Abstract
Analytical observations (in silico) indicate molecular features of SARS-Cov2 genome that potentially explains the high prevalence of asymptomatic cases in Covid-19 pandemic. We observed that the virus maintains a low preference for ‘GGG’ codon for glycine (3%) in its genome. We also observed multiple putative introns of 26–44 nucleotide (nt) length in the genomic region between the coding regions of Nsp10 and RPol in the viral ORF1ab, like several other beta-coronaviruses of similar infectivity levels. It appears that the virus employs a dual strategy to ensure unhindered replication within the host. One of the strategies employ a (− )1 frameshift translation event through programmed ribosomal slippage at the ribosomal slippage site in the ORF1ab. The alternate strategy relies on intron excision to generate a read through frame. The presence of ‘GGG’ in this conserved ribosomal slippage site ensures adequate tRNA in cytoplasm to match the codon, implying no additional frameshift translation due to ribosomal stalling. With fewer replication events, viral load remains low and resulting in asymptomatic cases. We suggest that this strategy is the primary reason for the prevalence of asymptomatic cases in the disease, enabling the virus to spread rapidly.
Similar content being viewed by others
Introduction
The SARS-Cov2 during Covid-19 pandemic that has already infected 151 million people and claimed more than 3.1 million lives across the world in a span of 15 months is undeniably among the greatest crises facing mankind in the century. This novel coronavirus continues to challenge the health care and administrative systems of countries worldwide with its high rate of infectivity (spread) (Aguilar et al. 2020; He et al. 2020; Petersen et al. 2020).
Typical of influenza virus infections, majority of fatalities in SARS-Cov2 infections are observed in people of higher age group (over 65 years) with weakened immune systems. Among persons under 21 years of age, fatality was higher in individuals with preexisting medical conditions as well as very young children (Bixler et al. 2020). Similar to other influenza viruses such as human influenza A (H5N1), SARS-CoV2 infection also induces cytokines storm in host which may cause severe acute respiratory syndrome, multiple organ failure and death (Ratajczak and Kucia 2020; Song et al. 2020). These inflammatory responses are typical of infections with heavy viral load in the hosts for influenza viruses (Boon et al. 2011; De Jong et al. 2006).
Interestingly, a large proportion of the individuals infected with SARS-Cov2 virus are asymptomatic harboring relatively lower viral loads (Zhou et al. 2020) while simultaneously being capable of spreading the infection themselves. It is the strong prevalence of such asymptomatic carriers that make containment measures difficult in the Covid-19 pandemic (Yu and Yang 2020). As per a study on evacuated people from China to Japan, asymptomatic ratio was nearly 30% (Nishiura et al. 2020). Though SARS-Cov2 genome sequence, its mutant and their potential impact on disease management have been investigated (Wu et al. 2020; Leung et al. 2020; Li et al. 2021; Starr et al. 2021), the molecular mechanism behind the prevalence of low viral load and asymptomatic cases is largely unexplored. Here we attempted an in silico dissection of the molecular peculiarities of the SARS-Cov2 viral genome using bioinformatic tools to develop a theoretical hypothesis behind the prevalence of asymptomatic cases in Covid-19.
Materials and method
In silico analysis of the molecular architecture of ORF1ab was carried out for 27 coronaviruses including Middle East respiratory syndrome coronavirus, SARS coronavirus and multiple novel coronavirus isolates (Table 1). ORF1ab of coronaviruses were subjected to Simple Modular Architecture Research Tool (SMART) for identification & annotation of protein domains and architectures (Letunic et al. 2015). Online web server of Sequence Manipulation Suite (https://www.bioinformatics.org/sms2/codon_usage.html) was used to estimate codon usage frequency for each amino acid in each coronavirus genome. Identification of putative introns between ORF1a and ORF1b was done based on standard GT-AG rule, and the presence of branch site (Wu and Krainer 1996). Generunner software (http://www.generunner.net/) was used for in silico sequence analyses viz to check translation frame, identify putative introns and find their length. Excision of identified putative introns and verification of the correctness of the reading frame after rejoining ORF1a and ORF1b was performed in silico using Generunner software(http://www.generunner.net/). Protein Homology/analogy Recognition Engine V 2.0 (Phyre2) is a free web-based services for protein structure modeling, prediction and analysis (Kelley et al., 2015). In silico protein sequence derived from ORF1ab of SARS-Cov2 was subjected to Phyre2 for identification of putative enzymes encoded in genome for RNA splicing.
