Study on Sequence Mutation and Molecular Dynamics Simulation of D614G SARS-CoV-2 Spike Protein in Indonesia

doi:10.21203/rs.3.rs-2454770/v1

Amino acid mutations in the D614G spike protein of SARS-CoV-2 need to be studied more deeply for their influence on the pathogenesis of the virus. The purpose of this research was to analyze 1) the variation and kinship of the SARS-CoV-2 spike protein gene; 2) the effect of D614G spike protein mutation on ACE2 receptor binding and the molecular dynamics simulation. The nucleotide sequence of SARS-CoV-2 virus isolate was downloaded from GISAID and NCBI. Classification of SARS-CoV-2 clades and construction of the phylogenetic tree using MEGA-program. Molecular dynamics simulation was demonstrated with GROMACS to compare the stability of D614 and G614 S proteins. The result of this research showed that the most common GH clade SARS-CoV-2 virus isolates were be discovered in Indonesia, and the percentage of isolates with the D614G mutation reached 97% in Indonesia. The clade with the D614G mutation made the SARS-CoV-2 virus more varied with new mutations and increased human-to-human transmision. In this study, an increase in infectivity or spread of the SARS-CoV-2 D614G virus most likely resulted from an increase in the strength of the S protein and a slight change in RBD conformation seen from the molecular interactions around the mutation area and the RMSF value (0.3144 nm for D614G, and 0.3818 nm for D614). This is likely to be the cause of the massive spread of variants belonging to VOC and VBM, where all these variants have the D614G mutation.

Corona viruses, especially betacoronaviruses, have caused at least three outbreaks of zoonotic diseases in the last two decades (Korber et al. 2020). First, in early 2003, the zoonotic disease SARS caused by SARS-CoV infected around 8,000 people with a mortality rate of 10%. This disease was originally reported in Guangdong, China, in 2002. Then in 2012 more than 2,300 cases of MERS with a fatality rate of 35% occurred. At the end of 2019, there was an outbreak of a serious respiratory disease which was later called COVID-19 which was caused by SARS-CoV-2. The virus, which then spread sporadically to all countries in the world, was first identified in China. As of March 2020, COVID-19 has transmission in more than 200 countries. The WHO has officially announced that SARS-CoV-2 is the cause of the COVID-19 zoonotic disease which has caused 2.71 million deaths out of 123 million cases. In Indonesia, COVID-19 has claimed 39,447 lives out of 1.46 million cases of infection. According to a report by John Hopkins University and Medicine on March 21^st, 2021, the COVID-19 mortality in Indonesia is 2.7%, ranks the 6^th highest in Asia, and number 40 in the world.

The COVID-19 outbreak has also encouraged researchers throughout the world to look for cures and vaccinations to stop the transmission of SARS-CoV-2. Genetically, 79% of the SARS-CoV-2 genome is identical to SARS-CoV. The mechanism of infection of SARS-CoV-2 into host cells also uses the same receptor, namely human angiotensin converting enzyme 2 (ACE2) (Kaur and Gupta 2020). SARS-CoV-2 has the possibility to cross mucosal membranes, particularly the nasal and laryngeal mucosa, and then enter the respiratory tract to enter the lungs. Additionally, the virus will target organs like the kidneys, lungs, heart, and gastrointestinal tract that express ACE2 (Di Gennaro et al. 2020).

SARS-CoV-2 has four primary structural proteins, namely spike (S), envelope (E), membrane (M), and nucleocapsid (N) (Astuti and Ysrafil 2020; Chan et al. 2020; Shereen et al. 2020). The S protein is involved in mediating membrane fusion and receptor binding. The corona virus's S protein has two domains, i.e. S1 and S2. Five amino acid residues, namely Asn439, Asn501, Gln493, Gly485, and Phe4861 play a key role in the receptor binding domain so that the binding of SARS-CoV-2 to ACE2 is more effective. The S protein controls how viruses enter host cells and serves as the main antigenic target for host immune responses, hence it plays an important part in the infection activity (Tang et al. 2020; Yuan et al. 2017). These functions demonstrate the significance of S protein in the mechanism of viral entry and related to the host immune system.

The SARS-CoV-2 virus, the same as to other Corona viruses, has a gene proofreading mechanism (Al Khatib et al. 2020; Korber et al. 2020). However, even though mutations are rare, they can give rise to natural selection that favors the virus. It is necessary to study the effects of changes in single amino acids in vivo because they can affect both structural, biochemical, and physiological characteristics of the virus. Among the Corona viruses that attack humans, gene mutations have been shown to provide resistance to the immunoglobulins (MERS-CoV and SARS-CoV) (Korber et al. 2020).

D614G is one of the amino acid alterations in SARS-CoV-2. One of the notable alterations in the spike glycoprotein gene of SARS-CoV-2 is the substitution of the 614^th amino acid, from aspartate (D) to glycine (G) (Becerra-Flores and Cardozo 2020). To enter host cells, the spike protein must bind to ACE2. Thus, mutations in the spike protein may alter the receptor binding affinity and virus infectivity, as well as affect the ability of antibodies to recognize viruses (Volz et al. 2021). This necessitates a thorough examination of SARS-CoV-2 alterations that result in novel variations (clades), particularly on S-D614G.

