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Article

Functional and Evolutionary Characterization of the NSP6 Protein in SARS-CoV-2 Omicron Variants

by
Joyhare Barbosa Souza
and
Samir Mansour Moraes Casseb
*
Oncology Research Center, Federal University of Pará, Belém 66075-110, Brazil
*
Author to whom correspondence should be addressed.
SynBio 2025, 3(2), 7; https://doi.org/10.3390/synbio3020007
Submission received: 30 September 2024 / Revised: 22 April 2025 / Accepted: 23 April 2025 / Published: 27 April 2025
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Abstract

:
The SARS-CoV-2 virus, which causes COVID-19, has rapidly evolved, producing highly transmissible variants like Omicron. Non-structural protein 6 (NSP6) is essential for viral replication and immune evasion. This study analyzed the NSP6 protein of the Omicron variant, focusing on conserved motifs, mutations, and residual properties to better understand its structure, function, and potential for immune evasion. Sequences from humans in South America were obtained from GISAID and aligned using Clustal Omega 1.2.4, with mutations identified by a Python 3 algorithm and conserved motifs detected using the MEME tool. Sequence diversity was assessed with Shannon’s entropy, while hydrophilicity, flexibility, accessibility, and antigenicity were analyzed using EMBOSS PEPSTATS and Expasy’s ProtScale tools. Phylogenetic analysis was performed with IQ-TREE software. Analysis of 161 NSP6 protein sequences revealed significant divergence from the reference sequence, with mutations proximal to conserved regions indicating potential functional and structural changes. The analysis also identified distinct hydrophobic and hydrophilic regions, with specific amino acid positions showing high flexibility and antigenicity. Phylogenetic analysis identified three clades with varying degrees of similarity to the reference sequence. This comprehensive study of the NSP6 protein in the Omicron variant provides insights into its role in viral replication and immune evasion, contributing to the development of targeted interventions against COVID-19.

