Rahul Bhowmick

Rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of apoptosis during infection.

Background: Rotaviral nonstructural protein 4 (NSP4) disrupts Ca2+ ion homeostasis by translocating to the endoplasmic reticulum. Results: In this study, we show translocation of NSP4 to mitochondria, dissipation of mitochondrial potential, and initiation of apoptosis, which NSP1 counteracts during early infection. Conclusion: NSP4 and NSP1 regulate apoptosis during infection. Significance: Study signifies modulation of cellular survival and apoptotic machinery by rotavirus for their own benefit. Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca2+ homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61–83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.

Antiviral activity of baicalin against influenza virus H1N1-pdm09 is due to modulation of NS1-mediated cellular innate immune responses

OBJECTIVES Baicalin, a flavonoid, has been shown to have antiviral and anti-inflammatory activities, although the mechanism of action has been unknown. Therefore, attempts were made to analyse the mechanism behind the antiviral effects of baicalin using an influenza A virus (IAV) model in vitro and in vivo. METHODS Baicalins anti-influenza activity was elucidated (in vitro and in vivo) utilizing pandemic influenza strain A/H1N1/Eastern India/66/pdm09 (H1N1-pdm09). Anti-influenza activity was measured by plaque inhibition, fluorescent focus-forming units (ffu) and quantifying viral transcripts using quantitative real-time PCR following treatment with baicalin in a dose- and time-dependent manner. The role of the IAV non-structural protein 1 (NS1) gene in modulating host responses was measured by immunoblotting, co-immunoprecipitation and molecular docking. RESULTS Baicalin treatment following IAV infection revealed up-regulation of interferon (IFN)-induced antiviral signalling and decreased phosphoinositide 3-kinase/Akt (PI3K/Akt) activation compared with infected, untreated controls. Baicalin exerts its antiviral effects by modulating the function of the IAV-encoded NS1 protein. NS1 has been shown to counteract cellular antiviral responses by down-regulating IFN induction and up-regulating PI3K/Akt signalling. Baicalin disrupted NS1-p85β binding. Molecular docking predicted the binding site of baicalin in the RNA binding domain (RBD) of NS1. Site-directed mutagenesis within the RBD region of NS1 and the difference in the fluorescence quenching pattern of full-length NS1 and mutant NS1 proteins in the presence of baicalin confirmed the interaction of baicalin with the NS1 RBD. Amino acid residues 39-43 of the NS1 RBD were found to be crucial for the baicalin-NS1 interaction. CONCLUSIONS Overall, this study highlights that baicalin exerts its anti-influenza virus activity by modulating viral protein NS1, resulting in up-regulation of IFN-induced antiviral signalling and a decrease in PI3K/Akt signalling in cells.

Rotavirus-encoded nonstructural protein 1 modulates cellular apoptotic machinery by targeting tumor suppressor protein p53.

ABSTRACT p53, a member of the innate immune system, is triggered under stress to induce cell growth arrest and apoptosis. Thus, p53 is an important target for viruses, as efficient infection depends on modulation of the host apoptotic machinery. This study focuses on how rotaviruses manipulate intricate p53 signaling for their advantage. Analysis of p53 expression revealed degradation of p53 during initial stages of rotavirus infection. However, in nonstructural protein-1 (NSP1) mutant strain A5-16, p53 degradation was not observed, suggesting a role of NSP1 in this process. This function of NSP1 was independent of its interferon or phosphatidylinositol 3-kinase (PI3K)/AKT modulation activity since p53 degradation was observed in Vero cells as well as in the presence of PI3K inhibitor. p53 transcript levels remained the same in SA11-infected cells (at 2 to 14 h postinfection), but p53 protein was stabilized only in the presence of MG132, suggesting a posttranslational process. NSP1 interacted with the DNA binding domain of p53, resulting in ubiquitination and proteasomal degradation of p53. Degradation of p53 during initial stages of infection inhibited apoptosis, as the proapoptotic genes PUMA and Bax were downregulated. During late viral infection, when progeny dissemination is the main objective, the NSP1-p53 interaction was diminished, resulting in restoration of the p53 level, with initiation of proapoptotic signaling ensuing. Overall results highlight the multiple strategies evolved by NSP1 to combat the host immune response.