Although the D614G amino acid is not placed at the position of the receptor binding domain (RBD) that associates directly with ACE2, the D614G amino acid is located between the S1 and S2 domains, close to the S2 furin cleavage site. The furin cleavage site includes residues 682-685 and performs a function in the mechanism of entry of the SARS-CoV-2 virus through the Furin-TMPRSS2 proteolytic pathway. This proteolytic process is significant for SARS-CoV-2 viral entrance (Bestle et al. 2020; Shang et al. 2020). Most of the SARS-CoV-2 vaccines are patterned using the D614 variant from the SARS-CoV-2 S protein sequence of the Wuhan isolate without mutation (Lurie et al. 2020). Amino acid mutations in the D614G spike protein need to be studied more deeply for their influence on the pathogenesis of the virus.

One way to analyze the stability of the D614 S protein and its mutation (G614), requires molecular dynamics simulation (MDS). This simulation method with computer media can be used to determine protein stability, protein structure, conformational changes, ion transport in biological systems, drug designing, and others (Salo-Ahen et al. 2021). This technique makes it possible to represent the interactions of atomic molecules over a certain period. GROMACS is an application that can carry out MDS to find out the stability ratio of the D614 and G614 S proteins.

The purpose of this research was to 1) analyzing the variation of the isolate clades of the SARS-CoV-2 virus, as well as the proportion of the D614G mutation in Indonesia and the world; 2) Analyzing the kinship of SARS-CoV-2 in Indonesia which is visualized from phylogenetic constructs; 3) Analyzing the effect of D614G spike protein mutation on ACE2 receptor binding; 4) Comparing the results of the molecular dynamics simulation of the reference protein with the spike protein with the D614G mutation.

This investigation was an exploratory observational study that aimed to determine the following: (1) the The kinship of SARS-CoV-2 with the D614G mutation in Indonesia with several virus samples from other countries; (2) the proportion of SARS-CoV-2 isolates with the D614G mutation in Indonesia and around the world; (3) the impact of D614G spike protein mutation on ACE2 receptor binding; and (4) the comparison of the MDS results of the mutant and comparion proteins. This study was performed in a computer laboratory in the Faculty of Mathematics and Natural Sciences, Department of Computer Science, Universitas Negeri Semarang.

Whole genome sequences that were taken from 2 open service sites served as the study's samples. The all nucleotide sequence of the SARS-CoV-2 spike protein was obtained from GISAID (gisaid.org), whereas the sequence of the Wuhan-Hu-1 virus (GeneBank reference NC 045512.2) was collected from NCBI (ncbi.nlm.nih.gov/). The samples from comparator countries were collected from January 2020 to August 19^th, 2021, whereas the samples from Indonesia were collected from March 2020 to August 19^th, 2021.

Data Mining

The nucleotide sequence of SARS-CoV-2 virus isolate was downloaded from GISAID (gisaid.org/) in March 2020-August 19th, 2021 (Elbe and Buckland-Merrett 2017). The sequence used was the whole genome, and there was no misreading at the 614^th residue of the S protein. The samples used as comparisons came from several countries in the world which were grouped into 6 continents, namely Oceania, Asia, Africa, Europe, North America, and South America. The Wuhan-Hu-1 (NC_045512.2) sample from NCBI (ncbi.nlm.nih.gov) was used as a reference.

Spike Protein Extraction and Analysis of The Proportion Number of D614G Isolates

S protein (ORF2) was checked by using the NCBI ORFfinder (ncbi.nlm.nih.gov/orffinder/). Then, the S sequence was translated into amino acids and MSA (Multiple Sequence Alignment) by using the ClustalW algorithm on Mega-X, with Wuhan-Hu-1 as a standart. Gap opening and extension penalties were set at 10.0 and 0.20, respectively. The negative matrix option was disabled, and the delay divergence cut off was set at 30%. The alignment results were then used to see the D614G point mutation status of the newly extracted SARS-CoV-2 protein sequence. Then, the proportion of D614G mutations in Indonesia was then determined using the fraction of isolates with both D and G amino acids in the S protein sequence. The same method was carried out for other samples.

Classification of SARS-CoV-2 Clades and Construction of the Phylogenetic Tree

Phylogenic construction was made in Mega-X from previous alignment results, using the Neighbor-Joining (NJ) Bootstrap 1000 replication method (Gaffar et al. 2021; Ghorbani et al. 2021; Tabibzadeh et al. 2020) from 18 random high coverage sequences from Indonesian representative for each type of GISAID clade, as well as Wuhan-Hu-1 as a reference. Then, the sequences were also analyzed for the type of PANGOLIN variant, as well as the status of the variant using the COG-UK web application (https://pangolin.cog-uk.io/) (O'Toole 2021; Rambaut et al. 2020). Other sequences from neighboring countries namely, Malaysia, Singapore, Thailand, and Vietnam were used as comparisons. The samples were taken randomly, and must had a sequence length of >29,000 bp.