1. Introduction

SARS-CoV-2 is a highly infectious virus that emerged in Wuhan, China, at the end of 2019 and rapidly spread to become a global pandemic. It causes the disease COVID-19, which can range in symptom severity from mild to severe and may be fatal in some cases. COVID-19 has already affected more than 460 million people and caused over 6 million deaths worldwide [1].
SARS-CoV-2 is a member of the Coronaviridae family, which includes other viruses that have caused outbreaks in the past, such as SARS-CoV and MERS-CoV. Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with a genome approximately 27–32 kb in length [2].
The SARS-CoV-2 viral particle consists of a helical nucleocapsid enclosed by an envelope that contains three structural proteins: spike (S), envelope (E), and membrane (M). The S protein projects from the viral surface, binding to the host cell receptor and facilitating viral entry into the cell. The E protein is involved in virus assembly and release, while the M protein is the most abundant protein in the virus and plays a critical role in virus assembly. The nucleocapsid consists of the viral RNA genome tightly bound to the nucleocapsid protein (N) [3].
The S protein is a large glycoprotein that projects from the viral surface and is responsible for binding to the host cell receptor and facilitating viral entry into the cell. It consists of two subunits, S1 and S2, responsible for receptor binding and fusion, respectively. The S protein mediates the virus’s attachment to host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, which is expressed on the surface of human cells in various tissues, including the respiratory tract, heart, kidneys, and intestines. The binding of the S protein to ACE2 is the first step for the virus to enter host cells, a process essential for establishing infection [4].
The E protein is a small transmembrane protein involved in virus assembly and release. It plays a critical role in the formation of viral particles, including the budding of the virus from the host cell membrane. It is also believed that the E protein may modulate the host immune response to the virus [5].
The M protein is the most abundant protein in the virus and is critical for virus assembly. It is a transmembrane protein that interacts with the nucleocapsid and envelope proteins and is necessary for forming the viral particle. The M protein also plays a role in viral entry by promoting the fusion of viral and host cell membranes [6].
The N protein is a highly conserved protein essential for the packaging and replication of the viral genome. It binds to the viral RNA genome and forms the helical nucleocapsid surrounded by the viral envelope. The N protein is also thought to play a role in suppressing the host’s immune response to the virus [7].
In addition to structural proteins, SARS-CoV-2 produces several non-structural proteins (NSPs). NSPs are a group of proteins produced by SARS-CoV-2 that are not part of the viral particle’s structure but are essential for viral replication and evasion of the host immune system. The virus produces these proteins during infection, and they are critical to the viral life cycle. The SARS-CoV-2 genome encodes 16 NSPs, which are translated from a polyprotein precursor cleaved by viral proteases during replication. NSPs are categorized based on their role in viral replication and evasion of the host immune system [8,9].
The RdRp (RNA-Dependent RNA Polymerase) is a key NSP responsible for the synthesis of viral RNA during replication; the RNA-synthesizing machinery in coronaviruses requires incorporation of RdRP together with other key NSPs to form a fully functional polymerase complex. It catalyzes the synthesis of new RNA strands using the viral RNA genome as a template. RdRp is a critical target for antiviral drugs, and several medications, including remdesivir. It was initially developed to treat hepatitis C, later explored for its potential against Ebola virus disease and Marburg virus infections, and eventually studied as a treatment for COVID-19 after infection [10].
Helicase is another essential NSP for viral replication. It unwinds double-stranded RNA and DNA structures to allow the synthesis of new RNA strands. It is necessary for viral RNA synthesis, and inhibitors of this enzyme are being investigated as potential treatments for COVID-19 [11]. The NSP6 is a small protein with a molecular weight of approximately 8 kDa. It is encoded by the ORF1a and ORF1b genes of the viral genome and is a transmembrane protein essential for viral replication, believed to play a critical role in the formation of the viral replication complex [12].
A NSP6 protein can be found in the endoplasmic reticulum (ER) and Golgi apparatus of infected cells from SARS-CoV-2 and has two transmembrane domains anchoring it to the ER membrane, with N- and C-terminal domains located in the cytosol. The cytosolic domain of NSP6 is highly conserved among different coronaviruses and is believed to be critical for its role in viral replication [13].
Recent studies have shown that NSP6 plays a key role in forming the viral replication complex. This complex is a structure formed within infected cells that facilitates viral genome replication and the production of new viral particles. Studies suggest that NSP6 is involved in forming double-membrane vesicles (DMVs) that comprise the viral replication complex. It is believed that the DMVs are formed from the ER, and NSP6 has been shown to interact with other viral proteins, including NSP3 and NSP4, to drive DMV formation [14,15,16,17].
NSP6 has also been associated with SARS-CoV-2’s evasion of the host immune system. It has been shown to interact with the host protein Beclin-1, which is involved in the autophagy pathway [18,19,20]. Autophagy is a process by which cells break down damaged or unwanted cellular components, including invading viruses. NSP6 has been shown to interfere with the autophagy pathway by interacting with Beclin-1, which may allow the virus to evade detection and elimination by the host immune system [21,22,23,24].
Overall, NSP6 is an essential protein for SARS-CoV-2 replication and pathogenesis. It is involved in forming the viral replication complex and is believed to play a role in evading the host immune system [25,26,27,28,29,30]. Research is needed to fully understand the function of NSP6 and develop treatments and vaccines targeting this essential protein [31,32,33,34]. Additionally, studies demonstrate how mutations in the NSP6 region are necessary for viral replication [35,36,37].
It is expected that the results of this in silico proteomic analysis will provide important insights into the implications of NSP6 mutations in terms of pathogenicity and immune response. This could assist in developing more effective therapeutic strategies and in the ongoing monitoring of the spread and evolution of the SARS-CoV-2 virus.

