Saumendra N. Sarkar

Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling.

Double-stranded RNA (dsRNA), a frequent byproduct of virus infection, is recognized by Toll-like receptor 3 (TLR3) to mediate innate immune response to virus infection. TLR3 signaling activates the transcription factor IRF-3 by its Ser/Thr phosphorylation, accompanied by its dimerization and nuclear translocation. It has been reported that the Ser/Thr kinase TBK-1 is essential for TLR3-mediated activation and phosphorylation of IRF-3. Here we report that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways. One involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells. Thus, the PI3K-Akt pathway plays an essential role in TLR3-mediated gene induction.

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1β Modulates Host Innate Immune Response by Antagonizing IRF3 Activation

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-β) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1α and NSP1β (self-cleavage products of NSP1 during virus infection) and NSP11, three NSPs with strong inhibitory activity. All of three proteins, when expressed stably in cell lines, strongly inhibited double-stranded RNA (dsRNA) signaling pathways. NSP1β was found to inhibit both IFN regulatory factor 3 (IRF3)- and NF-κB-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1β. Moreover, when tested in a porcine myelomonocytic cell line, NSP1β inhibited Sendai virus-mediated activation of porcine IFN-β promoter activity. We propose that this NSP1β-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.

Human placental trophoblasts confer viral resistance to recipient cells

Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal–fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections.

The Interferon-Stimulated Genes: Targets of Direct Signaling by Interferons, Double-Stranded RNA, and Viruses

The interferon system plays a profound role in determining the outcome of viral infection in mammals. Viruses induce the synthesis of interferon, which, in turn, blocks virus replication by inducing the expression of antiviral proteins encoded by interferon-stimulated genes. It is not widely appreciated that without the participation of interferon, many of the same genes can also be induced by a variety of virus-related agents, such as double-stranded RNA and viral proteins. In this chapter, we discuss different signaling pathways, activated by these agents, that lead to the induction of partially overlapping sets of genes, including the interferon-stimulated genes. We also review the biochemical and cellular properties of the protein products of a selected number of these genes including ISG56, ISG54, and ISG15.

Double-stranded RNA signaling by Toll-like receptor 3 requires specific tyrosine residues in its cytoplasmic domain.

Double-stranded (ds) RNA, a common product of viral infection, can induce transcription of many cellular genes, including the 561 gene that encodes P56, a regulator of protein synthesis. Here, we report that induction of the 561 mRNA by exogenous dsRNA is mediated by Toll-like receptor 3 (TLR3), and it requires no new protein synthesis. Because gene induction by dsRNA is blocked by inhibitors of tyrosine kinases, we investigated the potential roles of the five tyrosine residues present in the cytoplasmic domain of TLR3 by their individual and combinatorial mutations. Transfection assays, using a reporter gene driven by the 561 promoter, identified specific tyrosine residues to be essential for TLR3 signaling. This conclusion was further validated in permanent cell lines expressing tyrosine-mutant TLR3 proteins; in some of these cell lines dsRNA failed to induce the 561 mRNA. Our results provide the first demonstration of the importance of TLR3 cytoplasmic tyrosine residues in dsRNA signaling.

A Specific Isozyme of 2′-5′ Oligoadenylate Synthetase Is a Dual Function Proapoptotic Protein of the Bcl-2 Family

2-5(A) synthetases are a family of interferon-induced enzymes that polymerize ATP into 2′-5′ linked oligoadenylates that activate RNase L and cause mRNA degradation. Because they all can synthesize 2-5(A), the reason for the existence of so many synthetase isozymes is unclear. Here we report that the 9-2 isozyme of 2-5(A) synthetase has an additional activity: it promotes apoptosis in mammalian cells. The proapoptotic activity of 9-2 was isozyme-specific and enzyme activity-independent. The 9-2-expressing cells exhibited many properties of cells undergoing apoptosis, such as DNA fragmentation, caspase activation, and poly ADP-ribose polymerase and lamin B cleavage. The isozyme-specific carboxyl-terminal tail of the 9-2 protein was shown, by molecular modeling, to contain a Bcl-2 homology 3 (BH3) domain, suggesting that it may be able to interact with members of the Bcl-2 family that contain BH1 and BH2 domains. Co-immunoprecipitate assays and confocal microscopy showed that 9-2 can indeed interact with the anti-apoptotic proteins Bcl-2 and BclxL in vivo andin vitro. Mutations in the BH3 domain that eliminated the 9-2-Bcl-2 amd 9-2-BclxL interactions also eliminated the apoptotic activity of 9-2. Thus, we have identified an interferon-induced dual function protein of the Bcl-2 family that can synthesize 2-5(A) and promote cellular apoptosis independently. Moreover, the cellular abundance of this protein is regulated by alternative splicing; the other isozymes encoded by the same gene are not proapoptotic.

