Epidemiology and Infection ORF3a mutation associated with higher mortality rate in SARS-CoV-2 infection cambridge.org/hyg Parinita Majumdar and Sougata Niyogi Original Paper Independent Researcher Cite this article: Majumdar P, Niyogi S (2020). Abstract ORF3a mutation associated with higher mortality rate in SARS-CoV-2 infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has recently caused acute Epidemiology and Infection 148, e262, 1–6. respiratory distress syndrome affecting more than 200 countries with varied mortality rate. https://doi.org/10.1017/S0950268820002599 Successive genetic variants of SARS-CoV-2 become evident across the globe immediately after its complete genome sequencing. Here, we found a decent association of SARS-CoV-2 Received: 3 June 2020 ORF3a mutation with higher mortality rate. Extensive in silico studies revealed several Revised: 19 September 2020 Accepted: 14 October 2020 amino acid changes in ORF3a protein which ultimately leads to diverse structural modifica- tions like B cell epitope loss, gain/loss of phosphorylation site and loss of leucine zipper motif. Key words: We could further relate these changes to the enhanced antigenic diversity of SARS-CoV-2. COVID-19; ORF3a; mortality; mutation; SARS- Through protein−protein network analysis and functional annotation studies, we obtained CoV-2 a close federation of ORF3a protein with host immune response via divergent signal transduc- Abbreviations: tion pathways including JAK-STAT, chemokine and cytokine-related pathways. Our data not ARDS: acute respiratory distress syndrome; only unveil the fairly appreciable association of ORF3a mutation with higher mortality rate, CAV1: caveolin 1; CCND1: cyclin D1; CoV: but also suggest a potential mechanistic insight towards the immunopathogenic manifestation coronavirus; COVID-19: coronavirus disease 2019; DAVID: database for annotation, of SARS-CoV-2 infection. visualisation and integrated discovery; EGFR: epidermal growth factor receptor; IEDB-AR: immune epitope database-analysis resource; IL-6: interleukin 6; JAK: janus kinase; JNK: c- Introduction Jun N-terminal kinase; KEGG: Kyoto encyclopedia for gene and genomes; MAPK1: The betacoronaviruses of Coronaviridae family are potent human pathogens of zoonotic ori- mitogen-activated protein kinase 1; MERS: gin [1, 2]. Till now, human coronaviruses (CoVs) have been associated with three major out- middle-east respiratory syndrome; NF-κB: nuclear factor kappa-light-chain-enhancer of breaks of acute respiratory disorders namely severe acute respiratory syndrome (SARS) in activated B cells; NOS3: nitric oxide synthase 2002, middle-east respiratory syndrome (MERS) in 2012 and COVID-19 in 2019 3; NSPs: non-structural proteins; ORF: open (Coronavirus disease 2019) with an overall mortality rate of 9.6%, 40% and 6.9%, respectively reading frame; RdRp: RNA-dependent RNA [1, 3-5, https://www.worldometers.info/coronavirus/]. COVID-19 originated at Wuhan city of polymerase; SARS: severe acute respiratory Central China in mid-November 2019, gradually spread to more than 200 countries with a syndrome; SOCS3: suppressor of cytokine signalling 3; STAT3: signal transducer and death toll of 2,44,664 as on 2 May 2020 [5, https://www.worldometers.info/coronavirus/]. activator of transcription 3 The complete genome sequence of the causative organism was first reported from China and found to be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which Author for correspondence: bears 79% and 50% genetic similarities with SARS-CoV and MERS-CoV [2, 5]. The unusually Dr. Sougata Niyogi, ∼ E-mail: [email protected] larger, 30 kb genome of SARS-CoV-2 has similar genomic organisation with SARS-CoV and encodes 16 non-structural proteins (NSP1−NSP16), 4 structural proteins including Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) proteins and 6 accessory proteins (ORF3a, ORF6, ORF7a, ORF7b, ORF8 and ORF10) [2, 4-6]. The non-structural proteins of CoVs are indispensable for viral replication and transcription whereas the structural proteins constitute the mature virion [2, 5]. The role of accessory proteins has just begun to be evident in viral pathogenesis [7-9]. Among the accessory proteins, ORF3a is the largest one containing 274 amino acids in SARS-CoV [9]. Cell surface localisation of ORF3a in SARS-CoV potentiates viral entry within the host and has immunogenic properties [3, 9]. Moreover, it is involved in pro-inflammatory cytokine and chemokine production by activating various signal transduction pathways including C-Jun N-terminal kinase (JNK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [10]. An uncontrolled release