Analysis of SARS-CoV-2 nucleocapsid protein sequence variations in ASEAN countries

Mochammad Rajasa Mukti Negara,¹ Ita Krissanti,2,3 Gita Widya Pradini³

Background

Nucleocapsid (N) protein is one of four structural proteins of SARS-CoV-2 which is known to be more conserved than spike protein and is highly immunogenic. This study aimed to analyze the variation of the SARS-CoV-2 N protein sequences in ASEAN countries, including Indonesia.

 

Methods

Complete sequences of SARS-CoV-2 N protein from each ASEAN country were obtained from Global Initiative on Sharing All Influenza Data (GISAID), while the reference sequence was obtained from GenBank. All sequences collected from December 2019 to March 2021 were grouped to the clade according to GISAID, and two representative isolates were chosen from each clade for the analysis. The sequences were aligned by MUSCLE, and phylogenetic trees were built using MEGA-X software based on the nucleotide and translated AA sequences.

 

Results

98 isolates of complete N protein genes from ASEAN countries were analyzed. The nucleotides of all isolates were 97.5% conserved. Of 31 nucleotide changes, 22 led to amino acid (AA) substitutions; thus, the AA sequences were 94.5% conserved. The phylogenetic tree of nucleotide and AA sequences shows similar branches. Nucleotide variations in clade O (C28311T); clade GR (28881–28883 GGG>AAC); and clade GRY (28881–28883 GGG>AAC and C28977T) lead to specific branches corresponding to the clade within both trees.

 

Conclusions

The N protein sequences of SARS-CoV-2 across ASEAN countries are highly conserved. Most isolates were closely related to the reference sequence originating from China, except the isolates representing clade O, GR, and GRY which formed specific branches in the phylogenetic tree.

 

Keywords

nucleocapsid proteins, phylogeny, SARS-CoV-2, sequence alignment

Coronaviruses are a large virus family that cause infectious diseases in animals and humans.1 Coronavirus has been recorded to cause pandemics several times, including severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome (MERS) caused by MERS coronavirus (MERS-CoV) in 2012.2 It is an enveloped virus with a spherical virions structure and diameter of 70−160 nm. It has a single-stranded, non-segmented, positive-sense RNA genome. Its nucleocapsid has a helical shape and diameter of 11−13 nm.3

SARS-CoV-2 is the seventh discovery of coronavirus that causes infectious disease in humans and is classified into Betacoronavirus.1 It has a 79% similarity to SARS-CoV and 50% to MERS-CoV.4 Having 29,903 nucleotides in the genome makes it the second-largest RNA virus. This viral genome has 11 open reading frames (ORFs) encoding 27 proteins. The first ORF (ORF 1/ab) covers two-thirds of the viral genome and encodes 16 nonstructural proteins, while the other one-third of the genome encodes four structural proteins and six additional proteins (accessory).5,6

SARS-CoV-2 has a structural protein named nucleocapsid (N) protein. This protein has 1,259 nucleotides located from nucleotides 28,274 to 29,533 and is encoded by 419 amino acids (AAs).7,8 It has two domains, which are the N-terminal domain (46–176 AA) and C-terminal domain (247–364 AA). Between these two domains, there is a linkage region (182–247 AA) containing the serine/arginine-rich domain (184–204 AA). These three domains have electrostatic interactions with the RNA genome.9,10

The N protein of SARS-CoV-2 has a vital role during the transcription and replication of viral RNA. It forms a ribonucleoprotein helix during RNA genome packaging and regulates viral RNA synthesis during replication, transcription, and modulation of infected cells’ metabolism.11 It is widely expressed during infection as the interferon antagonists.10 It is also a highly immunogenic protein. The anti-N protein antibodies (IgG, IgA, and IgM) were detected in confirmed coronavirus disease 2019 (COVID-19) patients’ sera.9 This region is conserved, with only three variations found (S194L, K249I, and P344S).12 Hence, this protein has been used as a protein target for SARS-CoV-2 diagnostic and future vaccine.10,13 There are limited data analyzing the sequence variation of SARS-CoV-2 N protein along with the translated AAs in ASEAN countries. This study aimed to analyze the variation of the SARS-CoV-2 N protein sequences in ASEAN countries, including Indonesia. The phylogenetic tree was also generated to describe phylogenetic relatedness among N sequence isolates in ASEAN countries.

