Abstract

Autophagy is a natural cellular mechanism in which cellular components such as long-lived proteins and damaged organelles are degraded in response to starvation by forming autophagosomes. Viruses activate the autophagy process, which generates innate immune protection in the host against infection. While the actual molecular mechanism of this contagious viral infection remains unknown, studies on some other betacoronavirus show that their infection of host cells inhibits the autophagy process, resulting in autophagosome accumulation inside the cells. Non-structural protein 6 (NSP6) is crucial in blocking autophagosomes/autolysosome vesicle formation, which are more numerous and smaller than autophagosomes formed upon starvation. Because of its vital role in autophagy, NSP6 can be used as an effective drug target to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Therefore, this study aims to detect the mutations in the NSP6 of Indian isolates compared to Wuhan-type isolates. The NSP6 protein of Indiantype SARS-CoV-2 isolates contained 654 point mutations. Furthermore, secondary structure, energy change upon mutation, physicochemical properties, and hydropathy index of wild and mutated proteins were compared, clearly showing that mutations altered NSP6 stability. An immunoinformatics approach was also attempted to identify the B-cell and interferon (IFN)-inducing epitopes for using NSP6 as a probable vaccine candidate. Therefore, this study explored an important drug target (NSP6) essential for autophagy and assembly of coronavirus replicase proteins.

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic virus that caused this current devastating pandemic disease. The SARS-CoV-2 originated in Wuhan, China, and now can be found in > 210 countries across the globe. The World Health Organization classified SARS-CoV-2 as a pandemic of global concern on March 11, 20201, 2, 3. Since the emergence of this contagious disease, numerous novel studies and several research findings have been published on approaches to curb this pandemic. However, we are still behind in developing effective and durable antiviral therapeutics to combat the COVID-19 outbreak.

SARS-CoV-2 belongs to the family Coronaviridae and order Nidovirales. This virus is supposed to be the third zoonotic coronavirus originating from bats after SARS-CoV and the Middle East respiratory syndrome-related coronavirus (MERS-CoV). However, SARS-CoV-2 is the only novel coronavirus with pandemic potential4, 5, 6, 7, 8. This coronavirus is an enveloped single-stranded RNA virus with a genome of 29,891 nucleotides encoding 9680 amino acids9, 10, 11, 12. The genome of SARS-CoV-2 encodes structural or non structural proteins (NSP1 to NSP16)11, 13, 14. The NSPs are involved in RNA replication and processing, such as the RNA-dependent RNA polymerase (RDRP/NSP12) and helicase (NSP13). However, the functions of some NSPs remain unknown. Among the NSPs, NSP6 is a crucial component of membrane protein (34 KDa), comprising seven transmembrane helices and a conserved C terminus domain. NSP6 induces the autophagy process through the omegasome activation pathway15. NSP6 plays an important role in blocking autophagosomes/autolysosome vesicle formation that are more numerous and smaller than the autophagosomes induced upon starvation16. This condition might be favorable for SARS-CoV-2 to limit the autophagosome delivery of viral components to the lysosome for degradation.

Present study aim to investigate the effect of mutations on the structure of the NSP6 protein from Indian-type SARS-CoV-2 isolates and compare them with the Wuhan-type isolates. Significant alterations were found in the biochemical, immunological, and structural properties of SARS-CoV-2 NSP6 protein. Therefore, this study explored an important protein (NSP6) essential for autophagy and assembly of SARS-CoV-2 replicase proteins.

Methods Collection of protein sequences

The sequence data for the SARS-CoV-2 NSP6 protein (290 amino acids long) was retrieved from The US National Center for Biotechnology Information (NCBI) virus database. These sequences were filtered for those originating from India from the onset of this disease to August 15, 2022. The Wuhan SARS-CoV-2 virus sequence was used as the reference (accession number: YP_009724389) in the mutational analysis17.

Multiple sequence alignment and mutant identification

NSP6 protein sequences were aligned using CLUSTAL Omega, an online server performing alignment using hidden Markov model profiling18, with the Wuhan-type virus sequence as the reference. Jalview, an alignment viewing software, was used to detect the differences in the NSP6 region with the accession number of the Indian SARS-CoV-2 isolates. The nonsynonymous amino acid substitutions were analyzed using the Protein Variation Effect Analyzer (PROVEAN; v1.1.3) software with a cutoff score of −2.5019. The effect of NSP6 protein mutation on its stability could be inferred from the generated score.