The Prediction of Mutation Point and Visualization of Spike Protein and D614G

Prior to the MDS analysis, mutation point prediction was carried out to see the impact of structural and chemical changes using the HOPE web service tool (www3.cmbi.umcn.nl/hope/). Visualization of 3D structures was carried out using the SWISS-MODEL web server and PyMOL v2.4. This study used 2 models, namely the SARS-CoV-2 S protein (pdb code: 7DWZ) and the S protein that had a mutation at residue 614 (pdb code: 7DX1). The two cryo-EM model results were acquired from the Protein Data Bank (rcsb.org/).

Molecular Dynamics Simulation

Molecular Dynamics Simulation (MDS) was demonstrated with GROMACS to compare the stability of D614 and G614 S proteins. The software used was open source GROMACS v.2021.1. OPLS was chosen as a force field with a cube-shaped water box filled with water surrounding the complex with the TIP3P water model. Na⁺ and Cl^- were used to neutralize the protein. A derivation algorithm with a tolerance of 1000 kJ/mol/nm was used to minimize the system energy. After the convergence, equilibration, and simulation of NVT (temperature) at 1000 ps was performed, followed by NPT (pressure) at 1000 ps. Barostat and Berendsen thermostat were used to take care of the temperature and pressure stable at 300K and 1 bar. The dynamics simulation was carried out in 10-ns.

Data Analysis

The data obtained from this study were analyzed descriptively. The data is RMSF (Root-mean-square Fluctuation), which is a quantity of the deviation between the particle position and several references. In contrast to RMSD (Root-mean-square Deviation), RMSF is calculated for each residue making up the protein to see how far the movement of each residue fluctuates during the simulation. RMSF serves to evaluate the rising and falling of the sequence number of residues that make up the proteins during simulation (Elengoe et al. 2014).

The Composition of the SARS-CoV-2 Virus Isolate Clades

Data of virus clade composition in this study is divided into 2, i.e. Indonesia and the comparison countries. The composition of the SARS-CoV-2 virus clade from Indonesia was taken from data on service sites gisaid.org network from March 2020 to August 19^th, 2021 totaling 5,266 sequences (Table 1).

Table 1. The number of SARS-CoV-2 virus isolates in Indonesia based on the clades and residue of 614 of S protein

No.	Clade	614 amino acid residue	Amount
1	O	D	78 (1.48%)
2	L	D	63 (1.12%)
3	S	D	9 (0.17%)
4	V	D	0
5	G	G	208 (3.95%)
6	GH	G	2,801 (53.19%)
7	GR	G	407 (7.72%)
8	GK	G	1,646 (31.25%)
9	GV	G	7 (0.13%)
10	GRY	G	47 (0.89%)
			5,266 (100%)

Of the 5,266 isolates identified in Indonesia, 9 clades are detected, namely clades O, L, S, G, GH, GR, GK, GV, and GRY. While clade V is not found in Indonesia. Virus isolates in Indonesia are dominated by the GH clade (2,801 isolates), followed by the GK (1,646 isolates), and the GR (407 isolates). It can also be seen in Table 1 that there are far more clades with G614 (5116 isolates; 97.15%) than clades with D614 (150 isolates; 2.85%).

Protein S gene sequences from SARS-CoV-2 virus isolates in comparison countries using sequence data from GISAID (gisaid.org) until 19 August 2021, with a total sample of 2,904,279 sequences. Sequence data were analyzed and grouped into 7 continents, namely: Oceania (including Australia and the Pacific countries), Asia, Africa, Europe, North America, and South America. The clade composition of the comparison countries can be seen in Table 2.

Table 2. Number of SARS-CoV-2 virus isolates of 7 continents based on clades and residue of 614 of S proteins

Clade	614 amino acid residue	Continent
Clade	614 amino acid residue	Oceania	Asia	Africa	Europe	North America	South America
L	D	70	1.089	66	3.786	825	20
O	D	567	5.913	609	14.496	4.684	1.596
S	D	896	1,464	1.051	3.858	7.392	277
V	D	335	454	29	5.490	583	37
G	G	1.800	25.808	6.085	167.274	92.620	4.617
GH	G	2.482	22.579	12.941	77.912	328.244	3.976
GR	G	14.254	69.706	6.599	174.530	147.823	40.396
GK	G	4.800	28.384	4.913	389.837	117.790	978
GV	G	80	485	366	169.350	847	53
GRY	G	622	35.192	1.633	681.856	209.807	2.072
Total		25.906	191.075	35.013	1.688.414	909.899	53.972

The composition of the SARS-CoV-2 clade around the world varies in types and numbers, compared to Indonesian virus isolates. The most common virus isolates are found in Europe. The highest GR clades are found on the continents of Oceania, Asia, and South America. The GH clade is the most abundant in Africa and North America, and the GRY is the most abundant in Europe. In the S protein with D614 amino acid residue, there are 2 dominant clades, namely the O clade (Asia, Europe, and South America) and the S clade (Oceania, Africa, and North America). There are 3 dominant clades represent S protein with G614 amino acid residue, i.e. the GR, GH, and GRY. Meanwhile, the least number clades are L (Oceania, Europe, and South America), and V (Asia, Africa, North America) (Table 2). The illustration of the percentages of the clades can be seen in Figure 1.