of pro-inflammatory cytokines and chemokines results in an exacerbated host immune response in SARS-CoV-2 infected patients leading to cytokine storm [11]. ORF3a is also implicated in © The Author(s), 2020. Published by ion channel formation and modulates release of virus from the host cell [9]. Cambridge University Press. This is an Open Soon after the isolation of Wuhan strain (EPI_ISL_402123), an increasing number of Access article, distributed under the terms of SARS-CoV-2 variants with genetic diversity has started to emerge across the globe [4-6, the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), 12]. The large genome size of SARS-CoV-2 and low fidelity RNA-dependent RNA polymerase which permits unrestricted re-use, (RdRp) are the contributing factors for considerable higher rate of adaptive mutations leading distribution, and reproduction in any medium, to the evolution and coexistence of pathogenic viral strains worldwide [4, 6, 13]. Several inde- provided the original work is properly cited. pendent recurrent mutations in NSP6, NSP7, NSP12, NSP13 encoded by ORF1ab and spike protein resulting in non-synonymous amino acid mutations are identified as mutational hot- spot which are closely associated with inter-species transmission and virulence [4, 6, 14-15]. Thus it is intriguing to understand the functional implications of different mutations in the diverse infection and mortality rate of SARS-CoV-2. Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.219, on 27 Sep 2021 at 16:14:10, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002599 2 Parinita Majumdar and Sougata Niyogi Fig. 1. SARS-CoV-2 ORF3a mutations is associated with higher infection and mortality rate: (a) Overall screening scheme of the study. (b) Tornedo plot showing differential infection and death rate (%) of COVID-19 for indicated countries. (c) Genomic organisation of SARS-CoV-2 depicting protein coding ORFs. (d) Stacked bar chart showing mutation frequency at indicated ORFs for respective countries. Methods Sequence alignment Study design and data collection Amino acid sequences of ORF3a protein (NP_828852.2 and YP_009724391.1 for SARS-CoV and SARS-CoV-2, respectively) All data were collected from coronavirus worldometer section in FASTA format were downloaded from NCBI protein database (https://www.worldometers.info/coronavirus/) as on 2 May and pair-wise sequence alignment was performed in EMBOSS 2020. Initially, 23 countries were selected with a minimum posi- Needle web server with default parameters [16]. tive 20,000 case reports. Percent infection was defined as (total cases/total tests) × 100 and % death was calculated as (total deaths/total cases) × 100 (online Supplementary table 1). Fifteen Epitope and biological function prediction countries with either lower (group I) or higher (group II) values (both % infection and % death) were shortlisted for further ana- Amino acid sequences of concerned proteins were given to lysis (Fig. 1a and b). Wuhan strain from China (EPI_ISL_402123) IEDB-AR (Immune Epitope Database Analysis Resource) for B was included as a reference for most of the analysis. cell epitope prediction using default parameters and 0.55 thresh- old value [17]. Biological significance of any mutation was deter- mined using PROVEAN web tool (v1.1.3). FASTA sequence of a Mutation frequency calculation wild-type query protein and mutant variants were uploaded. Amino acid changes in SARS-CoV-2 strains for each country were Functional relevance was predicted with default settings [18]. extracted from NextStrain open source project (https://nextstrain. org/ncov)[12]. Mutation frequency was calculated by dividing the Protein structure prediction number of mutations for a particular protein with total number of mutations corresponding to all the proteins for a given country, Amino acid sequences of ORF3a protein (NP_828852.2 and multiplied by 100. The mutation frequency was represented by YP_009724391.1 for SARS-CoV and SARS-CoV-2, respectively) stacked bar chart which was created using Microsoft Excel. in FASTA format were further scanned in ExPASy-prosite Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.219, on 27 Sep 2021 at 16:14:10, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268820002599 Epidemiology and Infection 3 (prosite.expasy.org) portal for motif analysis. Tertiary structures differential infection and mortality rate of SARS-CoV-2 infection for ORF3a protein were obtained from Phyre2 web portal upon (Fig. 1b). amino acid sequence submission (FASTA). Biological significance of ORF3a mutations in SARS-CoV-2 Viral protein interactome study The association