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

TNF 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 Cellular Calcium Binding Protein Calmodulin as a Regulator of Rotavirus A Infection during Comparative Proteomic Study

Rotavirus (RV) being the major diarrhoegenic virus causes around 527000 children death (<5years age) worldwide. In cellular environment, viruses constantly adapt and modulate to survive and replicate while the host cell also responds to combat the situation and this results in the differential regulation of cellular proteins. To identify the virus induced differential expression of proteins, 2D-DIGE (Two-dimensional Difference Gel Electrophoresis) based proteomics was used. For this, HT-29 cells were infected with RV strain SA11 for 0 hours, 3 hours and 9 hours post infection (hpi), differentially expressed spots were excised from the gel and identified using MALDI-TOF/TOF mass spectrometry. 2D-DIGE based proteomics study identified 32 differentially modulated proteins, of which 22 were unique. Some of these were validated in HT-29 cell line and in BALB/c mice model. One of the modulated cellular proteins, calmodulin (CaM) was found to directly interact with RV protein VP6 in the presence of Ca2+. Ca2+-CaM/VP6 interaction positively regulates RV propagation since both CaM inhibitor (W-7) and Ca2+ chelator (BAPTA-AM) resulted in decreased viral titers. This study not only identifies differentially modulated cellular proteins upon infection with rotavirus in 2D-DIGE but also confirmed positive engagement of cellular Ca2+/CaM during viral pathogenesis.

Rotavirus infection induces G1 to S phase transition in MA104 cells via Ca+2/Calmodulin pathway

Abstract Viruses, obligate cellular parasites rely on host cellular functions and target the host cell cycle for their own benefit. In this study, effect of rotavirus infection on cell cycle machinery was explored. We found that rotavirus (RV) infection in MA104 cells induces the expression of cyclins and cyclin dependent kinases and down-regulates expression of CDK inhibitors, resulting in G1 to S phase transition. The rotavirus induced S phase accumulation was found to be concurrent with induction in expression of calmodulin and activation of CaMKI which is reported as inducer of G1–S phase transition. This cell cycle manipulation was found to be Ca+2/Calmodulin pathway dependent. The physiological relevance of G1 to S phase transition was established when viral gene expressions as well as viral titers were found to be increased in S phase synchronized cells and decreased in G0/G1 phase synchronized cells compared to unsynchronized cells during rotavirus infection.

Identification of common human host genes involved in pathogenesis of different rotavirus strains: An attempt to recognize probable antiviral targets

Although two rotavirus vaccines have been licensed and approved by WHO and FDA; other parallel therapeutic strategies are needed to reduce the mortality and morbidity of rotavirus induced diarrhea worldwide. Since rotaviruses utilize the host cell machinery for their replication, study was initiated to identify host proteins which positively regulate rotavirus infection. To overcome the possible variations in host response due to existence of large variety of genotypes and human-animal reassortants, the total gene expression profile of HT29 cells infected with either simian (SA11) or bovine (A5-13) or human (Wa) rotavirus strains was analyzed using genome microarrays. Even though cells infected with human strain revealed some differences compared to the viruses of animal origin, 131 genes were similarly induced by all three strains. Genes involved in innate immune response, stress response, apoptosis and protein metabolism were induced by all viral strains. Results were validated by immunoblotting or RT-PCR. Role of some host genes in rotavirus infection was analyzed by using specific siRNAs.

