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

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

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 microsecond. 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/451329v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1b6e8aborg.highwire.dtl.DTLVardef@165564eorg.highwire.dtl.DTLVardef@551f7org.highwire.dtl.DTLVardef@1485518_HPS_FORMAT_FIGEXP M_FIG C_FIG The schematic representation of NSP6 membrane topology and conformational dynamics of residue 91-112. The N-terminal and C-terminal are shown in cytoplasmic side based on the experimental evidence on coronaviruses reported by Oostra et al., 2008. The membrane anchoring domain are shown based on the TMHMM server prediction.

Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes

The phenomenon of protein aggregation is 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 this behaviour 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 SARS-CoV-2 proteins, 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 results show that these peptides and proteins form aggregates via a nucleation-dependent mechanism. 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. SignificanceThe aggregation of proteins is linked with human disease in a variety of ways. In the case of viral infections, one could expect that the aberrant aggregation of viral proteins may damage the host cells, and also that viral particles may trigger the misfolding and aggregation of host proteins, resulting in damage to the host organism. Here we investigate the aggregation propensity of SARS-CoV-2 proteins and show that many of them can form aggregates that are potentially cytotoxic. In perspective, these results suggest that a better understanding of the effects of viruses on the human protein homeostasis system could help future therapeutic efforts.

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

All available SARS-CoV-2 spike protein crystal and cryo-EM structures have shown missing electron densities for cytosolic C-terminal regions. Generally, the missing electron densities point towards the intrinsically disordered nature of the protein region. This curiosity has led us to investigate the C terminal cytosolic region of the spike glycoprotein of SARS-CoV-2 in isolation. The cytosolic regions is supposed to be from 1235-1273 residues or 1242-1273 residues depending on what prediction tool we use. Therefore, we have demonstrated the structural 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. Also, in temperature dependent replica-exchange molecular dynamics simulations it has shown its conformational dynamics. Further, we have validated our computational observations with circular dichroism (CD) spectroscopy-based experiments and found its signature spectra at 198 nm which is also adding the analysis as its intrinsically disordered nature. We believe that our findings will surely help us understand the structure-function relationship of the spike proteins cytosolic region. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

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

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. HighlightsO_LIThe E-CTD of SARS-CoV-2 is intrinsically disordered in solution C_LIO_LIThe E-CTD folds into an ordered structure in presence of membrane mimetics C_LIO_LIThe E-CTD displays conformational changes in the presence of osmolytes C_LIO_LIProlonged incubation of the E-CTD leads to its self-assembly into amyloid-like fibrils C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/424646v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a2d39aorg.highwire.dtl.DTLVardef@1f2581aorg.highwire.dtl.DTLVardef@17639eaorg.highwire.dtl.DTLVardef@d43ba1_HPS_FORMAT_FIGEXP M_FIG Structural heterogeneity of the E-CTD. The E-CTD shows a disordered secondary structure in an aqueous solution and converts into an ordered structure in the presence of membrane mimetics (neutral and negative lipids) and natural osmolytes (TMAO). Incubation at physiological condition shows typical amyloid-like fibrils. The yellow-colored structure represents a predicted structure of the E-CTD by PEP-FOLD. C_FIG

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

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, we tried to understand the interaction of membrane proteins interaction with envelope, spike, and nucleocapsid proteins using protein-protein docking. Further, simulation studies performed up to 100 ns to examine the stability of protein-protein complexes of Membrane-Envelope, Membrane-Spike, and Membrane-Nucleocapsid. Prime MM-GBSA showed high binding energy calculations than the docked complex. The interactions identified in our study will be of great importance, as it provides valuable insight into the protein-protein complex, which could be the potential drug targets for future studies.

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

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

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

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

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.