As in Indonesia, the sequences of isolates with the D614G S protein mutation are more dominate compared to those without the point mutation (Figure 1B). D614G S protein was first detected in Europe in February 2020 from a patient's swab sample. With only a span of a few days from when it was first discovered, this mutation dominated in several countries in Europe. This huge number makes the D614G mutation the main differentiator of clade G from S, L, O, and V. In addition to S-D614G mutation, the other markers for clade G are C241T, C3037T, and A23403G.

The multiple sequence alignment (MSA) results of the Spike protein in this study are shown in Table 3. Of the 18 high-coverage random samples from Indonesia that was taken randomly from each GISAID clade and 4 random comparison samples, there were 2 samples that were 100% identical to the sequence from NC_045512.2 (Wuhan-Hu-1), namely EPI_ISL_888982 (clade L) and EPI_ISL_3401924 (clade O). There were found 30 mutations that cause changes in amino acids. The most mutation is D614G, namely 16 samples. This mutation is classified as a non-conservative type of amino acid change because it has a PAM value of 11, as well as different properties and characteristics (Table 5) (Balasco et al. 2021).

Of the 22 samples used, there were 5 samples of the 4 variants that are commonly found in Indonesia, namely B.1.1.398, B.1.466.2, AY.23, and AY.127 (B.1.617.2-like). Two of the three strains are not listed in the dangerous status category according to the CDC (Table 4), but the rest (AY.23 and AY.24) belong to the Delta variant. Another Delta variant found in Indonesian samples is AY.127, which predominates in India. The Alpha variant that is also included in the VOC CDC list was also found in two Indonesian samples. This variant was first identified and became the dominant variant in England (Ramesh et al. 2021; Shiehzadegan et al. 2021). These two variants are suspected as the trigger of the 1^st and 2^nd peaks of the COVID-19 pandemic (Mlcochova et al. 2021).

In the comparison samples, three of the four samples taken randomly were the Delta variant. AY.23 that is now dominant in Indonesia is the variant that previously dominated in Singapore. This shows that the inclusion of the Delta AY.23 variant originated in Singapore and spread to Indonesia widely.

ades can be seen in Figure 1.

Figure 1. Illustration of the composition of SARS-CoV-2 virus clade in continental group A) Based on the clade. B) Based on a point mutation at 614.

As in Indonesia, the sequences of isolates with the D614G S protein mutation are more dominate compared to those without the point mutation (Figure 1B). D614G S protein was first detected in Europe in February 2020 from a patient's swab sample. With only a span of a few days from when it was first discovered, this mutation dominated in several countries in Europe. This huge number makes the D614G mutation the main differentiator of clade G from S, L, O, and V. In addition to S-D614G mutation, the other markers for clade G are C241T, C3037T, and A23403G.

The multiple sequence alignment (MSA) results of the Spike protein in this study are shown in Table 3. Of the 18 high-coverage random samples from Indonesia that was taken randomly from each GISAID clade and 4 random comparison samples, there were 2 samples that were 100% identical to the sequence from NC_045512.2 (Wuhan-Hu-1), namely EPI_ISL_888982 (clade L) and EPI_ISL_3401924 (clade O). There were found 30 mutations that cause changes in amino acids. The most mutation is D614G, namely 16 samples. This mutation is classified as a non-conservative type of amino acid change because it has a PAM value of 11, as well as different properties and characteristics (Table 5) (Balasco et al. 2021).

Of the 22 samples used, there were 5 samples of the 4 variants that are commonly found in Indonesia, namely B.1.1.398, B.1.466.2, AY.23, and AY.127 (B.1.617.2-like). Two of the three strains are not listed in the dangerous status category according to the CDC (Table 4), but the rest (AY.23 and AY.24) belong to the Delta variant. Another Delta variant found in Indonesian samples is AY.127, which predominates in India. The Alpha variant that is also included in the VOC CDC list was also found in two Indonesian samples. This variant was first identified and became the dominant variant in England (Ramesh et al. 2021; Shiehzadegan et al. 2021). These two variants are suspected as the trigger of the 1^st and 2^nd peaks of the COVID-19 pandemic (Mlcochova et al. 2021).

In the comparison samples, three of the four samples taken randomly were the Delta variant. AY.23 that is now dominant in Indonesia is the variant that previously dominated in Singapore. This shows that the inclusion of the Delta AY.23 variant originated in Singapore and spread to Indonesia widely.

