Rotavirus NSP1 inhibits interferon induced non-canonical NFκB activation by interacting with TNF receptor associated factor 2

AbstractTNF receptor associated factor 2 (TRAF2) plays a very important role in cellular innate immune as well as inflammatory responses. Previous studies have reported TRAF2 mediated regulation of TNF and Interferon (IFN) induced canonical and non-canonical activation of NFκB. In this study, we show that rotavirus NSP1 targets TRAF2 to regulate IFN induced non-canonical NFκB activation. Here we found that rotavirus Non-Structural Protein-1 (NSP1) interacts with TRAF2 and degrades it in a proteasome dependent manner. C-terminal part of NSP1 was sufficient for interacting with TRAF2 but it alone could not degrade TRAF2. This inhibition of interferon mediated non-canonical NFκB activation by NSP1 may modulate inflammatory cytokine production after rotavirus infection to help the virus propagation

Identification of potential inhibitor against Leishmania donovani mitochondrial DNA primase through in-silico and in vitro drug repurposing approaches

Abstract Leishmania donovani is the causal organism of leishmaniasis with critical health implications affecting about 12 million people around the globe. Due to less efficacy, adverse side effects, and resistance, the available therapeutic molecules fail to control leishmaniasis. The mitochondrial primase of Leishmania donovani (LdmtPRI1) is a vital cog in the DNA replication mechanism, as the enzyme initiates the replication of the mitochondrial genome of Leishmania donovani. Hence, we target this protein as a probable drug target against leishmaniasis. The de-novo approach enabled computational prediction of the three-dimensional structure of LdmtPRI1, and its active sites were identified. Ligands from commercially available drug compounds were selected and docked against LdmtPRI1. The compounds were chosen for pharmacokinetic study and molecular dynamics simulation based on their binding energies and protein interactions. The LdmtPRI1 gene was cloned, overexpressed, and purified, and a primase activity assay was performed. The selected compounds were verified experimentally by the parasite and primase inhibition assay. Capecitabine was observed to be effective against the promastigote form of Leishmania donovani, as well as inhibiting primase activity. This study's findings suggest capecitabine might be a potential anti-leishmanial drug candidate after adequate further studies

Viperin, an IFN-Stimulated Protein, Delays Rotavirus Release by Inhibiting Non-Structural Protein 4 (NSP4)-Induced Intrinsic Apoptosis

Viral infections lead to expeditious activation of the host’s innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin’s antiviral action in addition to the intricacy of viperin–virus interaction

NSP1 inhibits IFN-β induction irrespective of IRF3 degradation.

<p><b>A)</b> HEK293 cells were transfected with FLAG-MAVS and pcD-OSU-NSP1 vector in order to assess the MAVS mediated inhibition of IRF3 phosphorylation. Cell lysates were analyzed for pIRF3, IRF3, Anti-His, Anti-FLAG and GAPDH specific antibodies. <b>B)</b> Fold change of IFN-β transcripts was assessed in cells overexpressing human TBK1 and pFLAG-MAVS vectors, in presence or absence of pcD-NSP1. The data shown are means ± the SD (n = 3). * Significantly different in comparison to human TBK1 and NSP1 transfected and pFLAG-MAVS untransfected condition. P<0.05 <b>C)</b> Activation of IRF3 was assessed in absence or presence of pcD-NSP1 in cells transfected with TBK1 and FLAG-MAVS. <b>D)</b> Association of MAVS with TBK1 was studied by Co-IP in presence or absence of pcD-NSP1 in cells overexpressing TBK1 and MAVS. The MAVS degradation was controlled by proteosomal inhibitor MG132 Results reveal reduced interaction between MAVS-TBK1 in presence of NSP1.</p

Correction: MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1

<p>Correction: MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1</p

Proteosome-mediated MAVS degradation.

<p><b>A)</b> NSP1 induced MAVS degradation is prevented in presence of MG132. HEK293 cells were either transfected with FLAG-MAVS or co transfected with pFLAG-MAVS and pcD-NSP1 in presence or absence of 20 μM MG132 followed by immunoblot analysis with anti-FLAG, anti-GAPDH and anti-His. <b>B)</b> NSP1 induces ubiquitinylation of MAVS. HEK293 cells were transfected with expression vector encoding FLAG-tagged ubiquitin with presence or absence of pcD-NSP1. Cells were grown in DMEM containing MG132 (20 μM) for 6 h. Anti-MAVS immunoprecipitates were analyzed by immunoblotting His-tagged ubiquitin with anti-His Ab. Whole-cell lysates were subjected to immunoblotting with anti-His and anti-GAPDH was used as an equal loading control. Figure also showed that MG132 affects MAVS degradation but not ubiquitinylation.</p

Formation of MAVS aggregates during Rotavirus infection.

<p><b>A)</b> Crude mitochondrial extracts were prepared from HEK293 cells infected with A5-16 strain (3 M.O.I.) at increasing time points (4, 8 and 12). Extracts were analyzed by SDD-AGE to assess the MAVS aggregate formation. Results revealed MAVS aggregate formation from 4 hpi. <b>B)</b> Role of NSP1 on MAVS aggregation and ubiquitinylation was observed by overexpressing FLAG-MAVS in absence or presence of NSP1. Infection of A5-16 was used for inducing MAVS aggregation, as overexpression of MAVS alone is insufficient for inducing aggregate formation. Results show inhibition of MAVS aggregates in presence of NSP1, which gets restored following MG132 (20 μM) treatment. A fraction of SDD-AGE lysate was analyzed by SDS-PAGE followed by immunoblotting of MAVS, p-IRF3 and COX IV.</p

MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1

<div><p>Rotavirus is the single, most important agent of infantile gastroenteritis in many animal species, including humans. In developing countries, rotavirus infection attributes approximately 500,000 deaths annually. Like other viruses it establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Among various pattern recognition receptors (PAMPs) of the host, the cytosolic RNA helicases interact with viral RNA to activate the Mitochondrial Antiviral Signaling protein (MAVS), which regulates cellular interferon response. With an aim to identify the role of different PAMPs in rotavirus infected cell, MAVS was found to degrade in a time dependent and strain independent manner. Rotavirus non-structural protein 1 (NSP1) which is a known IFN antagonist, interacted with MAVS and degraded it in a strain independent manner, resulting in a complete loss of RNA sensing machinery in the infected cell. To best of our knowledge, this is the first report on NSP1 functionality where a signaling protein is targeted unanimously in all strains. In addition NSP1 inhibited the formation of detergent resistant MAVS aggregates, thereby averting the antiviral signaling cascade. The present study highlights the multifunctional role of rotavirus NSP1 and reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit by various back up strategies.</p></div

Role of a Kinesin Motor in Cancer Cell Mechanics

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