2. Results

A total of 161 amino acid sequences of the NSP6 protein from Omicron variants isolated from humans in eight South American countries were aligned (Figure 1). After alignment curation, 20 representative sequences were obtained to assess the level of conservation and amino acid divergence compared to the reference sequence. Among these 20 sequences, those with the highest divergence were selected for further analysis. A Python algorithm was developed to identify the number of mutated amino acids and their positions relative to the reference sequence.
The analysis of conserved motifs showed that the mutations occur near conserved regions, which may cause functional and structural modifications in these regions. The hydrophilicity assessment indicated that only the amino acids between positions 100 and 150, and at position 250, were considered hydrophilic. The hydrophobicity analysis revealed that a large part of the sequences has an affinity with lipid environments, suggesting an association with cell membranes (Figure 2).
The flexibility of the sequences was mainly observed between positions 4 and 50, and at position 250. The analysis also showed a strong tendency for the formation of transmembrane structures across most of the sequence length, suggesting a high capacity for the protein to interact with its environment (Figure 1).
In the analysis of residual properties, indices such as molecular weight and average molecular weight assess amino acid density, while the isoelectric point determines the pH value at which the protein has a net zero charge, meaning the quantity of positive charges is equal to the quantity of negative charges (Figure 3).
In the phylogenetic analysis, three clades were generated, corresponding to different levels of similarity with the reference sequence: one group with over 98% similarity, another with less than 94%, and a third with less than 80%, representing the most divergent sequences. The antigenicity analysis identified the positions of amino acids with the highest potential for B-cell recognition within a 150-amino acid window.
Antigenicity analysis was conducted based on the prediction of potential linear B-cell epitopes. A sliding window of 150 amino acid positions was established across the sequences. By converging the scores of all previously elucidated physicochemical properties, we were able to identify amino acids and their corresponding positions with the highest antigenicity potential.

3. Discussion

In Figure 1A, hydrophilicity is calculated according to scale, which follows the principle that higher values indicate greater hydrophilicity, meaning a higher affinity for aqueous environments. The Hopp and Woods scale assigns numerical values to each amino acid based on its hydrophilic properties. According to the values found, only the amino acids between positions 100 and 150, and at position 250, were considered hydrophilic on the scale, which evaluated eight amino acid sequences from eight South American countries compared to the reference sequence from Wuhan [35,36,37,38,39].
These data support the information from graph C, where hydrophobicity is assessed using the same scale, and a large portion of the amino acid sequences is considered hydrophobic, suggesting a greater affinity of the NSP6 amino acids for lipid environments such as cell membranes [40,41,42,43,44,45,46,47].
The flexibility property of a protein sequence refers to the sequence’s ability to bend, twist, or adjust its three-dimensional conformation in response to environmental conditions or specific interactions [48,49,50,51,52,53]. According to the information from graph B, most sequences showed high flexibility scores between positions 4 and 50, and at position 250.
The tendency to form transmembrane structures received higher scores in the amino acids throughout most of the analyzed amino acid window. The tendency to form transmembrane structures in amino acid sequences relates to the accessibility of the protein’s three-dimensional structure, with high scores in this residual analysis suggesting a high potential for interaction with the surrounding environment [54,55].
In the context of the COVID-19 vaccine, the NSP6 protein plays an important role in regulating the immune response and modulating cellular functions. Changes in the hydrophobicity and hydrophilicity of NSP6 may impact its ability to interact with cell membranes and, consequently, influence the efficacy of vaccines that use viral proteins as a base. Therefore, it is essential to understand how the biochemical characteristics of this protein evolve, especially in critical regions such as those highlighted in the graphs [54].
Mutations observed in NSP6, such as those in the regions between positions 100 and 150, and position 250, may alter its functionality and pathogenic potential. Alterations that increase hydrophobicity may favor its association with cell membranes, impacting its ability to form transmembrane structures and interfering with intracellular trafficking, a mechanism often associated with immune evasion. These changes may require adjustments in vaccine formulation to ensure that immune responses are adequate to emerging variants [55].
Studies suggest that specific mutations in NSP6 can enhance the virus’s ability to evade the host’s immune system. By interfering with the immune response, these mutations can render the virus more virulent, i.e., capable of causing more severe disease. Additionally, mutations in NSP6 can also affect the virus’s ability to replicate and spread within the host, contributing to increased viral load and exacerbating COVID-19 symptoms [20,21,22].
Previous studies on other coronaviruses reinforce the idea that viral proteins play crucial roles in manipulating the host to favor viral replication. For instance, in the Infectious Bronchitis Virus (IBV), the nucleocapsid protein (N) localizes to the nucleolus of infected cells, suggesting interference with cellular functions, such as RNA processing, which may benefit the viral cycle [56]. Although the NSP6 protein of SARS-CoV-2 operates in a distinct compartment, the endoplasmic reticulum, where it facilitates the formation of double-membrane vesicles (DMVs) and interacts with Beclin-1 to inhibit autophagy [14], both proteins exemplify a conserved strategy among Coronaviridae to subvert host cellular mechanisms. This comparison underscores the importance of historical studies on coronaviruses in contextualizing the evolution of viral protein functions, such as NSP6, and suggests that understanding these interactions may guide the development of targeted antiviral therapies.
It is important to note that the relationship between NSP6 mutations and increased SARS-CoV-2 virulence is complex and not yet fully understood. Other factors, such as the presence of other mutations in the viral genome and individual host characteristics, can also influence disease severity. Ongoing research in this area is crucial for a better understanding of the molecular mechanisms underlying SARS-CoV-2 evolution and for developing novel strategies to combat COVID-19 [38,57,58].