Results and discussion
This study attempted an in silico exploration of the novel coronavirus genomic features underlying the high prevalence of asymptomatic carriers. A basic feature of the ORF1ab of coronaviruses appears to be the presence of a conserved ribosomal slippage site. Closer examination also reveals that the ribosomal slippage junction of all the studied coronaviruses consistently features a ‘GGG’ codon (Table 1). Now, though this GGG codon at the ribosomal slippage site presents itself within the correct frame, the translating machinery reading through it would invariably encounter a pre-mature termination codon (PTC). In other words, reading through the ‘GGG’ at the ribosomal slippage site disrupts the translation of key proteins such as viral RNA polymerase (RPol), RNA-dependent RNA polymerase (RdRP), helicase, non-structural protein 11 (Nsp11) and Nsp13 located downstream of this junction in ORF1b (Fig. 1). In addition, while introns in ORF1ab are not reported in coronaviruses, we observed multiple putative introns in silico between the coding regions of Nsp10 and RPol based on the standard GT-AG rule (Wu and Krainer 1996). In silico excision of the observed putative introns in this region (that would also remove this 'GGG' codon from the ribosomal slippage site) could place ORF1b with ORF1a in correct frame without affecting the size of the preceding and succeeding domains (Nsp10 and RPol). Intriguingly, reading the viral RNA in a (− )1 frame at this ribosomal slippage site also produces the same result. This molecular position binds the virus to exercise one of the two options for successful translation of ORF1ab for replication in the host— either intron excision by RNA splicing or reading the template from a (− )1 frame at the ribosomal slippage site to generate a read through ORF (Fig. 2).
An interesting twist to this simplistic model is that SARS coronaviruses are known to replicate in the host cell cytoplasm (Klein et al. 2020; Knoops et al. 2008; Snijder et al. 2006; Stertz et al. 2007), while the spliceosome complex required for intron removal reside inside the cell nucleus (Pessa et al. 2008). However, proteins homologous to enzymes of intron excision pathways have been identified from coronaviruses including SARS-Cov (Snijder et al. 2003). Curiously, in silico protein folding prediction models for ORF1b segment of SARS-Cov2 (Accession number: MN908947) polypeptide trained on 2’-O-MT, intron binding protein and pre-mRNA splicing factors also indicate 100% probability of homology (Suppl. file 1, 2, 3 & 4). Read together, based on bioinformatics analysis there is scope to speculate that these viral genomes encode their own splicing enzyme, albeit with limited experimental evidence.
Frameshift translation in eukaryotic systems occurs either by a programmed ribosomal slippage or due to stalling of the ribosomes during a translation event when faced with unavailability of specific tRNA matching the RNA template codon. Programmed ribosomal slippage in association with an RNA pseudoknot has been reported in coronaviruses (Brierley et al. 1989). Curiously, we also observed that coronavirus genomes have a low frequency (10%) of GGG codon usage for glycine (Table 1) compared to other common human viruses (Table 2). The GGG codon usage frequency was especially low for SARS group of viruses, with the lowest inSAS-Cov2 (3%). This would imply that the tRNA corresponding to the ‘GGG’ codon in the viral genome would be abundant in the tRNA pool of the host cell, leading to extremely low probability of ribosomal slippage events. Thus the viral replication in the host would continue to remain at low levels. Intense inflammatory response to influenza-like viral infections leading to clinical disease manifestations is significantly correlated with viral load in the hosts (Boon et al. 2011; De Jong et al. 2006). Thus basal level replication would ensure that the virus triggers negligibly low immune reaction in otherwise healthy hosts, resulting in asymptomatic cases. Indeed, SARS-CoV2 viral load in nasopharyngeal swabs, have been observed to be several fold less in 'asymptomatic patients' than the 'asymptomatic patients in the incubation period' (Zhou et al. 2020). At the same time, these asymptomatic patients also demonstrate a period of viral shedding (Zhou et al. 2020), during which viral transmission is a strong possibility and complicates containment (Yu and Yang 2020).
