Coronaviruses Spike glycoprotein endodomains: the sequence and structure-based comprehensive study (preprint)

The flexibility of proteins makes them available to interact with many biomolecules in the cell. Specifically, such interactions in viruses help them to perform more functions despite having a smaller genome. Therefore, these flexible regions can be exciting and essential targets to be explored for their role in pathogenicity and therapeutic developments as they achieve essential interactions. In the continuation with our previous study on disordered analysis of SARS-CoV-2 spike cytoplasmic tail (CTR), or endodomain, here we have explored the disordered potential endodomains of six other coronaviruses using multiple bioinformatics approaches and molecular dynamics simulations. Based on the comprehensive analysis of its sequence and structural composition, we report the varying disorder propensity in endodomains of spike proteins of coronaviruses. The observations of this study may help to understand the importance of spike glycoprotein endodomain and creating therapeutic interventions against them.

Met58 and di-acidic motif located at C-terminal region of SARS-CoV-2 ORF6 plays a crucial role in its structural conformations (preprint)

Despite being mostly neglected in structural biology, the C-terminal Regions (CTRs) are studied to be multifunctional in humans as well as in viruses. Their role in cellular processes such as trafficking, protein-protein interactions, and protein-lipid interactions are known due to their structural properties. In our previous findings on SARS-CoV-2 Spike and NSP1 proteins, the C-terminal regions (CTRs) are observed to be disordered and experimental evidence showed a gain of structure properties in different physiological environments. In this line, we have investigated the structural dynamics of CTR (residues 38-61) of SARS-CoV-2 ORF6 protein, disrupting bidirectional transport between the nucleus and cytoplasm. Like Spike and NSP1-CTR, the ORF6-CTR is also disordered in nature but possesses gain of structure properties in minimal physiological conditions. As per studies, the residue such as Methionine at 58th position in ORF6 is critical for interaction with Rae1-Nup98. Therefore, along with M58, we have identified a few other mutations from the literature and performed extensive structure modelling and dynamics studies using computational simulations. The exciting revelations in CTR models provide evidence of its structural flexibility and possible capabilities to perform multifunctionality inside the host.

Mitoxantrone dihydrochloride, an FDA approved drug, binds with SARS-CoV-2 NSP1 C-terminal (preprint)

One of the major virulence factors of SARS-CoV-2, NSP1, is a vital drug target due to its role in host immune evasion through multiple pathways. NSP1 protein is associated with inhibiting host mRNA translation by binding to the small subunit of ribosome through its C-terminal region. Previously, we have shown the structural dynamics of NSP1 C-terminal region (NSP1-CTR) in different physiological environments. So, it would be very interesting to investigate the druggable compounds that could bind with NSP1-CTR. Here, in this article, we have performed the different spectroscopic technique-based binding assays of an anticancer drug Mitoxantrone dihydrochloride (MTX) against the NSP1-CTR. We have also performed molecular docking followed by computational simulations with two different forcefields up to one microsecond. Overall, our results have suggested good binding between NSP1-CTR and MTX and may have implications in developing therapeutic strategies targeting NSP1 protein of SARS-CoV-2.

Investigating the conformational dynamics of SARS-CoV-2 NSP6 protein with emphasis on non-transmembrane 91-112 & 231-290 regions (preprint)

The NSP6 protein of SARS-CoV-2 is a transmembrane protein, with some regions lying outside the membrane. Besides, a brief role of NSP6 in autophagosome formation, this is not studied significantly. Also, there is no structural information available till date. Based on the prediction by TMHMM server for transmembrane prediction, it is found that the N-terminal residues (1-11), middle region residues (91-112) and C-terminal residues (231-290) lies outside the membrane. Molecular Dynamics (MD) simulations showed that NSP6 consisting of helical structures, whereas membrane outside lying region (91-112) showed partial helicity, which further used as model and obtain disordered type conformation after 1.5 microseconds. Whereas, the residues 231-290 has both helical and beta sheet conformations in its structure model. A 200ns simulations resulted in the loss of beta sheet structures, while helical regions remained intact. Further, we have characterized the residue 91-112 by using reductionist approaches. The NSP6 (91-112) was found disordered like in isolation, which gain helical conformation in different biological mimic environmental conditions. These studies can be helpful to study NSP6 (91-112) interactions with host proteins, where different protein conformation might play significant role. The present study adds up more information about NSP6 protein aspect, which could be exploited for its host protein interaction and pathogenesis.

Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes (preprint)

The phenomenon of protein aggregation is widespread and associated with a wide range of human diseases. Our knowledge on the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated the distribution of aggregation-prone regions in the the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions in these proteomes, and revealed an enhanced aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone regions in the of SARS-CoV-2 proteome, including the signal sequence peptide and fusion peptide 1 of the spike protein, a peptide from the NSP6 protein (NSP6-p), the ORF10 protein, and the NSP11 protein. Our re-sults show that these peptides and proteins form aggregates via a nucleation-dependent mecha-nism. Moreover, we demonstrated that the aggregates of NSP11 are toxic to mammalian cell cultures. These findings provide evidence about the aggregation of proteins in the SARS-CoV-2 proteome.

Microsecond simulation unravel the structural dynamics of SARS-CoV-2 Spike-C-terminal cytoplasmic tail (residues 1242-1273) (preprint)

Spike protein of human coronaviruses has been a vital drug and vaccine target. The multifunctionality of this protein including host receptor binding and apoptosis has been proved in several coronaviruses. It also interacts with other viral proteins such as membrane (M) protein through its C-terminal domain. The specific dibasic motif signal present in cytosolic region at C-terminal of spike protein helps it to localize within the endoplasmic reticulum (ER). However, the structural conformation of cytosolic region is not known in SARS-CoV-2 using which it interacts with other proteins and transporting vesicles. Therefore, we have demonstrated the conformation of cytosolic region and its dynamics through computer simulations up to microsecond timescale using OPLS and CHARMM forcefields. The simulations have revealed the unstructured conformation of cytosolic region (residues 1242-1273). Also, in temperature dependent replica-exchange molecular dynamics simulations it has shown to form secondary structures. We believe that our findings will surely help us understand the structure-function relationship of the spike protein's cytosolic region.

The hyperlipidaemic drug fenofibrate significantly reduces infection by SARS-CoV-2 in cell culture models (preprint)

The SARS-CoV-2 pandemic has caused a significant number of fatalities and worldwide disruption. To identify drugs to repurpose to treat SARS-CoV-2 infections, we established a screen to measure dimerization of ACE2, the primary receptor for the virus. This screen identified fenofibric acid, the active metabolite of fenofibrate. Fenofibric acid also destabilized the receptor binding domain (RBD) of the viral spike protein and inhibited RBD binding to ACE2 in ELISA and whole cell binding assays. Fenofibrate and fenofibric acid were tested by two independent laboratories measuring infection of cultured Vero cells using two different SARS-CoV-2 isolates. In both settings at drug concentrations which are clinically achievable, fenofibrate and fenofibric acid reduced viral infection by up to 70%. Together with its extensive history of clinical use and its relatively good safety profile, these studies identify fenofibrate as a potential therapeutic agent requiring urgent clinical evaluation to treat SARS-CoV-2 infection.

Environmental Dependence of the Structure of the C-terminal Domain of the SARS-CoV-2 Envelope Protein (preprint)

The SARS-CoV-2 envelope protein (E) is involved in a broad spectrum of functions in the cycle of the virus, including assembly, budding, envelope formation, and pathogenesis. To enable these activities, E is likely to be capable of changing its conformation depending on environmental cues. To investigate this issue, here we characterised the structural properties of the C-terminal domain of E (E-CTD), which has been reported to interact with host cell membranes. We first studied the conformation of the E-CTD in solution, finding characteristic features of a disordered protein. By contrast, in the presence of large unilamellar vesicles and micelles, which mimic cell membranes, the E-CTD was observed to become structured. The E-CTD was also found to display conformational changes with osmolytes. Furthermore, prolonged incubation of the E-CTD under physiological conditions resulted in amyloid-like fibril formation. Taken together, these findings indicate that the E-CTD can change its conformation depending on its environment, ranging from a disordered state, to a membrane-bound folded state, and an amyloid state. Our results thus provide insight into the structural basis of the role of E in the viral infection process.