 

 

This study analyzed the SARS-CoV-2 N protein gene sequences from GenBank and Global Initiative on Sharing All Influenza Data (GISAID) from December 2019 to March 2021. The SARS-CoV-2 N protein reference sequence (GenBank accession number: NC_045512.2) was downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank). SARS-CoV-2 N protein gene sequences from ASEAN countries were downloaded from the GISAID website under the EpiCoV™ platform (www.gisaid.org). Data collection from GISAID were adjusted to the countries; two of each GISAID clade were taken as representative isolates. The collected sequences were aligned by MUSCLE using the MEGA-X software version 10.1.8.14

Multiple sequence alignment of nucleotide was done to analyze nucleotide substitution mutations and their impact on translated AA (synonymous and non-synonymous mutation) compare with the reference sequence. Non-synonymous mutation, which led to AA substitution, was then subsequently checked for the AA polarity changes based on the AA classification.15 The phylogenetic trees of nucleotide sequences were built with MEGA-X software using the maximum likelihood method and the Tamura-Nei model with a 1,000 replications of the bootstrap test. The translated protein sequences used the maximum likelihood method and the Jones-Taylor-Thornton model with 1,000 replications of the bootstrap test to construct the AA phylogenetic tree. Analysis of variations in the multiple sequence alignment was carried out on nucleotide and translated protein sequences. Nucleotide and AA phylogenetic trees were built to determine the relationship of the N gene of SARS-CoV-2 from ASEAN countries.

 

 

Ninety eight isolates represented the SARS-CoV-2 N protein complete coding sequences (each sequence was 1,259 nucleotides in length) were collected (Table 1). No isolate was found in the clade GV from ASEAN countries, and there were no data from Laos in the GISAID database (data were collected up to March 2021). Among all isolates, there were 31 nucleotide changes (2.4%) and 97.5% of sequences were being conserved. All of the clade O isolates had substitution mutation at C28311T. Isolates from clade S had substitution mutation at G28378C. All isolates in the GR clade had substitution mutations at position 28881 (GGG to AAC). These mutations caused two AA substitutions (R203K and G204R). All isolates from clade GRY had four substitutions, making them branched out from the GR clade. The substitutions were at position 28280–28282 (GAT to CTA) and C28977T (Table 1). Other isolates which do not undergo any nucleotide changes can be found in Table 2.

 

 

 

 

The nucleotide sequences were then translated into AAs with appropriate codon and displayed the similarities and positions between all clades (Figure 1). Of all AA sequences, 94.5% were conserved with 22 sites of AA substitution (non-synonymous mutation) (Table 1).

Twelve out of 22 non-synonymous mutations resulted in polarity changes of the corresponding AAs (Table 1). Of 12 non-synonymous mutations, 9 were changed from polar AA to non-polar AA, and 3 substitutions changed from non-polar AA to polar AA. Ten isolates in the clade O had the P13L (substitution of proline to leucine at position 13), which both were non-polar AAs.

Based on the phylogenetic tree of the N gene nucleotide sequence (Figure 1a), all isolates from clade G, clade GH, clade L, clade O, clade S, and clade V, together with the reference sequence, did not form specific branches. All isolates originated from clade O formed one specific branch due to substitution mutations at position 28311. Moreover, isolate Malaysia | EPI_ISL_528743 | O had additional nucleotides changes at position 29086 and 29218 (Table 1), making additional branch arose from clade O. All isolates in clade GR formed a separate branch (Figure 1a) due to nucleotide substitutions at position 28881 to 28883 (Table 1). The entire isolates of clade GRY were branched out from the clade GR (Figure 1a), thus formed one branch due to nucleotide substitutions at position 28280 to 28282 (GAT to CTA) and position 28977 (C to T) (Table 1). The AA phylogenetic tree formed similar branches to the phylogenetic tree of nucleotide sequences (Figure 1b). The entire sequences in clade O formed a branch due to the P13L AA substitution (Table 1). Meanwhile, all isolates within the GR clade formed a branch due to AA substitutions in R203K and G204R (Table 1). The isolates of the GRY clade was branched out from the GR clade (Figure 1b) because of AA substitutions of D3L and S235F (Table 1).