A similar strategy is observed in Rous sarcoma virus (RSV) where the frameshift site features a stop codon (Jacks et al. 1988). However, by placing a functional codon that has been used sparsely in the genome at the frameshift site, the probability of frameshift translation is further reduced, as in the case of SARS-Cov2. We speculate that this is the reason for the high prevalence of asymptomatic carriers for SARS-CoV2. Strengthening our hypothesis, the closely related MERS beta-coronavirus (GGG codon usage 7%) exhibits quicker progression of disease in infected individuals (Hilgenfeld and Peiris 2013).
In yeast model, natural modification by addition of methyl derivatives on uridines at wobble position promotes decoding of G-ending codon (Johansson et al. 2008). In silico analysis of the ORF1b segment of SARS-Cov2 (Accession number: MN908947) polypeptide predict the presence of an S-adenosyl-L-methionine-dependent methyltransferases domain in the viral genome. Assuming a phenomenon similar to yeast in human cells, this could potentially help in unhindered decoding of other GGG codons in the SARS coronavirus genome despite poor abundance of cytoplasmic tRNA corresponding to the ‘GGG’ codon. On the other hand, same can also assist SARS coronaviruses for avoiding ribosomal slippage and producing more asymptomatic cases. Coronavirus replication in vitro gets inhibited after supplementation of 'D, L-lysine acetylsalicylate and glycine' (Muller et al. 2016). These two studies invite further investigations to understand the evolution of molecular mechanisms for coronavirus replication strategies and their relation with the prevalence of asymptomatic carriers.
Multiple introns of lower (26 & 44 nucleotide) size ranges in this genomic region were also characteristic of coronaviruses with lower GGG codon usage preference such as SARS and SARS-Cov2. In addition, the intron sizes also appeared to be conserved among several viruses in our study, suggesting a definite selective basis to these molecular features. On the other hand, we could observe only a single, 89 nucleotide- long putative intron in MERS. In fact, in silico excision of even this putative intron in MERS using SMART resulted in the disruption of either Nsp10 or RPol domain. Notably, SARS-Cov2 possesses more infectivity (transmission ability) than the SARS and MERS (Chu et al. 2020; Petersen et al. 2020).
Since multiple introns offer a wider probability for generation of correct reading frames, it may be argued that the SARS and SARS-Cov2 viruses should preferably resort to the intron excision method for rapid replication over frameshift translation. In fact, influenza viruses are known to hijack host splicing machinery to process some of their own RNA (Dubois et al. 2014) as well as possess features aiding in programmed ribosomal frameshifting (Firth et al. 2012). Through subgenomic RNAs (sgRNA) quantification from the SARS-CoV2 infected people, it has been learned that transcription is repressed in asymptomatic cases compared to symptomatic cases (Wong et al. 2021). The study also revealed, higher prevalence of structural deletions in SARS-CoV2 RNAs in symptomatic cases. Together, these two observations support our hypothesis of more active transcription and splicing of the viral RNA in symptomatic cases. However, it needs to be remembered that the ssRNA genome of coronaviruses is the positive sense strand for viral protein translation (Wu et al. 2020). Therefore, excision of the introns from the initial viral particles would literally destroy the true copies of the original genetic material from the host system, thus eliminating raw material for further mutation and evolution. With this logic it is tempting to suggest the presence of a molecular switch that dictates which of the two mechanisms would be adopted by the virus for replication at a given time or tissue location. We also suggest that the presence of these combined hindrances to replication is in fact the major selective advantage to the SARS-Cov2 virus, resulting in the rapid spread of the disease. Indeed, viruses that replicate rapidly, sending the host immune systems into overdrive in a short duration are at a disadvantage, since rapid development of symptoms help elimination of infected individuals before the virus has a chance to spread in the population (Fig. 2).