Enzymatic Activity of 2′–5′-Oligoadenylate Synthetase Is Impaired by Specific Mutations that Affect Oligomerization of the Protein

Previous studies from our laboratory have shown that deletion of residues 321 to 344 of the 9-2 isozyme of 2′–5′-oligoadenylate (2–5(A)) synthetase causes a loss of its enzyme activity (Ghosh, S. K., Kusari, J., Bandyopadhyay, S. K., Samanta, H., Kumar, R., and Sen, G. C. (1991) J. Biol. Chem. 266, 15293–15299). Sequence comparison of this region among the different isozymes of 2–5(A) synthetases revealed that the residues at positions 330 to 333 are highly conserved. Alanine-scanning mutagenesis of these residues demonstrated that the residues present at 331, 332, and 333 are important for activity but the proline at position 330 was dispensable. The triple mutant containing Ala residues at 331, 332, and 333 was completely inactive. Different double mutants were slightly active, and the three single mutants were partially active. The triple mutant was further characterized for delineating the nature of its defect. The mutant protein was enzymatically inactive irrespective of whether it was synthesized in rabbit reticulocyte lysate, Escherichia coli or Trichoplusia niinsect cells. It could bind double-stranded RNA and ATP as efficiently as the wild type protein. It was, however, defective in oligomerization. Gel filtration and sedimentation velocity analyses ofin vitro synthesized proteins revealed that the wild type protein, but not the triple mutant, formed tetramers. The tetrameric fraction, but not the monomeric fraction of the wild type protein was enzymatically active. The failure of the triple mutant to participate in homomeric protein-protein interaction was confirmed byin vivo assays in insect cells. These results indicate that tetramerization of the protein is required for the enzymatic activity of the small 2–5(A) synthetases.

The Nature of the Catalytic Domain of 2′-5′-Oligoadenylate Synthetases

2′-5′-Oligoadenylate (2-5(A)) synthetases are a family of interferon-induced enzymes that are activated by double-stranded RNA. To understand why, unlike other DNA and RNA polymerases, they catalyze 2′-5′ instead of 3′-5′ phosphodiester bond formation, we used molecular modeling to compare the structure of the catalytic domain of DNA polymerase β (pol β) to that of a region of the P69 isozyme of 2-5(A) synthetase. Although the primary sequence identity is low, like pol β, P69 can assume an αββαβββ structure in this region. Moreover, mutation of the three Asp residues of P69, which correspond to the three catalytic site Asp residues of pol β, inactivated the enzyme without affecting its substrate and activator binding capacity, providing further credence to the concept that this region is the catalytic domain of P69. This domain is highly conserved among all 2-5(A) synthetase isozymes. Biochemical and mutational studies demonstrated that dimerization of the P69 protein is required for its enzyme activity. However, a dimer containing a wild type subunit and an inactive catalytic domain mutant subunit was also active. The rate of catalysis of the heterodimer was half of that of the wild type homodimer, although the two proteins bound double-stranded RNA and ATP equally well.

PKC alpha regulates Sendai virus-mediated interferon induction through HDAC6 and β-catenin

Recognition of viral RNA by cytoplasmic retinoic acid inducible gene I (RIG‐I)‐like receptors initiates signals leading to the induction of type I interferon (IFN) transcription via transcription factors such as interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF‐κB). Here, we describe a new signalling pathway that involves protein kinase C alpha (PKCα), histone deacetylase 6 (HDAC6) and beta‐catenin (β‐catenin), which is essential for IFN gene induction following virus infection. Knockdown of PKCα in various human cells, including primary cells, inhibited Sendai virus (SeV)‐mediated IFN induction and enhanced virus replication. In the absence of this pathway IRF3 becomes activated, but does not bind to its promoter and is thus unable to support transcription. Mechanistically, SeV infection induced the activation of PKCα, which promoted its interaction with HDAC6 and enhanced its deacetylation activity in a phosphorylation‐dependent manner. Further downstream, HDAC6 caused deacetylation of β‐catenin and enhanced its nuclear translocation and promoter binding. In the nucleus, β‐catenin acted as a co‐activator for IRF3‐mediated transcription. Our findings suggest an important role of a novel signalling pathway mediated by PKCα–HDAC6–β‐catenin in controlling IRF3‐mediated transcription.

Enzymatic characteristics of recombinant medium isozyme of 2'-5' oligoadenylate synthetase.

P69 is an isozyme of the medium size class of human 2′-5′ oligoadenylate synthetases. In this study, recombinant P69 was expressed and used for enzymological and structural investigations. Bacterially expressed P69 was inactive whereas the same protein expressed in insect cells was highly active. Whether this difference could be due to differential post-translational modifications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme activity. In contrast, inhibition of glycosylation of P69, by tunicamycin treatment of the insect cells, produced an enzymatically inactive protein. Recombinant P69 produced in insect cells was purified by affinity chromatography. It was a dimeric glycoprotein, very stable and completely dependent on double stranded (ds) RNA for activity. The enzyme catalyzed the non-processive synthesis of 2′-5′-linked oligoadenylate products containing up to 30 residues. 2′-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural studies.