Prediction of NSP6 protein structure

The model of NSP6 protein was predicted using Swiss-Model online web servers. The I-Tasser software was also used to predict the NSP6 protein model. The secondary structure of the NSP6 protein was predicted using the PSIPRED online web tool. The NSP6 protein sequences were examined using the Chou and Fasman secondary structure prediction (CFSSP) online software to detect differences in the secondary structure due to a mutation20. Secondary structure prediction tools were used to analyze alterations in the formation or loss of secondary structures.

Determination of the NSP6 protein’s physicochemical parameters

The NSP6 protein’s physicochemical parameters were determined using ExPASy’s online ProtParam tool. The NSP6 protein’s hydrophobicity was calculated using ExPASy’s Protscale tool21.

Variation in protein stability after mutation

The changes in protein stability after mutation were predicted using the I-Mutant 2.0 online program, which predicts stability in terms of the Gibbs free energy change (ΔG) values, where the negative ΔG values suggest a decrease in flexibility and positive values indicate a gain in flexibility after mutation of the wild-type protein.

B-cell epitope and interferon (IFN)-inducing epitope prediction

The B-cell epitopes of the NSP6 protein were estimated using the Immune Epitope Database (IEDB)22 and Bepipred 2.0 server23. The IFN-inducing epitopes were predicted by IFN epitope24.

T-cell epitope prediction and major histocompatibility complex (MHC) restriction

The IEDB17 Tepitool server was used to predict the T-cell binding epitope and the MHC allele. The binding of processed antigen to an MHC molecule is essential for T-cell recognition and eliciting an immune response.

Determination of the NSP6 protein’s antigenicity and allergenicity

The NSP6 protein’s antigenicity was predicted using the Vaxijen v2.0 server25, and its allergenic properties were determined using the AllerTOP server26.

Results Collection of target protein sequences for mutational analysis

The NSP6 protein sequences of Indian-type SARS-CoV-2 isolates from the onset of this disease to August 15, 2022, were retrieved from the NCBI database along with the first SARS-CoV-2 virus sequence. In the above period, 548 NSP6 protein sequences were submitted of Indian-type isolates. These sequences were aligned to detect variations in the NSP6 protein region of Indian-type isolates relative to a Wuhan-type isolate. Those mutations in the NSP6 region were recorded with the accession number of the isolate and the mutated base in the wild-type and mutated sequences. The NSP6 region contained 654 point mutations in Indian-type SARS-CoV-2 (Supplementary Table 1). Nine mutations were frequently observed in the NSP6 protein: T77A, V149A, L37F, T181I, V190F, L125F, M183I, and V153C (Supplementary Figure S1).

Nonsynonymous mutations in the SARS-CoV-2 NSP6 protein

Of these 654 mutations, five (I49T, M86I, L142F, I162T, and V190I) were selected for further characterization. The five nonsynonymous amino acid substitutions showed similar effects on the structure of the NSP6 protein. The five NSP6 protein mutants showed a neutral impact at a cutoff PROVEAN score of −2.5 (Table 1).

Table 1.

List of 5 nonsynonymous amino acid substitutions in nsp6 protein (cut off= -2.5)

Amino acid substitution in the helicase region PROVEAN score Variation effect on protein I49T -1.698 Neutral M86I -0.427 Neutral L142F -1.244 Neutral I162T -1.474 Neutral V190I -0.486 Neutral

Table 2.

Showing the effect of mutation on energy change (ΔG) in the mutated nsp6 as compared to wild type nsp6

S.No. Accession No. Position of mutation Wild sequence Mutated sequence ΔG Protein Stability 1. QKY59987 49 A T -2.80 Decreases 2. QKV26075 86 M I -1.02 Decreases 3. QKV26087 142 L F -0.66 Decreases 4. QKJ68675 162 I T -4.61 Decreases 5. QKJ68699 162 I T -4.61 Decreases 6. QKV25895 190