Japanese encephalitis virus capsid protein interacts with non-lipidated MAP1LC3 on replication membranes and lipid droplets

Sarkar, Riya; Sharma, Kiran Bala; Kumari, Anita; Asthana, Shailendra; Kalia, Manjula

doi:10.1099/jgv.0.001508

Cited by 19 publications

(16 citation statements)

References 65 publications

(130 reference statements)

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“…VCP showed strong colocalization with NS5 and NS1, suggesting its potential role in virus replication. In contrast, very little overlap was seen between VCP and capsid, which is known to be in close proximity, but distinct from the replication complex (41). Further, the specificity of the replication complex localization was confirmed by staining with the ER marker calnexin (Fig.…”

Section: Fig 5 Vcp Depletion Delays the Internalized Virus Capsid Degradation (A)mentioning

confidence: 90%

Valosin-Containing Protein/p97 Plays Critical Roles in the Japanese Encephalitis Virus Life Cycle

Sehrawat

Khasa

Deb

et al. 2021

J Virol

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Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle – nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses. Importance JEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.

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Section: Fig 5 Vcp Depletion Delays the Internalized Virus Capsid Degradation (A)mentioning

confidence: 90%

Valosin-Containing Protein/p97 Plays Critical Roles in the Japanese Encephalitis Virus Life Cycle

Sehrawat

Khasa

Deb

et al. 2021

J Virol

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“…Here, we have explored four systems, APO-protein of FXR (System A), APO+agonist (System B), APO + 'co-activator' (System C), and APO+Agonist + 'co-activator' (System D), to identify the transition dynamics between the different conformational states of FXR with its binding partners. Before MD simulation, each targeted protein structure was prepared using the Protein Preparation Wizard encoded in the Schrodinger 3.5 suite (Sastry et al, 2013;Anang et al, 2018;Sarkar et al, 2021). The crystal waters were also removed, and hydrogens were added.…”

Section: Protein Structure Preparationmentioning

confidence: 99%

Conformational Characterization of the Co-Activator Binding Site Revealed the Mechanism to Achieve the Bioactive State of FXR

Kumari

Mittal

Srivastava

et al. 2021

Front. Mol. Biosci.

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FXR bioactive states are responsible for the regulation of metabolic pathways, which are modulated by agonists and co-activators. The synergy between agonist binding and ‘co-activator’ recruitment is highly conformationally driven. The characterization of conformational dynamics is essential for mechanistic and therapeutic understanding. To shed light on the conformational ensembles, dynamics, and structural determinants that govern the activation process of FXR, molecular dynamic (MD) simulation is employed. Atomic insights into the ligand binding domain (LBD) of FXR revealed significant differences in inter/intra molecular bonding patterns, leading to structural anomalies in different systems of FXR. The sole presence of an agonist or ‘co-activator’ fails to achieve the essential bioactive conformation of FXR. However, the presence of both establishes the bioactive conformation of FXR as they modulate the internal wiring of key residues that coordinate allosteric structural transitions and their activity. We provide a precise description of critical residue positioning during conformational changes that elucidate the synergy between its binding partners to achieve an FXR activation state. Our study offers insights into the associated modulation occurring in FXR at bound and unbound forms. Thereafter, we also identified hot-spots that are critical to arrest the activation mechanism of FXR that would be helpful for the rational design of its agonists.

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“…The protein structure was prepared using the Protein Preparation Wizard module of Maestro (Sastry et al, 2013;Anang et al, 2018) (Schrödinger release 2020-1: Maestro, Schrödinger, LLC, New York, NY, 2020.) (Sarkar et al, 2021). OPLS3 (Jorgensen et al, 1996) force field model was used for preparation (Jorgensen et al, 1996).…”

Section: System Preparationmentioning

confidence: 99%

Molecular Dynamics Simulations Reveal the Interaction Fingerprint of Remdesivir Triphosphate Pivotal in Allosteric Regulation of SARS-CoV-2 RdRp

Srivastava

Mittal

Kumari

et al. 2021

Front. Mol. Biosci.

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The COVID-19 pandemic has now strengthened its hold on human health and coronavirus’ lethal existence does not seem to be going away soon. In this regard, the optimization of reported information for understanding the mechanistic insights that facilitate the discovery towards new therapeutics is an unmet need. Remdesivir (RDV) is established to inhibit RNA-dependent RNA polymerase (RdRp) in distinct viral families including Ebola and SARS-CoV-2. Therefore, its derivatives have the potential to become a broad-spectrum antiviral agent effective against many other RNA viruses. In this study, we performed comparative analysis of RDV, RMP (RDV monophosphate), and RTP (RDV triphosphate) to undermine the inhibition mechanism caused by RTP as it is a metabolically active form of RDV. The MD results indicated that RTP rearranges itself from its initial RMP-pose at the catalytic site towards NTP entry site, however, RMP stays at the catalytic site. The thermodynamic profiling and free-energy analysis revealed that a stable pose of RTP at NTP entrance site seems critical to modulate the inhibition as its binding strength improved more than its initial RMP-pose obtained from docking at the catalytic site. We found that RTP not only occupies the residues K545, R553, and R555, essential to escorting NTP towards the catalytic site, but also interacts with other residues D618, P620, K621, R624, K798, and R836 that contribute significantly to its stability. From the interaction fingerprinting it is revealed that the RTP interact with basic and conserved residues that are detrimental for the RdRp activity, therefore it possibly perturbed the catalytic site and blocked the NTP entrance site considerably. Overall, we are highlighting the RTP binding pose and key residues that render the SARS-CoV-2 RdRp inactive, paving crucial insights towards the discovery of potent inhibitors.

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Japanese encephalitis virus capsid protein interacts with non-lipidated MAP1LC3 on replication membranes and lipid droplets

Cited by 19 publications

References 65 publications

Valosin-Containing Protein/p97 Plays Critical Roles in the Japanese Encephalitis Virus Life Cycle

Valosin-Containing Protein/p97 Plays Critical Roles in the Japanese Encephalitis Virus Life Cycle

Conformational Characterization of the Co-Activator Binding Site Revealed the Mechanism to Achieve the Bioactive State of FXR

Molecular Dynamics Simulations Reveal the Interaction Fingerprint of Remdesivir Triphosphate Pivotal in Allosteric Regulation of SARS-CoV-2 RdRp

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