Dipanjan Dutta

Constitutive interferon-inducible protein 16-inflammasome activation during Epstein-Barr virus latency I, II, and III in B and epithelial cells.

ABSTRACT Epstein-Barr virus (EBV), etiologically linked with human B-cell malignancies and nasopharyngeal carcinoma (NPC), establishes three types of latency that facilitate its episomal genome persistence and evasion of host immune responses. The innate inflammasome responses recognize the pathogen-associated molecular patterns which lead into the association of a cytoplasmic sensor such as NLRP3 and AIM2 proteins or nuclear interferon-inducible protein 16 (IFI16) with adaptor ASC protein (apoptosis-associated speck-like protein with a caspase recruitment domain) and effector procaspase-1, resulting in active caspase-1 formation which cleaves the proforms of inflammatory interleukin-1β (IL-1β), IL-18, and IL-33 cytokines. Whether inflammasome responses recognize and respond to EBV genome in the nuclei was not known. We observed evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β, -IL-18, and -IL-33, in EBV latency I Raji cells, latency II NPC C666-1 cells, and latency III lymphoblastoid cell lines (LCL). Interaction between ASC with IFI16 but not with AIM2 or NLRP3 was detected in all three latencies and during EBV infection of primary human B cells. IFI16 and cleaved caspase-1, IL-1β, IL-18, and IL-33 were detected in the exosomes from Raji cells and LCL. Though EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) are common to all forms of EBV latency, caspase-1 cleavage was not detected in cells expressing EBNA1 alone, and blocking EBER transcription did not inhibit caspase-1 cleavage. In fluorescence in situ hybridization (FISH) analysis, IFI16 colocalized with the EBV genome in LCL and Raji cell nuclei. These studies demonstrated that constant sensing of latent EBV genome by IFI16 in all types of latency results in the constitutive induction of the inflammasome and IL-1β, IL-18, and IL-33 maturation.

Rotavirus Nonstructural Protein 1 Suppresses Virus-Induced Cellular Apoptosis To Facilitate Viral Growth by Activating the Cell Survival Pathways during Early Stages of Infection

ABSTRACT Following virus infection, one of the cellular responses to limit the virus spread is induction of apoptosis. In the present study, we report role of rotavirus nonstructural protein 1 (NSP1) in regulating apoptosis by activating prosurvival pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and NF-κB (nuclear factor κB) during early hours of infections (2 to 8 hpi). The NSP1 mutant strain A5-16 induces weak and transient activation of Akt (protein kinase B) and p65 NF-κB compared to the isogenic wild-type strain A5-13 in MA104 or HT29 cells. The weak NF-κB promoter activity or Akt phosphorylation after A5-16 infection could be complemented in cells transfected with plasmid expressing NSP1 after infection with the rotavirus A5-16 strain. In cells either infected with A5-13 or transfected with pcD-NSP1, coimmunoprecipitation of NSP1 with phosphoinositide 3-kinase (PI3K) was observed, indicating that strong activation of PI3K/Akt could be due to its interaction with NSP1. In addition, after infection with same multiplicity of infection, A5-16 showed reduced number of viral particles compared to the A5-13 strain at the end of the replication cycle. A lower growth rate could be due to weak induction of PI3K/Akt and NF-κB, since the A5-13 strain also showed reduced growth in the presence of PI3K or NF-κB inhibitors. This effect was interferon independent; however, it was partly due to significantly higher caspase-3 activity, poly-ADP ribose polymerase (PARP) cleavage, and apoptosis during earlier stages of infection with the NSP1 mutant. Thus, our data suggest that NSP1 positively supports rotavirus growth by suppression of premature apoptosis for improved virus growth after infection.

Surveillance and molecular characterization of rotavirus strains circulating in Manipur, North-Eastern India: Increasing prevalence of emerging G12 strains.

To determine the frequency and genotypes of rotavirus strains, samples were collected from children hospitalized with acute diarrhea at the Regional Institute of Medical Sciences, Manipur. The globally common genotypes G1P[8] and G2P[4] constituted 58% of the total positive strains, while 3% and 8% strains were emerging genotypes, G9P[6] and G12P[6]. This is the first report of genotype G12 in Manipur. The G12 strains clustered with lineage III strains and had >98% identity with corresponding rotaviruses from Bangladesh, Thailand and the USA. Other uncommon G-P combinations including G4P[4], G4P[6], G10P[6] and G9P[19], along with a few strains that could not be typed were also found. The VP7 genes of G4 and G10 strains clustered with porcine and bovine strains, indicating possible zoonotic transmission. High frequency (36-62%) of rotavirus infection and predominance of G1P[8] and G2P[4] among children with acute diarrhea emphasized the need for implementation of currently available vaccines to reduce the burden of rotavirus induced diarrhea in India.

Interactions between exosomes from breast cancer cells and primary mammary epithelial cells leads to generation of reactive oxygen species which induce DNA damage response, stabilization of p53 and autophagy in epithelial cells.

Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment.

Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses

The IL-1β and type I interferon-β (IFN-β) molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16) involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1) episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the increased nuclear acetylation of IFI16 as a dynamic essential post-genome recognition event in the nucleus that is common to the IFI16-mediated innate responses of inflammasome induction and IFN-β production during herpesvirus (KSHV, EBV, HSV-1) infections.