Table 3. Point mutation of the spike protein encoding gene from the SARS-CoV-2 virus isolate in Indonesia which was alignment with isolate NC_045512.2 (Wuhan-Hu-1)

*C =Conservative, NC=Non-Conservative

**Comparison sequences

No.		19	76	80	102	142	147	156	157	222	352	367	439	452	478	501	521	570	613	614	655	681	701	716	939	950	982	1078	1118	1251	1264	∑
No.	NC_045512.2	T	T	D	R	G	K	E	F	A	A	V	N	L	T	N	P	A	Q	D	H	P	A	T	S	D	S	A	D	G	V	∑
1.	EPI_ISL_435283	.	I	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1
2.	EPI_ISL_458081	.	.	.	.	.	.	.	.	.	S	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1
3.	EPI_ISL_888982	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0
4.	EPI_ISL_1257824	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.	.	V	.	.	.	.	.	.	.	.	2
5.	EPI_ISL_1257844	.	.	.	I	.	.	.	L	.	.	F	.	.	.	.	.	.	H	.	.	R	.	.	.	.	.	.	.	.	.	5
6.	EPI_ISL_1284152	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.	.	.	.	.	.	.	.	.	.	.	1
7.	EPI_ISL_1416191	.	.	.	.	.	.	.	.	.	.	.	.	.	.	Y	.	D	.	G	.	H	.	I	.	.	A	.	H	.	.	7
8.	EPI_ISL_1660413	.	.	.	.	.	.	.	.	.	.	.	K	.	.	.	.	.	.	G	.	.	.	.	.	.	.	.	.	.	.	2
9.	EPI_ISL_1824607	R	.	.	.	D	.	K	.	.	.	.	.	R	K	.	.	.	.	G	.	R	.	.	.	.	.	.	.	.	.	7
10.	EPI_ISL_2047525	.	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.	.	.	G	.	.	.	.	.	.	.	.	.	.	.	2
11.	EPI_ISL_2364585	.	.	Y	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.	.	.	.	.	.	.	S	.	.	.	3
12.	EPI_ISL_2382411	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	T	.	.	.	Y	.	.	.	.	.	.	.	.	.	.	3
13.	EPI_ISL_2835650	R	.	.	.	D	.	.	.	.	.	.	.	R	K	.	.	.	.	G	.	R	.	.	.	N	.	.	.	.	L	8
14.	EPI_ISL_2987628	R	.	.	.	.	.	.	.	V	.	.	.	.	K	.	.	.	.	G	.	R	.	.	.	N	.	.	.	.	.	6
15.	EPI_ISL_3070877	.	.	.	.	.	.	.	.	.	.	.	.	.	.	Y	.	D	.	G	.	H	.	I	.	.	A	.	H	.	.	7
16.	EPI_ISL_3160359	.	.	.	.	.	.	.	.	.	.	.	K	.	.	.	.	.	.	G	.	R	.	.	.	.	.	.	.	.	.	3
17.	EPI_ISL_3383438	.	.	.	.	.	N	.	.	.	.	.	K	.	.	.	.	.	.	G	.	R	.	.	.	.	.	.	.	.	.	4
18.	EPI_ISL_3401924	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0
19.	EPI_ISL_5052185 MALAYSIA**	.	.	.	.	.	.	.	.	.	.	.	K	.	.	.	.	.	.	G	.	R	.	.	.	.	.	.	.	V	.	4
20.	EPI_ISL_3426997 SINGAPORE**	R	.	.	.	D	.	.	.	.	.	.	.	R	K	.	.	.	.	G	.	R	.	.	.	N	.	.	.	.	L	8
21.	EPI_ISL_5695468 THAILAND**	R	.	.	.	D	.	.	.	.	.	.	.	R	K	.	.	.	.	G	.	R	.	.	F	N	.	.	.	.	.	8
22.	EPI_ISL_4503976 VIETNAM**	R	.	.	.	D	.	.	.	V	.		.	R	K	.	.	.	.	G	.	R	.	.	.	N	.	.	.	.	.	8
	∑	6	1	1	1	5	1	1	1	3	1	1	4	5	6	3	1	2	1	16	1	12	1	2	1.	5	2	1	2	1	2
	Type of mutation	NC	NC	NC	NC	NC		NC*	C*	C	NC	C	NC	NC	NC	NC	NC	NC	NC	NC	NC	NC	C	NC		NC	NC	NC	NC	NC	C
DOMAIN			NTD								RBD															HR1				HR2
DOMAIN		S1																								S2

Table 4. Pangolin strain, status, and countries with the highest Indonesian samples

