Widespread amyloid aggregates formation by Zika virus proteins and peptides.

Viral pathogenesis typically involves numerous molecular mechanisms. Protein aggregation is a relatively unknown characteristic of viruses, despite the fact that viral proteins have been shown to form terminally misfolded forms. Zika virus (ZIKV) is a neurotropic one with the potential to cause neurodegeneration. Its protein amyloid aggregation may link the neurodegenerative component to the pathogenicity associated with the viral infection. Therefore, we investigated protein aggregation in the ZIKV proteome as a putative pathogenic route and one of the alternate pathways. We discovered that it contains numerous anticipated aggregation-prone regions in this investigation. To validate our prediction, we used a combination of supporting experimental techniques routinely used for morphological characterization and study of amyloid aggregates. Several ZIKV proteins and peptides, including the full-length envelope protein, its domain III (EDIII) and fusion peptide, Pr N-terminal peptide, NS1 ß-roll peptide, membrane-embedded signal peptide 2K, and cytosolic region of NS4B protein, were shown to be highly aggregating in our study. Because our findings show that viral proteins can form amyloids in vitro, we need to do a thorough functional study of these anticipated APRs to understand better the role of amyloids in the pathophysiology of ZIKV infection.

Investigating the aggregation perspective of Dengue virus proteome.

Dengue viruses are human pathogens that are transmitted through mosquitoes. Apart from the typical symptoms associated with viral fevers, DENV infections are known to cause several neurological complications such as meningitis, encephalitis, intracranial haemorrhage, retinopathies along with the more severe, and sometimes fatal, vascular leakage and dengue shock syndrome. This study was designed to investigate, in detail, the predicted viral protein aggregation prone regions among all serotypes. Further, in order to understand the cross-talk between viral protein aggregation and aggregation of cellular proteins, cross-seeding experiments between the DENV NS1 (1-30), corresponding to the ß-roll domain and the diabetes hallmark protein, amylin, were performed. Various techniques such as fluorescence spectroscopy, circular dichroism, atomic force microscopy and immunoblotting have been employed for this. We observe that the DENV proteomes have many predicted APRs and the NS1 (1-30) of DENV1-3, 2K and capsid anchor of DENV2 and DENV4 are capable of forming amyloids, in vitro. Further, the DENV NS1 (1-30), aggregates are also able to cross-seed and enhance amylin aggregation and vice-versa. This knowledge may lead to an opportunity for designing suitable inhibitors of protein aggregation that may be beneficial for viral infections and comorbidities.

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 of the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated this behaviour in 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 (APRs) in these proteomes and revealed a strong aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone SARS-CoV and SARS-CoV-2 proteins and protein regions, including the signal sequence peptide and fusion peptides 1 and 2 of the spike protein, a peptide from the NSP6 protein, and the ORF10 and NSP11 proteins. Our results show that these peptides and proteins can form amyloid aggregates. We used circular dichroism spectroscopy to reveal the presence of ß-sheet rich cores in aggregates and X-ray diffraction and Raman spectroscopy to confirm the formation of amyloid structures. Furthermore, we demonstrated that SARS-CoV-2 NSP11 aggregates are toxic to mammalian cell cultures. These results motivate further studies about the possible role of aggregation of SARS proteins in protein misfolding diseases and other human conditions.

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 to 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 consists of helical structures. In contrast, the membrane outside lying region (91-112) showed partial helicity, which was further used as a model and obtained disordered type conformation during 1.5 µs. Additionally, a 200ns simulation study of residues 231-290 have shown significant conformational changes. As compared to helical and beta-sheet conformations in its structure model, the 200ns simulation resulted in the loss of beta-sheet structures while helical regions remained intact. Further, we have experimentally characterized the residue 91-112 by using reductionist approaches. CD spectroscopy suggests that the NSP6 (91-112) is disordered-like region in isolation, which gains 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 conformations might play a significant role. The present study adds up more information about the NSP6 protein aspect, which could be exploited for its host protein interaction and pathogenesis.

Zika virus capsid anchor forms cytotoxic amyloid-like fibrils.

Capsid-anchor (CA) of Zika virus (ZIKV) is a small, single-pass transmembrane sequence that separates the capsid (C) protein from downstream pre-membrane (PrM) protein. During polyprotein processing, CA is cleaved-off from C and PrM and left as a membrane-embedded peptide. CA plays an essential role in the assembly and maturation of the virus. However, its independent folding behavior is still unknown. Therefore, in this study, we investigated the amyloid-forming propensity of CA at physiological conditions. We observed the aggregation behavior of CA peptide using dye-binding assays and ThT kinetics. The morphological analysis of CA aggregates explored by high-resolution microscopy (TEM, AFM) and Far-UV CD spectroscopy revealed characteristic amyloid-like fibrils rich in ß-sheet secondary structure. Further, the effect on mammalian cells exhibited the cytotoxic nature of the CA amyloid-fibrils. Our findings collectively shed light on the amyloidogenic phenomenon of flaviviral protein, which may contribute to their infection.

Unlike dengue virus, the conserved 14-23 residues in N-terminal region of Zika virus capsid is not involved in lipid interactions.