4. Materials and Methods

4.1. Database and Alignment

All 148 available sequences of Omicron variants of SARS-CoV-2 isolated in humans were downloaded from the GISAID database https://www.gisaid.org/ (accessed on 4 March 2024); a dataset was constructed including human sequences and excluding sequences from animals (such as bats or pangolins). The dataset was aligned and manually edited using the Clustal Omega amino acid alignment tool. The complete dataset was subjected to analysis of conserved motifs through the MEME tool (https://meme-suite.org/meme/tools/meme, accessed on 4 March 2024).

4.2. Inclusion and Exclusion Criteria

As inclusion criteria, only Omicron variants (BA.1) from human samples isolated in populous countries of South America, whose complete nucleotide coding sequence of the NSP6 protein has been deposited in the GISAID genetic database (available at https://gisaid.org/, accessed on 4 March 2024), will be considered. Regarding exclusion criteria, viral isolates that have not been classified by the Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN) system, sequences isolated from animals (bats or pangolins), and those with partial and/or one or more unresolved amino acids in the NSP6 protein sequence will be disregarded.

4.3. Mutation Identification

From the alignment of the 161 amino acid sequences, a distance matrix was constructed, elucidating the percentage divergence of the sequences in relation to the reference sequence (Wuhan) and their divergence from each other, aiming to identify the most divergent sequences compared to the reference sequence. These were selected for further analyses. A Python algorithm was implemented to identify the number of mutated amino acids in each sequence.

4.4. Conserved Motif Identification

To identify conserved amino acid patterns in the sequences, the MEME tool (Multiple Em for Motif Elicitation), available at (https://meme-suite.org/, accessed on 4 March 2024), was adopted. These patterns can represent functional motifs, such as protein binding sites or regulatory regions that play a specific functional role. These motifs are basic units in biological sequences that are often preserved throughout evolution due to their functional importance.

4.5. Entropy Analysis

A Python script was developed for entropy analysis of the sequences, implementing Shannon’s entropy to assess the diversity or complexity of amino acid sequences in proteins. Shannon’s entropy is a measure of uncertainty or average information in a system. In biological sequence contexts, such as proteins, it is used to evaluate the variability or conservation at amino acid positions.

4.6. Determination of Residual Properties of NSP6 Protein

For the next step, the amino acid sequences obtained through GISAID will be used in another digital tool, EMBOSS PEPSTATS (available at the site https://www.ebi.ac.uk/Tools/seqstats/emboss_pepstats/, accessed on 4 March 2024), to obtain the residual properties of the NSP6 protein of the Omicron variant (BA.1) of SARS-CoV-2, providing data through numerical values. As parameters for the analysis of these sequences, terminal charges of amino acids will be included without considering monoisotopic weights.
Residual properties refer to the type, quantity, and polarity of amino acids composing the protein in question and will be presented as bar graphs, considering the different physicochemical properties of amino acids (apolar, neutral polar, acidic polar, and basic polar).