No.	ID	GISAID	Pangolin	Country
1.	EPI_ISL_435283	O	B	America
2.	EPI_ISL_458081	L	B	America
3.	EPI_ISL_888982	L	B	America
4.	EPI_ISL_1257824	G	B.1.524	Malaysia
5.	EPI_ISL_1257844	S	A.23.1	Canada
6.	EPI_ISL_1284152	GR	B.1.1.398	Indonesia
7.	EPI_ISL_1416191	GRY	B.1.1.7-like (Alpha)*	England
8.	EPI_ISL_1660413	GH	B.1.466.2	Indonesia
9.	EPI_ISL_1824607	GK	AY.127 (B.1.617.2-like, Delta)*	India
10.	EPI_ISL_2047525	GV	B.1.177.43	Switzerland
11.	EPI_ISL_2364585	GR	B.1.1.398	Indonesia
12.	EPI_ISL_2382411	S	A.28	France
13.	EPI_ISL_2835650	GK	AY.23 (B.1.617.2-like, Delta)*	Indonesia
14.	EPI_ISL_2987628	GV	AY.24 (B.1.617.2-like, Delta)*	Indonesia
15.	EPI_ISL_3070877	GRY	B.1.1.7-like (Alpha)*	England
16.	EPI_ISL_3160359	GH	B.1.466.2	Egypt
17.	EPI_ISL_3383438	G	B.1.1.519	America
18.	EPI_ISL_3401924	O	B	America
19.	EPI_ISL_5052185/MALAYSIA	GH	AU.2 (B.1.466.2.2)	Malaysia
20.	EPI_ISL_3426997/SINGAPORE	GK	AY.23 (B.1.617.2-like, Delta)*	Indonesia
21.	EPI_ISL_5695468/THAILAND	GK	AY.30 (B.1.617.2-like, Delta)*	Thailand
	EPI_ISL_4503976/VIETNAM	GK	AY.57 (B.1.617.2-like, Delta)*	Vietnam

*Status CDC: VOC (Variant of Concern)

Figure 2 shows that more viral isolates with the D614G mutation were found than the D614 WT (wild type) sequence. It can also be seen that the highest number of D614 WT was only in the initial months of the outbreak and tended to continue to decrease to even 0 at the end of 2020. In early 2021, WT cases were detected again (8 isolates) and gradually decreased until July to 0 case. Although the first confirmed case of COVID-19 in Indonesia was on March 2, 2020 (Indonesian COVID-19 Handling Task Force (2021), GISAID data shows that the D614G mutation has existed since March 27, 2020. Only a few months after its appearance, in September to be precise, the G614 (mutant) sequence has dominated all sequences taken each month. The significant increase in COvid-19 cases was caused by SARS-CoV-2 which had the S-D614G mutation. The same information was also reported by Korber et al. (2020).

Construction of the SARS-CoV-2 Phylogeny in Indonesia

The construction of the Neighbor-Joining phylogeny was made from nucleotide sequence data of the Indonesian SARS-CoV-2 S protein. Two isolates that had high coverage were randomly selected from each of the GISAID clades, as well as 4 sequences from several neighboring countries, such as Malaysia, Singapore, Thailand, and Vietnam. The phylogeny construction of the Indonesian SARS-CoV-2 spike sequence sample in this study can be seen in Figure 3. The first cluster (1) is a small cluster that has similarities with the original reference sequence Wuhan-Hu-1 (NC_045512.2). While cluster 2 is a large cluster consisting of 2 sub-clusters.

The figure shows that cluster 1 does not have much difference from the SARS-CoV-2 S sequence that initial emerged in Wuhan City, China. EPI_ISL_3401924 sample has 100% similarity with the spike nucleotide sequence of the reference sample, while EPI_ISL_435283, EPI_ISL_458081, and EPI_ISL_888982 only has 1 base difference. However, EPI_ISL_888982 show changes of 1 base at 213^th sequence (point mutation) that results in a silent mutation of the TCT>TCC codon, that still encodes serine (S71). Whereas the sequence EPI_ISL_2382411 in the same cluster has 7 different bases which result in a change of 3 amino acids. This condition explains the distance of the branches in that sequence from the other sequence in the same cluster. EPI_ISL_435283 and EPI_ISL_3401924 belong to clade O, EPI_ISL_458081 and EPI_ISL_888982 to clade L, while EPI_ISL_2382411 to clade S. These three clades are included in the "early" clade category because many were found in the early months since the pandemic appeared in December 2019. This information explains about 4 out of 5 samples in this cluster that were taken from March to August 2020. However, EPI_ISL_2382411 was collected in April 2021, indicating that the initiation of this virus entry occurred at different times. Even so, this sequence has far more mutations than the other sequences which explains the difference in the branch spacing of the sequence. In cluster 1, there was no misssense mutation of D614G.

Cluster 2 is formed by 2 subclusters, subcluster 1 and 2. EPI_ISL_1284152 (GISAID clade: G) in particular, only has one different base compared to the comparison sample at 1841 (GAT>GGT). This change results in a different amino acid and phenotypic character expression at 614^th residue, this non-conservative missense mutation commonly referred to as D614G. D614G is one of the markers used by GISAID to differentiate the G clade and its derivatives, from the S, L, O, and V clades. There were 16 out of 17 samples that had the D614G mutation in cluster 2, and it was the only mutation in the SARS-Cov-2 S protein with elevated consistency and frequency (Ghorbani et al. 2021; Korber et al. 2020; Shiehzadegan et al. 2021; Yurkovetskiy et al. 2020).