 

 

 

 

As the COVID-19 pandemic continues, there is a fast, ongoing transmission of SARS-CoV-2 across the world.¹⁶ This study analyzed 98 sequences of the N protein gene of SARS-CoV-2 from ASEAN countries. The association of 10 countries in Southeast Asia (ASEAN countries) has enabled the ease of mobility within the internal regions. Thus, it promotes the virus to spread across the countries. Since human migration is a significant factor in viral evolution, investigating the possible mutations of the circulating SARS-CoV-2 in ASEAN countries is crucial.¹³ The phenomena of human migration naturally drive the virus to adapt to various host immune system and geographic condition with several mechanisms, including mutations, deletions, and/or recombinations.16

Overall, the nucleotides and AAs sequences of SARS-CoV-2 N protein (1,259 nucleotides and 419 AAs) across ASEAN countries are highly conserved (97.5% of conserved nucleotides and 94.5% of conserved AAs). These findings support the previous report on the N protein gene as a more conserved and stable gene.10 Meanwhile, a study of 61,485 N protein gene sequences across the world by Rahman et al17 found that the mutations of the N protein gene sequences occurred more frequently, with 75.66% of AA positions underwent evolutionary changes in the SARS-CoV‐2 nucleocapsid. However, as we specifically analyze the SARS-CoV-2 N protein sequences within ASEAN countries, our finding might raise the question of whether any specific region in which N protein is more conserved compared with other countries. Although the mutation frequency on N gene sequences is high, Rahman et al17 found no substitution of AAs compared with the reference in 29 countries and/or regions. These contrary findings highlight a challenge in designing SARS-CoV-2 N protein for vaccine candidates and diagnostic tools. The nucleotide variations within N sequences of concern were the substitutions at position 28881 (GGG to AAC), which were found in clade GR and GRY. Additionally, nucleotide substitution C28887T was found in some clade G and GH isolates. All of these variations were included in the target for forwarding primer of the N gene from China Centers for Disease Control and Prevention.18 A study has found mismatches in the primer binding region because of nucleotide variations, leading to a false-negative result of N gene detection by polymerase chain reaction assay.19

The phylogenetic tree of the SARS-CoV-2 N protein across ASEAN countries showed a similar branching pattern between the nucleotide and AA tree. The phylogenetic tree of AA sequences showed a more even branching pattern, suggesting variations within the nucleotide sequences mostly did not impact AA substitutions. Thus, the sequences of the N protein gene analyzed in this study are conserved. There were three clades that consistently grouped as one clade: clade O (n = 10), clade GR (n = 13), and clade GRY (n = 7) (Figure 1a), whereas the remaining isolates (n = 68) consisted of clades G, GH, L, S, and V along with the reference sequence or “wild type” did not form specific branches according to the original clade. This pattern suggests the N gene sequence insufficiently represents the nucleotide polymorphisms that composed the grouping of each clade. The O clade forms its branch characterized by AA substitution P13L, while the GR clade exhibits two AA substitutions R203K and G204R. The R203K and G204R mutations were known to be implicated in destabilized and decreased general structural flexibility of the N protein.19 Clade GRY was separated from the GR clade by other AA substitutions D3L and S235F.

Multiple sequence alignment of AA sequences found 12 non-synonymous mutations which contribute to AA’s polarity changes (Table 1). The R-groups of non-polar AAs have either aliphatic or aromatic groups, making them to have hydrophobic features. Hydrophobic AAs tend to repel water, making them structurally buried in the hydrophobic core of the protein and less exposed to the surface, thus less antigenic.20 AA changes in the antibody-antigen interactions play an important role in the maturation of antibody affinity responses and antigenic variations. AA substitutions in the antibody-antigen interaction interface are important in a biological context. In particular, antigen changes can affect the entire antigen interaction with the antibody, providing an effective means of antigenic variation.21 Moreover, antigenic variation within N protein could affect the performance of the antigen-based rapid diagnostic test since this protein is often used as the target analyte.22 Thus, any site with changes in the AA polarity warrants further attention to study its impact on the antigenicity of the corresponding site.

This study has limitations since there were only 98 sequences from ASEAN countries. A more robust study with larger samples that cover many regions would better describe the N gene variations. Furthermore, a study describing the structural prediction of the N protein is suggested to confirm the impact of AA variations on antigenicity.

In conclusion, the N protein sequence of SARS-CoV-2 across ASEAN countries showed high similarity and indicated the N protein gene conservation within ASEAN countries. Based on the nucleotide and AA phylogenetic tree, most isolates were closely related to the reference sequence (China).

 

 

 

 

 

 

 

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