The molecular chaperone heat shock protein-90 positively regulates rotavirus infection

Rotaviruses are the major cause of severe dehydrating gastroenteritis in children worldwide. In this study, we report a positive role of cellular chaperone Hsp90 during rotavirus infection. A highly specific Hsp90 inhibitor, 17-allylamono-demethoxygeldanamycin (17-AAG) was used to delineate the functional role of Hsp90. In MA104 cells treated with 17-AAG after viral adsorption, replication of simian (SA11) or human (KU) strains was attenuated as assessed by quantitating both plaque forming units and expression of viral genes. Phosphorylation of Akt and NFkappaB observed 2-4 hpi with SA11, was strongly inhibited in the presence of 17-AAG. Direct Hsp90-Akt interaction in virus infected cells was also reduced in the presence of 17-AAG. Anti-rotaviral effects of 17-AAG were due to inhibition of activation of Akt that was confirmed since, PI3K/Akt inhibitors attenuated rotavirus growth significantly. Thus, Hsp90 regulates rotavirus by modulating cellular signaling proteins. The results highlight the importance of cellular proteins during rotavirus infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies.

BRCA1 Regulates IFI16 Mediated Nuclear Innate Sensing of Herpes Viral DNA and Subsequent Induction of the Innate Inflammasome and Interferon-β Responses

The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

Kaposi's Sarcoma-Associated Herpesvirus Induces the ATM and H2AX DNA Damage Response Early during De Novo Infection of Primary Endothelial Cells, Which Play Roles in Latency Establishment

ABSTRACT The DNA damage response (DDR) that evolved to repair host cell DNA damage also recognizes viral DNA entering the nucleus during infections. Here, we investigated the modulation of DDR signaling during de novo infection of primary endothelial cells by Kaposis sarcoma-associated herpesvirus (KSHV). Phosphorylation of representative DDR-associated proteins, such as ataxia telangiectasia mutated (ATM) and H2AX, was induced as early as 30 min (0.5 h) postinfection and persisted during in vitro KSHV latency. Phosphorylated H2AX (γH2AX) colocalized at 30 min (0.5 h) with the KSHV genome entering the nuclei. Total H2AX protein levels also increased, and the increase was attributed to a decrease in degradative H2AX Lys48-linked polyubiquitination with a concomitant increase in Lys63-linked polyubiquitination that was shown to increase protein stability. ATM and H2AX phosphorylation and γH2AX nuclear foci were also induced by UV-inactivated KSHV, which ceased at later times of infection. Inhibition of ATM kinase activity by KU-55933 and H2AX knockdown by small interfering RNA significantly reduced the expression of the KSHV latency-associated nuclear antigen 1 (LANA-1; ORF73) and LANA-1 nuclear puncta. Knockdown of H2AX also resulted in a >80% reduction in the nuclear KSHV DNA copy numbers. Similar results were also observed in ATM-negative cells, although comparable levels of viral DNA entered ATM-negative and ATM-positive cell nuclei. In contrast, knockdown of CHK1 and CHK2 did not affect ORF73 expression. Collectively, these results demonstrate that KSHV induces ATM and H2AX, a selective arm of the DDR, for the establishment and maintenance of its latency during de novo infection of primary endothelial cells. IMPORTANCE Eukaryotic cells mount a DNA damage response (DDR) to sense and repair different types of cellular DNA damage. In addition, DDR also recognizes exogenous genetic material, such as the viral DNA genome entering the nucleus during infections. The present study was undertaken to determine whether de novo Kaposis sarcoma-associated herpesvirus (KSHV) infection modulates DDR. Our results demonstrate that early during de novo infection of primary endothelial cells, KSHV induces a selective arm of DDR signaling, such as the ATM kinase and its downstream target, H2AX, which are essential for KSHVs latent gene expression and the establishment of latency. These studies suggest that targeting ATM and H2AX could serve as an attractive strategy to block the establishment of KSHV latent infection and the associated malignancies.

Interaction of KSHV with Host Cell Surface Receptors and Cell Entry

Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry.

Analysis of p53 and NF-κB signaling in modulating the cardiomyocyte fate during hypertrophy.

Cardiac hypertrophy leading to eventual heart failure is the most common cause of mortality throughout the world. The triggering mechanisms for cardiac hypertrophy are not clear but both apoptosis and cell proliferation have been reported in sections of failing hearts. In this study, we utilized both angiotensin II (AngII) treatment of cardiomyocytes and aortic ligation in rats (Rattus norvegicus, Wistar strain) for induction of hypertrophy to understand the cellular factors responsible for activation of apoptotic or anti‐apoptotic pathway. Hypertrophy markers (ANF, β‐MHC), apoptotic proteins (Bax, Bad, Fas, p53, caspase‐3, PARP), and anti‐apoptotic or cell proliferation marker proteins (Bcl2, NF‐κB, Ki‐67) were induced significantly during hypertrophy, both in vitro as well as in vivo. Co‐localization of both active caspase‐3 and Ki‐67 was observed in hypertrophied myocytes. p53 and NF‐κBp65 binding to co‐activator p300 was also increased in AngII treated myocytes. Inhibition of p53 resulted in downregulation of apoptosis, NF‐κB activation, and NF‐κB–p300 binding; however, NF‐κB inhibition did not inhibit apoptosis or p53–p300 binding. Blocking of either p53 or NF‐κB by specific inhibitors resulted in decrease in cell proliferation and hypertrophy markers, suggesting that p53 initially binds to p300 and then this complex recruits NF‐κB. Thus, these results indicate the crucial role of p53 in regulating both apoptotic and cell proliferation during hypertrophy. J. Cell. Physiol. 226: 2543–2554, 2011.