All samples in cluster 2 were collected in 2021, 16 of the 17 samples included in this cluster encode the D614G mutation. Subcluster 1, which is a small subcluster, only has 1 homologous mutation, namely D614G. while subcluster 2 has more variations, and consists of small groups. The samples with D614G + N439K + P681R mutation gathered in group 1 subcluster 2 except for the EPI_ISL_1660413 sample (without P681R mutation). Samples belonging to the Alpha strain gathered in group 2 subcluster 2, the S protein sequences of these samples were 100% identical to each other (including D614G + P681R, without N439K). All samples in group 3 subcluster 2 encode for P681R. Six of the 7 samples belonging to this group are called Delta strains, having 3 mutations highlighted in several studies related to the Delta variant, namely the D614G + P681R + L452R mutation, and the presence of deletions at residues 157-158. In this study, the frequency of both P681R and L452R was quite high. In fact, according to research by Starr et al. (2021), L452R indicates the possibility of increasing the affinity of the S protein with ACE2, because it is present in the RBD domain which binds directly to the host cell receptor.

The Alpha variant in this study appeared earlier, while the Delta variant appeared later in the April-June 2021. This condition is in accordance with Figure 2 which shows the trend of increasing and decreasing cases in Indonesia. The first peak of Indonesia's COVID-19 cases due to the Alpha variant occurred in January 2021 and the peak of second due to the Delta variant happened in June 2021 (Covid-19 Handling Task Force, 2021). As a result of the emersion of a novel variant (Alpha B.1.1.7, clade GRY) in the UK in mid-October 2020, the Indonesian government had taken anticipatory measures by launching a strict PPKM (Pemberlakuan Pembatasan Kegiatan Masyarakat: implementation of restrictions on community activities). This policy was also carried out to address the christmas and new year holidays 2021. Even so, the mobility of the Indonesian people was still quite high, especially foreign travel, so the inclusion of this new variant was unavoidable.

The new variant appeared when Alpha GRY cases experienced a rapid increase in January 2021. It was identified for the earliest in India with the strain code B.1.617.2, clade GK, which was then named Delta variant. This strain is consideration to have more severity than the previous variant (Shiehzadegan et al. 2021). Previously, there had been several studies that predicted the development of COVID-19. Based on those studies, COVID-19 would continue to develop throughout the world if no control measures are implemented. This control measure will also minimize the number of new strains that will continue to appear along with the spread of COVID-19 (Hu et al. 2020; Rendana and Idris, 2021; Ribeiro et al. 2020). WHO, especially the Indonesian government, continues to limit activities and implement 3T (Testing, Tracing and Treatment) and encourage people to do 3M: Memakai masker (Wearing masks), Mencuci tangan (Washing hands), dan Menjaga jarak (Maintaining distance) (Ministry of Health RI, 2021).

Four comparison samples were taken from several countries around Indonesia. The SARS-CoV-2 S sequence sample from Malaysia belongs to the GH clade, while the other three samples from Singapore, Thailand, and Vietnam belong to the GK clade. Those three sample strains are included in the VOC category because they are derived from the Delta B.1.617.2 strain from India.

Effect of D614G Spike Protein Mutation on ACE2 Receptor Binding

S-D614G protein mutation changes the amino acid residue of aspartic acid to glycine. This raises the question whether the changes affect its binding to the ACE2 receptor, considering that the mutations that occur are located in the S protein which makes direct contact with the host cell. To answer that question, mutation point predictions were carried out using the service site in the HOPE network (www3.cmbi.umcn.nl/hope/). In addition, 3D visualization was also carried out to see the changes in more detail.

The results of the point mutation predictions in Table 5 show that the G614 amino acid has a smaller size and molecular weight. Moreover, there is a change in the residual charge from initially negative to neutral, and the mutant amino acid is more hydrophobic than the WT amino acid. The glycine structure, which is only a backbone without side chains, makes the mutant amino acids much more flexible. The lighter size and neutral charge of the mutant amino acids may result in the loss of some intermolecular interactions. The hydrophobicity of the amino acids which became more hydrophobic also resulted in the loss of the hydrogen bonds that were originally present in the WT amino acids. The five changes that occur can easily disrupt the rigidity of the protein in the mutation area.

Hence, Cryogenic electron microscopy (cryo-EM) of SARS-CoV-2 variants D614 and G614 was used in the RSCB Protein Data Bank, with accession numbers 7DWZ and 7DX1, to determine the structure of the S protein on the superficies of the virion in more detail. Differences in size and structure can be seen in detail by visualizing the 3D protein sticks of WT/D614 and mutant/G614 in Figure 5.

The S protein is an identical trimer structure, that protrudes from the virus. The D614G mutation that occurs in the spike protein changes the 3 location points in chains A, B, and C (Fig. 4.4. A and B). The D614G mutation is located in S1, close to the cleavage site.