Based on bioinformatic analyses of the SARS-Cov2 genome, we suggest that the SARS-Cov2 viral replication in host cells is strongly dependent on either a programmed frameshift translation at a specific ribosomal slippage site in the ORF1ab region or excision of introns within this region. The inherent presence of these two hindrances to viral replication appears to be the reason for its slower pace of replication, resulting in a high prevalence of asymptomatic carriers in the host population. Though our study provides an insight on molecular peculiarities of SARS-Cov2 underlying the high prevalence of asymptomatic cases, our observations are exclusively from in silico observations and require experimental testing and validation.
References
Aguilar JB, Faust JS, Westafer LM, Gutierrez JB (2020) Investigating the impact of asymptomatic carriers on COVID-19 Transmission. medRxiv
Bixler D (2020) SARS-CoV-2–associated deaths among persons aged 21 Years—United States, February 12–July 31, 2020. MMWR. Morbidity and mortality weekly report, 69
Boon AC, Finkelstein D, Zheng M, Liao G, Allard J, Klumpp K, Webster R, PeltzG Webby RJ (2011) H5N1 influenza virus pathogenesis in genetically diverse mice is mediated at the level of viral load. MBio. https://doi.org/10.1128/mBio.00171-11
Brierley I, Digard P, Inglis SC (1989) Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell 57(4):537–547
Chu H, Chan JF, Yuen TT, Shuai H, Yuan S, Wang Y, Hu B, Yip CC, Tsang JO, Huang X, Chai Y (2020) Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study. The Lancet Microbe 1(1):e14–e23
De Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, Chau TNB, Hoang DM, Chau NVV, Khanh TH, Dong VC, Qui PT (2006) Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 12(10):1203–1207
Dubois J, Terrier O, Rosa-Calatrava M (2014) Influenza viruses and mRNA splicing: doing more with less. MBio. https://doi.org/10.1128/mBio.00070-14
Firth AE, Jagger BW, Wise HM, Nelson CC, Parsawar K, Wills NM, Napthine S, Taubenberger JK, Digard P, Atkins JF (2012) Ribosomal frameshifting used in influenza a virus expression occurs within the sequence UCC_UUU_CGU and is in the+ 1 direction. Open Biol 2(10):120109
He G, Sun W, Fang P, Huang J, Gamber M, Cai J, Wu J (2020) The clinical feature of silent infections of novel coronavirus infection (COVID-19) in Wenzhou. J Med Virol 92(10):1761–1763
Hilgenfeld R, Peiris M (2013) From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses. Antivir Res 100(1):286–295
Jacks T, Madhani HD, Masiarz FR, Varmus HE (1988) Signals for ribosomal frameshifting in the rous sarcoma virus gag-pol region. Cell 55(3):447–458
Johansson MJ, Esberg A, Huang B, Björk GR, Byström AS (2008) Eukaryotic wobble uridine modifications promote a functionally redundant decoding system. Mol Cell Biol 28(10):3301–3312
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845
Klein S, Cortese M, Winter SL, Wachsmuth-Melm M, Neufeldt CJ, Cerikan B, Stanifer ML, Boulant S, Bartenschlager R, Chlanda P (2020) SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography. Nat Commun 11(1):1–10
Knoops K, Kikkert M, Van Den Worm SH, Zevenhoven-Dobbe JC, Van Der Meer Y, Koster AJ, Mommaas AM, Snijder EJ (2008) SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6(9):e226
Letunic I, Doerks T, Bork P (2015) SMART: recent updates, new developments and status in 2015. Nucleic Acids Res 43(D1):D257–D260
Leung K, Pei Y, Leung GM, Lam TT, Wu JT (2020) Empirical transmission advantage of the D614G mutant strain of SARS-CoV-2. medRxiv.