Zika virus capsid protein is involved in multiple essential steps of the viral life cycle. Many vital functionalities are attributed to the dynamic N- terminal domain of this protein, which is intrinsically disordered in ZIKV and among several flaviviruses too. Other than genome encapsulation, studies have shown interaction with host lipid droplets to be crucial for replication and maturation. In Dengue virus, the molecular basis of such interplay has been studied in detail, and residues within the capsid N-terminal disordered domain has been mapped. It revealed a new function of a conserved region in mediating capsid-lipid droplet association through a conformational transition. Therefore, in this study, we attempt to analyze the structural dynamics of Zika virus capsid's N- terminal domain and analyzed it through a reductionist approach by dividing the N-terminal domain into three truncated segments and studied them individually. Techniques such as Circular dichroism spectroscopy, Dynamic light scattering, Zeta potential and Molecular dynamic simulations were employed to identify the motif responsible for structural flexibility and ability to interact with membrane models. Our results confirm that the truncated segments 5-26 and 1-30 readily adopt an α-helical conformation in the presence of 2,2,2-trifluoro-ethanol, detergent and negatively charged phospholipids. However, in contrast to Dengue virus, we report the conserved residues 14-23 region to be unstructured and do not undergo a conformational switch in Zika virus. Thus, our study illustrates the possibility of conserved 14-23 region's non-involvement in ZIKV capsid-lipid droplet association, unlike DENV.

Exploring the SARS-CoV-2 structural proteins for multi-epitope vaccine development: an in-silico approach.

INTRODUCTION: The ongoing life-threatening pandemic of coronavirus disease 2019 (COVID-19) has extensively affected the world. During this global health crisis, it is fundamentally crucial to find strategies to combat SARS-CoV-2. Despite several efforts in this direction and continuing clinical trials, no vaccine has been approved for it yet. METHODS: To find a preventive measure, we have computationally designed a multi-epitopic subunit vaccine using immuno-informatic approaches. RESULTS: The structural proteins of SARS-CoV-2 involved in its survival and pathogenicity were used to predict antigenic epitopes. The antigenic epitopes were capable of eliciting a strong humoral as well as cell-mediated immune response, our predictions suggest. The final vaccine was constructed by joining the all epitopes with specific linkers and to enhance their stability and immunogenicity. The physicochemical property of the vaccine was assessed. The vaccine 3D structure prediction and validation were done and docked with the human TLR-3 receptor. Furthermore, molecular dynamics simulations of the vaccine-TLR-3 receptor complex are employed to assess its dynamic motions and binding stability in-silico. CONCLUSION: Based on this study, we strongly suggest synthesizing this vaccine, which further can be tested in-vitro and in-vivo to check its potency in a cure for COVID-19.

Japanese encephalitis virus - exploring the dark proteome and disorder-function paradigm.

Japanese encephalitis virus (JEV) is one of the major causes of viral encephalitis all around the globe. Approximately 3 billion people in endemic areas are at risk of Japanese encephalitis. To develop a wholistic understanding of the viral proteome, it is important to investigate both its ordered and disordered proteins. However, the functional and structural significance of disordered regions in the JEV proteome has not been systematically investigated as of yet. To fill this gap, we used here a set of bioinformatics tools to analyze the JEV proteome for the predisposition of its proteins for intrinsic disorder and for the presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs). We also analyzed all JEV proteins for the presence of the probable nucleic acid-binding (DNA and RNA) sites. The results of these computational studies are experimentally validated using JEV capsid protein as an illustrative example. In agreement with bioinformatic analysis, we found that the N-terminal region of the JEV capsid (residues 1-30) is intrinsically disordered. We showed that this region is characterized by the temperature response typical for highly disordered proteins. Furthermore, we have experimentally shown that this disordered N-terminal domain of a capsid protein has a noticeable 'gain-of-structure' potential. In addition, using DOPS liposomes, we demonstrated the presence of pronounced membrane-mediated conformational changes in the N-terminal region of JEV capsid. In our view, this disorder-centric analysis would be helpful for a better understanding of the JEV pathogenesis.

Identification of peptidomimetic compounds as potential inhibitors against MurA enzyme of Mycobacterium tuberculosis.

Increasing prevalence of resistance to anti-tubercular drugs has become the foremost challenge now. According to WHO, over half a million of multidrug resistance cases (rifampicin, isoniazid, etc.) were reported in 2017, mostly emerging from countries such as China, India, and Russia. Therefore, developing new drugs or repurposing existing ones is need of the hour. The Mycobacterium cell wall biogenesis pathway offers many attractive targets for drug discovery against Tuberculosis (TB). MurA, a transferase enzyme that catalyzes the initial step of peptidoglycan (PG) biosynthesis, is one among them. A peptidoglycan layer resides over the plasma membrane and is an integral component of the bacterial cell wall. Therefore, disruption of their formation through inhibition of MurA enzyme should lead to deficiency in Mycobacterium cell synthesis. Based on this strategy, we have designed this study where two libraries of peptidomimetic compounds (Asinex & ChemDiv) were first screened against our modeled MurA structure and then validated through molecular dynamic simulations. From our virtual screening, top four compounds (ChemDiv: D675-0102, D675-0217; Asinex: BDE25373574, BDE 26717803) were selected based on their docking scores, binding energies, and interactions with catalytic site residues, for further evaluation. Results revealed stable ligand-MurA interactions throughout 50 ns of MD simulation and also druggability acceptable pharmacokinetic profile for all four compounds. Thus, based on our findings, these compounds could be considered as potential inhibitors of Mycobacterium MurA enzyme and hence be further tested for in vitro experimental validation as TB therapeutic drug candidate.Communicated by Ramaswamy H. Sarma.