One of the prediction points of protein mutations shows a change in hydrophobicity in the area around the mutation. Proteins with the D614G mutation have higher hydrophobicity. Figure 4.6 is a 3D surface image with PyMoL, the surface is a representation of the protein surface which shows the surface area that can be accessed by the solvent (Solvent-Accessible Surface Area/SASA), in this case the solvent used is water.

Table 6. Solvent-Accessible Surface Area on the highlighted residue

	Res.
	D614	G614	Q613 (WT)	Q613 (mutant)
SASA (Ᾰ²)	57.102	55.888	28.964	39.593

The SASA value of the G614 residue in the mutant protein was 55.888Ᾰ2 that smaller than the WT protein was due to the change in residual size. Apart from the size, the hydrophobicity which changed to be higher in the mutant protein caused an increase in the SASA value in Q613 (Table 6), causing structural changes that can be physically seen in Figures 6 A and B.

D614 (S1/chain A) is adjacent to T859, which is part of the S2 domain of chain B. These two residues form short hydrogen bonds of 3.5Ᾰ. However, in the G614 mutant protein, the hydrogen bonds are lost because glycine is an amino acid without side chains. This change can shift the RBD to an 'up' conformation (RBD-up) (Korber et al. 2020). The negatively charged D614 also forms a salt bridge with the positively charged K854, a residue that is also in the S2 domain of chain B. The mutant protein does not have this interaction because the mutant amino acid is neutrally charged. However, according to Zhang et al. (2021), the loss of salt bridge in the mutant protein does not make major structural changes. The possibilities that might occur due to the loss of hydrogen bonds in the D614G mutant, namely 1) The loss of hinge to the -helix subunit S2, making it more flexible to interact with host cell receptors (hACE2); and 2) Elevated flexibility, so that furin cleavage sites are more accessible (Mohammad et al. 2021). These conditions make the hydrogen bonds of the D614-T859 residues significant for the stability of the protein's overall structure.

Molecular Dynamics Simulation of the D614 and G614 Spike Protein

The RMSF value of the WT spike protein (D614) and the mutant protein (G614) was used to determine the dynamic behavior of the residues that had the mutation. RMSF is the result of the MD simulation which is used as a means of describing the differences in flexibility between residues. The RMSF backbone of each WT and mutant S protein residue was calculated to analyze the flexibility of the backbone structure. The RMSF value has a positive correlation with movement flexibility. Higher RMSF values indicate more flexible movements while low RMSF values indicate limited movements during the simulation. This value is the calculation of the average position of each residue.

The RMSF range of the D614 is between 0.2487 nm to 0.9778 nm. High fluctuations can be seen at residue positions at 154, 351, 416, 455, 491, 704, 751, 985, 1085, 1128, and 1146. The RMSF fluctuation at residue 614 in D614 is 0.3818 nm. While the RMSF range of G614 is between 0.1190 nm to 0.6387 nm. From the figure presented, it can be seen clearly that the fluctuation of the mutant protein residues as a whole has decreased compared to the structure of the WT protein. High fluctuations can be seen at residues 445, 450, 556, 569, 1085, and 1124. The RMSF fluctuation at residue 614 in the mutant spike protein is 0.3144 (Figure 7B).

When compared to the structure of the mutant protein, the structure of the WT protein exhibits extremely large fluctuations in practically all residual positions, as can be seen from the RMSF plot in Figure 7A. The terminal residue of the WT protein showed greater fluctuations than the mutant protein. Based on RMSF data, the structure of the mutant protein (G614) is more stable than the WT protein (D614). The RMSF data demonstrate that the mutant/G614 protein structure is more stable than the WT/D614 structure.

The most common GH clade SARS-CoV-2 virus isolates were be discovered in Indonesia, and the percentage of isolates with the D614G mutation reached 97% in Indonesia. The clade with the D614 residue in the spike protein was the wildtype of SARSCoV-2 in the first phase of the pandemic, while the clade with the D614G mutation appeared later and made the SARS-CoV-2 virus more varied with new mutations and increased human-to-human transmision. In this study, an increase in infectivity or spread of the SARS-CoV-2 D614G virus most likely resulted from an increase in the strength of the S protein and a slight change in RBD conformation seen from the molecular interactions around the mutation area and the RMSF value (0.3144 nm for D614G, and 0.3818 nm for D614). This is likely to be the cause of the massive spread of variants belonging to VOC and VBM, such as B.1.351 (Beta variant, South African lineage), B.1.1.7 (Alpha variant, British lineage), and B.1.617.2 (Delta variant, India lineage) where all of these variants have the D614G mutation.

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No competing interests reported.

Study on Sequence Mutation and Molecular Dynamics Simulation of D614G SARS-CoV-2 Spike Protein in Indonesia

Status:

Version 1

Abstract

Figures

Introduction

Methods

Results And Discussion

Conclusion

References

Additional Declarations

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Version 1