4.7. Analysis of Hydrophilicity, Flexibility, Accessibility, and Antigenicity of NSP6 Protein

In the following step, the Expasy platform (SIB—Swiss Institute of Bioinformatics), available at the site (https://www.expasy.org//, accessed on 4 March 2024), will be used. Within this environment, for the investigation of hydrophilicity, flexibility, accessibility, and antigenicity of NSP6 protein amino acids, the ProtScale tool, available at the link (https://web.expasy.org/protscale/, accessed on 4 March 2024), will be utilized, providing data on such parameters through tables and graphs. A window size of seven amino acids will be used as a parameter for the analysis of these sequences.
Hydrophilicity represents the ability of polar structures to interact with an aqueous solution; flexibility comprises the ability of molecules to move and change their conformation; accessibility consists of the ability of a molecule to be accessible or not to other molecules for chemical interactions; antigenicity, on the other hand, is the ability of an antigen, in this case a protein, to be recognized by an antibody in the organism, ultimately. Such data will be represented in the form of line graphs, highlighting individual values for each grouping of amino acids in the protein sequence.

5. Conclusions

The present research revealed significant mutations in the amino acid sequences of the non-structural protein 6 (NSP6) of SARS-CoV-2, specifically in the Omicron variants. These mutations show a direct impact on the physicochemical properties of the protein, suggesting potential interference in the pathogenicity of the virus. A detailed analysis of the identified changes indicates possible modifications in the structure and function of NSP6, implying substantial effects on viral replication, immune evasion, or other critical host–pathogen interactions, as suggested by recent studies.
The understanding of these molecular changes provides guidelines for understanding the biology of the Omicron variant and may influence therapeutic and vaccination strategies. Moreover, the identification of specific regions of interest in NSP6 may guide future investigations into pathogenic mechanisms, enabling the development of more precise and effective therapeutic approaches. This research highlights the ongoing dynamics of viral evolution and the importance of monitoring and analyzing emerging variants for a rapid and effective public health response.

Author Contributions

Conceptualization, writing—review and editing, S.M.M.C.; methodology, writing and editing, J.B.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

I would like to express my sincere gratitude to the Federal University of Pará and the Oncology Research Center for their support and for providing the opportunity to conduct this research.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Analysis of the physicochemical properties of the NSP6 protein from SARS-CoV-2 Omicron variants in South America, representing the hydrophilicity of the NSP6 protein sequences. Clustal, EMBOSS PEPSTATS, and Expasy’s ProtScale were used for estimation calculations.
Figure 1. Analysis of the physicochemical properties of the NSP6 protein from SARS-CoV-2 Omicron variants in South America, representing the hydrophilicity of the NSP6 protein sequences. Clustal, EMBOSS PEPSTATS, and Expasy’s ProtScale were used for estimation calculations.
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Figure 2. Analysis of conserved motifs of the NSP6 protein of SARS-CoV-2 Omicron variants isolated from humans in South America from GISAID. Illustration of the conserved graphical motifs identified using the MEME tool, highlighting regions of functional significance.
Figure 2. Analysis of conserved motifs of the NSP6 protein of SARS-CoV-2 Omicron variants isolated from humans in South America from GISAID. Illustration of the conserved graphical motifs identified using the MEME tool, highlighting regions of functional significance.
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Figure 3. Figure containing residual property data for NSP6.
Figure 3. Figure containing residual property data for NSP6.
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Souza, J.B.; Casseb, S.M.M. Functional and Evolutionary Characterization of the NSP6 Protein in SARS-CoV-2 Omicron Variants. SynBio 2025, 3, 7. https://doi.org/10.3390/synbio3020007

AMA Style

Souza JB, Casseb SMM. Functional and Evolutionary Characterization of the NSP6 Protein in SARS-CoV-2 Omicron Variants. SynBio. 2025; 3(2):7. https://doi.org/10.3390/synbio3020007

Chicago/Turabian Style

Souza, Joyhare Barbosa, and Samir Mansour Moraes Casseb. 2025. "Functional and Evolutionary Characterization of the NSP6 Protein in SARS-CoV-2 Omicron Variants" SynBio 3, no. 2: 7. https://doi.org/10.3390/synbio3020007

APA Style

Souza, J. B., & Casseb, S. M. M. (2025). Functional and Evolutionary Characterization of the NSP6 Protein in SARS-CoV-2 Omicron Variants. SynBio, 3(2), 7. https://doi.org/10.3390/synbio3020007

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