An insight into SARS-CoV-2 Membrane protein interaction with Spike, Envelope, and Nucleocapsid proteins (preprint)

Intraviral protein-protein interactions are crucial for replication, pathogenicity, and viral assembly. Among these, virus assembly is a critical step as it regulates the arrangements of viral structural proteins and helps in the encapsulation of genomic material. SARS-CoV-2 structural proteins play an essential role in the self-rearrangement, RNA encapsulation, and mature virus particle formation. In SARS-CoV, the membrane protein interacts with the envelope and spike protein in Endoplasmic Reticulum Golgi Intermediate Complex (ERGIC) to form an assembly in the lipid bilayer, followed by membrane-ribonucleoprotein (nucleocapsid) interaction. In this study, using protein-protein docking, we tried to understand the interaction of membrane protein's interaction with envelope, spike and nucleocapsid proteins. Further, simulation studies performed up to 100ns agreed that protein complexes M-E, M-S, and M-N were stable. Moreover, the calculated free binding energy and dissociation constant values support the protein complex formation. The interaction identified in the study will be of great importance, as it provides valuable insight into the protein complex, which could be the potential drug targets for future studies.

NSP 11 of SARS-CoV-2 is an Intrinsically Disordered Protein (preprint)

The intrinsically disordered proteins/regions (IDPs/IDPRs) are known to be responsible for multiple cellular processes and are associated with many chronic diseases. In viruses, the existence of a disordered proteome is also proven and is related to its conformational dynamics inside the host. The SARS-CoV-2 has a large proteome, in which, structure and functions of many proteins are not known yet, along with nsp11. In this study, we have performed extensive experimentation on nsp11. Our results based on the CD spectroscopy gives characteristic disordered spectrum for IDPs. Further, we investigated the conformational behavior of nsp11 in the presence of membrane mimetic environment, -helix inducer, and natural osmolyte. In the presence of negatively charged and neutral liposomes, nsp11 remains disordered. However, with SDS micelle, it adopted an -helical conformation, suggesting the helical propensity of nsp11. Finally, we again confirmed the IDP behavior of nsp11 using MD simulations. In future, this conformational dynamic study could help to clarify its functional importance in SARS-CoV-2 infection.

SARS-CoV-2 NSP1 C-terminal region (residues 130-180) is an intrinsically disordered region (preprint)

Nonstructural protein 1 (NSP1) of SARS-CoV-2 plays a key role in downregulation of RIG-I pathways and interacts with 40 S ribosome. Recently, the cryo-EM structure in complex with 40S ribosome is deciphered. However, the structure of full length NSP1 without any partner has not been studies. Also, the conformation of NSP1-C terminal region in isolation is not been studied. In this study, we have investigated the conformational dynamics of NSP1C-terminal region (NSP1-CTR; amino acids 130-180) in isolation and under different solvent environments. The NSP1-CTR is found to be intrinsically disordered in aqueous solution. Further, we used alpha helix inducer, trifluoroethanol, and found induction of alpha helical conformation using CD spectroscopy. Additionally, in the presence of SDS, NSP1-CTR is showing a conformational change from disordered to ordered, possibly gaining alpha helix in part. But in presence of neutral lipid DOPC, a slight change in conformation is observed. This implies the possible role of hydrophobic interaction and electrostatic interaction on the conformational changes of NSP1. The changes in structural conformation were further studied by fluorescence-based studies, which showed significant blue shift and fluorescence quenching in the presence of SDS and TFE. Lipid vesicles also showed fluorescence-based quenching. In agreement to these result, fluorescence lifetime and fluorescence anisotropy decay suggests a change in conformational dynamics. The zeta potential studies further validated that the conformational dynamics is mostly because of hydrophobic interaction. In last, these experimental studies were complemented through Molecular Dynamics (MD) simulation which have also shown a good correlation and testify our experiments. We believe that the intrinsically disordered nature of the NSP1-CTR will have implications in disorder based binding promiscuity with its interacting proteins.

