To characterize the critical amino acid residues on ZIKV coat protein domain III required for host-GRP78 protein interaction. Recently, study has shown that GRP78 protein acts as a receptor for ZIKV entry into A549 cells.
The interaction between ZIKV E and GRP78 protein has been consistently demonstrated by Royle and colleagues (Royle et al., 2020). In addition, the interaction domain between ZIKV E and GRP78 protein was predicted by molecular docking and molecular dynamics simulations. They proposed that the amino acids at C308, T309 and A311 on ZIKV E domain III were the critical binding residues to substrate the binding domain of GRP78 protein.
Then the bacterial cells were spread in the selected plate (LB agar supplemented with 100 μg/ml ampicillin) and incubated for 15-17 hours at 37°C to allow them to grow. Initially, the old media was discarded and cells were washed with the sterile 1X PBS (approximately 15 ml).
FINDINGS
Result
The wild-type pcDNA-ZIKV E-HA plasmid was used as a template to synthesize the T309A mutated pcDNA-ZIKV E-HA using quikchange site-directed mutagenesis. Each line of the gel showed a band, its amplified plasmids were cut and the size of ZIKV E-HA pcDNA is 5957 bps. The nucleotide sequences of all selected clones (C1, C4, C6 and C9) were aligned with the wild-type ZIKV E nucleotide sequence using CLUSTAL O (1.2.4) multiple sequence alignment.
The red mark indicated that it could introduce nucleotide change in the plasmid where all the selected clones were changed from "A" to "G". Amino acid sequences of all selected clones (C1, C4, C6 and C9) were aligned with wild-type ZIKV E amino acid sequence using CLUSTAL O(1.2.4) multiple sequence alignment. HEK23T/17 cells were co-transfected with pEGFP-GRP78 and wild type pcDNA3.1-ZIKV E-HA plasmid.
HEK23T/17 cells were transfected with pEGFP-empty or pEGFP-GRP78 (EGFP) and co-transfected with wild-type or mutated A311S and T309A pcDNA3.1-ZIKV E-HA plasmid. HEK293T/17 cells were transfected with pEGFP-GRP78 and co-transfected with either wild-type or A311S- or T309A-mutated pcDNA-ZIKV E-HA plasmid. The immunoprecipitated level of wild-type and mutant ZIKV E protein was normalized against their EGFP-GRP78 protein and shown as the co-immunoprecipitation ratio.
Wild-type and mutant ZIKV E proteins were specifically co-immunoprecipitated with EGFP-tag GRP78 protein (Figures 10-12). From results, mutated A311S and T309A ZIKV E protein co-immunoprecipitated with EGFP tag GRP78 protein at the same level as wild-type ZIKV E protein (Figure 13). To optimize PCR conditions for introducing mutation at position T309A pcDNA ZIKV E-HA plasmid of the different gradient of annealing temperature by PCR-based site-directed mutagenesis to see which condition has appropriate temperature for all reagents.
At the first nucleotide, all sequences are compared to the wild type, which can be seen as the same. The wild type of the template must be “A” and all clones submitted for sequencing must be “G”. It can introduce a nucleotide change into a plasmid where all selected clones have been changed from "A" to "G".
This is achieved by cloning GFP at the N- or C-terminus of the amino acid chain in frame with the target protein (Palmer & Freeman., 2004). Introduces a nucleotide change into a plasmid where all selected clones have been changed from "A" to "G". In addition to the success of the proposed Quikchange site-directed mutagenesis technique, there were some limitations during the experiment, such as sample loss during pipetting into the gel electrophoresis, contamination of the DNA ladder with an irregular sample and the wrong sample, and the absence of a control in the gel electrophoresis.
However, I managed to overcome all this difficulty as time passed along with the guidance and help of the staff.