Phosphorylation Drives an Apoptotic Protein to Activate Antiapoptotic Genes PARADIGM OF INFLUENZA A MATRIX 1 PROTEIN FUNCTION

Background: Influenza A matrix 1 protein (M1) associates with nuclear domain 10 (ND10) early in infection. Results: Phosphorylated M1 upon nuclear translocation induces survival genes by inhibiting death domain-associated protein 6 (Daxx) of ND10. Conclusion: M1 in early infection exhibits phosphorylation-dependent antiapoptotic function. Significance: This study uncovers the antiapoptotic role of M1 and identifies a possible therapeutic target to limit virus infection. During infection, viral proteins target cellular pathways that regulate cellular innate immune responses and cell death. We demonstrate that influenza A virus matrix 1 protein (M1), an established proapoptotic protein, activates nuclear factor-κB member RelB-mediated survival genes (cIAP1, cIAP2, and cFLIP), a function that is linked with its nuclear translocation during early infection. Death domain-associated protein 6 (Daxx) is a transcription co-repressor of the RelB-responsive gene promoters. During influenza virus infection M1 binds to and stabilizes Daxx protein by preventing its ubiquitination and proteasomal degradation. Binding of M1 with Daxx through its Daxx binding motif prevents binding of RelB and Daxx, resulting in up-regulation of survival genes. This interaction also prevents promoter recruitment of DNA methyltransferases (Dnmt1 and Dnmt3a) and lowers CpG methylation of the survival gene promoters, leading to the activation of these genes. Thus, M1 prevents repressional function of Daxx during infection, thereby exerting a survival role. In addition to its nuclear localization signal, translocation of M1 to the nucleus depends on cellular kinase-mediated phosphorylation as the protein kinase C inhibitor calphostin C effectively down-regulates virus replication. The study reconciles the ambiguity of dual antagonistic function of viral protein and potentiates a possible target to limit virus infection.

Rotavirus disrupts cytoplasmic P bodies during infection.

Cytoplasmic Processing bodies (P bodies), the RNA-protein aggregation foci of translationally stalled and potentially decaying mRNA, have been reported to be differentially modulated by viruses. Rotavirus, the causative agent of acute infantile gastroenteritis is a double stranded RNA virus which completes its entire life-cycle exclusively in host cell cytoplasm. In this study, the fate of P bodies was investigated upon rotavirus infection. It was found that P bodies get disrupted during rotavirus infection. The disruption occurred by more than one different mechanism where deadenylating P body component Pan3 was degraded by rotavirus NSP1 and exonuclease XRN1 along with the decapping enzyme hDCP1a were relocalized from cytoplasm to nucleus. Overall the study highlights decay and subcellular relocalization of P body components as novel mechanisms by which rotavirus subverts cellular antiviral responses.

Tyrosine Phosphorylation Modulates Mitochondrial Chaperonin Hsp60 and Delays Rotavirus NSP4-Mediated Apoptotic Signaling in Host Cells.

Phosphoproteomics‐based platforms have been widely used to identify post translational dynamics of cellular proteins in response to viral infection. The present study was undertaken to assess differential tyrosine phosphorylation during early hours of rotavirus (RV) SA11 infection. Heat shock proteins (Hsp60) were found to be enriched in the data set of RV‐SA11 induced differentially tyrosine‐phosphorylated proteins at 2 hr post infection (hpi). Hsp60 was further found to be phosphorylated by an activated form of Src kinase on 227th tyrosine residue, and tyrosine phosphorylation of mitochondrial chaperonin Hsp60 correlated with its proteasomal degradation at 2–2.5hpi. Interestingly, mitochondrial Hsp60 positively influenced translocation of the rotaviral nonstructural protein 4 to mitochondria during RV infections. Phosphorylation and subsequent transient degradation of mitochondrial Hsp60 during early hours of RV‐SA11 infection resulted in inhibition of premature import of nonstructural protein 4 into mitochondria, thereby delaying early apoptosis. Overall, the study highlighted one of the many strategies rotavirus undertakes to prevent early apoptosis and subsequent reduced viral progeny yield.