Translation-associated mutational U-pressure in the first ORF of SARS-CoV-2 and other coronaviruses (preprint)

Within four months of the ongoing COVID-19 pandemic caused by SARS-CoV-2, more than 250 nucleotide mutations have been detected in the ORF1 of the virus isolated from different parts of the globe. These observations open up an obvious question about the rate and direction of mutational pressure for further vaccine and therapeutics designing. In this study, we did a comparative analysis of ORF1a and ORF1b by using the first isolate (Wuhan strain) as the parent sequence. We observed that most of the nucleotide mutations are C to U transitions. The rate of synonymous C to U transitions is significantly higher than the rate of nonsynonymous ones, indicating negative selection on amino acid substitutions. Further, trends in nucleotide usage bias have been investigated in 49 coronaviruses species. A strong bias in nucleotide usage in fourfold degenerated sites towards uracil residues is seen in ORF1 of all the studied coronaviruses. A more substantial mutational U pressure is observed in ORF1a than in ORF1b owing to the translation of ORF1ab via programmed ribosomal frameshifting. Unlike other nucleotide mutations, mutational U pressure caused by cytosine deamination, mostly occurring in the RNA-plus strand, cannot be corrected by the proof-reading machinery of coronaviruses. The knowledge generated on the direction of mutational pressure during translation of viral RNA-plus strands has implications for vaccine and nucleoside analogue development for treating covid-19 and other coronavirus infections.

Dark proteome of Newly Emerged SARS-CoV-2 in Comparison with Human and Bat Coronaviruses (preprint)

Recently emerged coronavirus designated as SARS-CoV-2 (also known as 2019 novel coronavirus (2019-nCoV) or Wuhan coronavirus) is a causative agent of coronavirus disease 2019 (COVID-19), which is rapidly spreading throughout the world now. More than 9,00,000 cases of SARS-CoV-2 infection and more than 47,000 COVID-19-associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. World Health Organization (WHO) has declared the SARS-CoV-2 spread as a global public health emergency and admitted that the COVID-19 is a pandemic now. The multiple sequence alignment data correlated with the already published reports on the SARS-CoV-2 evolution and indicated that this virus is closely related to the bat Severe Acute Respiratory Syndrome-like coronavirus (bat SARS-like CoV) and the well-studied Human SARS coronavirus (SARS CoV). The disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. Therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of SARS-CoV-2, bat SARS-like, and human SARS CoVs by analysing the prevalence of intrinsic disorder in their proteins. According to our findings, SARS-CoV-2 proteome contains very significant levels of structural order. In fact, except for Nucleocapsid, Nsp8, and ORF6, the vast majority of SARS-CoV-2 proteins are mostly ordered proteins containing less intrinsically disordered protein regions (IDPRs). However, IDPRs found in SARS-CoV-2 proteins are functionally important. For example, cleavage sites in its replicase 1ab polyprotein are found to be highly disordered, and almost all SARS-CoV-2 proteins were shown to contain molecular recognition features (MoRFs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. The results of our extensive investigation of the dark side of the SARS-CoV-2 proteome will have important implications for the structural and non-structural biology of SARS or SARS-like coronaviruses. SignificanceThe infection caused by a novel coronavirus (SARS-CoV-2) that causes severe respiratory disease with pneumonia-like symptoms in humans is responsible for the current COVID-19 pandemic. No in-depth information on structures and functions of SARS-CoV-2 proteins is currently available in the public domain, and no effective anti-viral drugs and/or vaccines are designed for the treatment of this infection. Our study provides the first comparative analysis of the order- and disorder-based features of the SARS-CoV-2 proteome relative to human SARS and bat CoV that may be useful for structure-based drug discovery.