Li R, Ma X, Deng J, Chen Q, Liu W, Peng Z, Qiao Y, LinY He X, Zhang H (2021) Differential efficiencies to neutralize the novel mutants B. 1.1. 7 and 501Y. V2 by collected sera from convalescent COVID-19 patients and RBD nanoparticle-vaccinated rhesus macaques. Cell Mol Immunol 18(4):1058–1060
Muller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-Lysine acetylsalicylate+ glycine impairs coronavirus replication. J Antivir Antiretrovir 8:142–150
Nishiura H, Kobayashi T, Miyama T, Suzuki A, Jung SM, Hayashi K, Kinoshita R, Yang Y, Yuan B, Akhmetzhanov AR, Linton NM (2020) Estimation of the asymptomatic ratio of novel coronavirus infections (COVID-19). Int J Infect Dis 94:154
Pessa HK, Will CL, Meng X, Schneider C, Watkins NJ, Perälä N, Nymark M, Turunen JJ, Lührmann R, Frilander MJ (2008) Minor spliceosome components are predominantly localized in the nucleus. Proc Natl Acad Sci 105(25):8655–8660
Petersen E, Koopmans M, Go U, Hamer DH, Petrosillo N, Castelli F, Storgaard M, Khalili AS, Simonsen L (2020) Comparing SARS-CoV-2 with SARS-CoV and influenza pandemics. Lancet Infect Dis 20(9):e238–e244
Ratajczak MZ, Kucia M (2020) SARS-CoV-2 infection and overactivation of Nlrp3 inflammasome as a trigger of cytokine “storm” and risk factor for damage of hematopoietic stem cells. Leukemia 34(7):1726–1729
Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LL, Guan Y, Rozanov M, Spaan WJ, Gorbalenya AE (2003) Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331(5):991–1004
Snijder EJ, Van Der Meer Y, Zevenhoven-Dobbe J, Onderwater JJ, van der Meulen J, Koerten HK, Mommaas AM (2006) Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J Virol 80(12):5927–5940
Song PL, Xie J, Hou Y, You C (2020) Cytokine storm induced by SARS-CoV-2. Clin Chim Acta 509:280–287
Starr TN, GreaneyAJ AA, Hannon WW, Choudhary MC, Dingens AS, Li JZ, Bloom JD (2021) Prospective mapping of viral mutations that escape antibodies used to treat COVID-19. Science 371(6531):850–854
Stertz S, Reichelt M, Spiegel M, Kuri T, Martínez-Sobrido L, García-Sastre A, Weber F, Kochs G (2007) The intracellular sites of early replication and budding of SARS-coronavirus. Virology 361(2):304–315
Yu X, Yang R (2020) COVID-19 transmission through asymptomatic carriers is a challenge to containment. Influenza Other Respir Viruses 14(4):474–475
Wong CH, Ngan CY, Goldfeder RL, Idol J, Kuhlberg C, Maurya R, Kelly K, Omerza G, Renzette N, De Abreu F, Li L (2021) Subgenomic RNAs as molecular indicators of asymptomatic SARS-CoV-2 infection. bioRxiv
Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, Hu Y, Tao ZW, Tian JH, Pei YY, Yuan ML (2020) A new coronavirus associated with human respiratory disease in China. Nature 579(7798):265–269
Wu Q, Krainer AR (1996) U1-mediated exon definition interactions between AT-AC and GT-AG introns. Science 274(5289):1005–1008
Zhou R, Li F, Chen F, Liu H, Zheng J, Lei C, Wu X (2020) Viral dynamics in asymptomatic patients with COVID-19. Int J Infect Dis 96:288–290
Acknowledgements
The authors express their humble gratitude to all warriors of the Covid-19 pandemic the world over.
Author information
Authors and Affiliations
Contributions
HP: Concept and Sequence Analysis; RD: Analysis, Literature survey, Manuscript Writing.
Corresponding author
Ethics declarations
Conflict of interest
This manuscript has been drafted purely based on theoretical bioinformatic analyses and has no experimental and/or clinical basis. The authors assume full responsibility and liability for the ideas and opinions expressed in this article. There are no conflicts of interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Priyadarshi, H., Das, R. Complexities in viral replication strategies as a potential explanation for prevalence of asymptomatic carriers in Covid-19 infections: analytical observation on SARS-Cov2 genome characteristics. Theory Biosci. 140, 241–247 (2021). https://doi.org/10.1007/s12064-021-00349-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12064-021-00349-3