Rotavirus NSP1 Mediates Degradation of Interferon Regulatory Factors through Targeting of the Dimerization Domain

Arnold, Michelle M.; Barro, Mario; Patton, John T.

doi:10.1128/jvi.01146-13

Cited by 60 publications

(66 citation statements)

References 65 publications

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“…Moreover, NSP1 seems to interact with the cellular transcription factor interferon regulatory factor 3 (IRF3) and targets it for degradation by the proteasome, thus acting to avoid the host antiviral defense by the blocking INF production [14][15][16][17][18] . However, NSP1 seems not to be necessary for rotavirus replication in vitro and its role is not fully clear 19 .…”

Section: Proteins Involved In Replication Pathogenesis and Immune Rementioning

confidence: 99%

Rotavirus Disease Mechanisms Diarrhea, Vomiting and Inflammation : How and Why

Hagbom¹

2015

Linköping University Medical Dissertations

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Rotavirus infections cause diarrhea and vomiting that can lead to severe dehydration. Despite extensive tissue damage and cell death, the inflammatory response is very limited. The focus of this thesis was to study pathophysiological mechanisms behind diarrhea and vomiting during rotavirus infection and also to investigate the mechanism behind the limited inflammatory response. An important discovery in this thesis was that rotavirus infection and the rotavirus toxin NSP4 stimulate release of the neurotransmitter serotonin from intestinal sensory enterochromaffin cells, in vitro and ex vivo. Interestingly, serotonin is known to be a mediator of both diarrhea and vomiting. Moreover, mice pups infected with rotavirus responded with central nervous system (CNS) activation in brain structures associated with vomiting, thus indicating a cross-talk between the gut and brain in rotavirus disease. Our finding that rotavirus infection activates the CNS led us to address the hypothesis that rotavirus infection not only activates the vagus nerve to stimulate vomiting, but also suppresses the inflammatory response via the cholinergic anti-inflammatory pathway, both of which are mediated by activated vagal afferent nerve signals into the brain stem. We found that mice lacking an intact vagus nerve, and mice lacking the α7 nicotine acetylcholine receptor (nAChR), being involved in cytokine suppression from macrophages, responded with a higher inflammatory response. Moreover, stimulated cytokine release from macrophages, by the rotavirus toxin NSP4, could be attenuated by nicotine, an agonist of the α7 nAChR. Thus, it seems most reasonable that the cholinergic anti-inflammatory pathway contributes to the limited inflammatory response during rotavirus infection. Moreover, rotavirus-infected mice displayed increased intestinal motility at the onset of diarrhea, which was not associated with increased intestinal permeability. The increased motility and diarrhea in infant mice could be attenuated by drugs acting on the enteric nervous system, indicating the importance and contribution of nerves in the rotavirus mediated disease. In conclusion, this thesis provides further insight into the pathophysiology of diarrhea and describe for the first time how rotavirus and host cross-talk to induce the vomiting reflex and limit inflammation. Results from these studies strongly support our hypothesis that serotonin and activation of the enteric nervous system and CNS contributes to diarrhea, vomiting and suppression of the inflammatory response in rotavirus disease

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Section: Proteins Involved In Replication Pathogenesis and Immune Rementioning

confidence: 99%

Rotavirus Disease Mechanisms Diarrhea, Vomiting and Inflammation : How and Why

Hagbom¹

2015

Linköping University Medical Dissertations

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“…Three different NSP1 proteins were investigated and compared to a pulldown with HaloTag alone in order to ascertain if there were similarities and differences among NSP1s. NSP1 from SA11-4F rotavirus has been shown to induce the degradation of IRF3, IRF5, IRF7, and IRF9 (21,25). NSP1 from SA11-5S rotavirus is identical to SA11-4F except for a C-terminal truncation of 17 amino acids, which interferes with its ability to induce IRF degradation (25).…”

Section: Resultsmentioning

confidence: 99%

“…IRF3 was first identified as a target of NSP1-mediated degradation and was also shown to directly interact at the C-terminal end of NSP1 (16)(17)(18)(19). IRF5, IRF7, and IRF9 were subsequently identified as additional targets of degradation by NSP1 due to the structural similarity of their IRF association domain (or dimerization domain) to that of IRF3 (20,21). ␤-TrCP has also been shown to be a target of some NSP1 proteins, and the loss of ␤-TrCP prevents the nuclear translocation of NF-B, thereby preventing IFN induction (22,23).…”

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confidence: 99%

Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

Lutz

Pace

Arnold

2016

J Virol

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The rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or ␤-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or ␤-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity. IMPORTANCEThe ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response. P rotein ubiquitination is among the most widely used posttranslational modifications and regulates many aspects of cell biology, including cell signaling, DNA damage responses, and protein degradation (1). Conjugation of ubiquitin to a target protein occurs by the sequential activities of three types of enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligating enzyme (E3). E3 ubiquitin ligases are classified on the basis of the presence of either a...

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“…NSP1 specifically targets the IRF association domain of these proteins, which mediates IRF homo-or heterodimerization. The constitutively activated form of IRF3 is targeted by NSP1, as are forms of IRF3 that are unable to dimerize, suggesting that NSP1 broadly inhibits IFN production (10).…”

Section: How Does Nsp1 Prevent Ifn Induction and Signaling?mentioning

confidence: 99%

“…IRF3, which is a transcription factor that binds to the IFN promoter to induce transcription, was the first host protein shown to be degraded when NSP1 was expressed in infected or transfected cells (8). IRF5, IRF7, and IRF9 are also targets of NSP1-mediated degradation, due to similarities with IRF3 in domain structure (9,10). NSP1 specifically targets the IRF association domain of these proteins, which mediates IRF homo-or heterodimerization.…”

Section: How Does Nsp1 Prevent Ifn Induction and Signaling?mentioning

confidence: 99%

The Rotavirus Interferon Antagonist NSP1: Many Targets, Many Questions

Arnold

2016

J Virol

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Rotavirus is a leading cause of death due to diarrhea among young children across the globe. Despite the limited coding capacity that is characteristic of RNA viruses, rotavirus dedicates substantial resources to avoiding the host innate immune response. Among these strategies is use of the interferon antagonist protein NSP1, which targets cellular proteins required for interferon production to be degraded by the proteasome. Although numerous cellular targets have been described, there remain many questions about the mechanism of NSP1 activity and its role in promoting replication in specific host species.A ll viruses must evolve ways to replicate in the face of the host immune response. When a virus begins its replication cycle within a cell, viral RNA may be recognized as foreign by the host pattern recognition receptors (PRRs). A cascade of signaling events is then initiated that stimulates the transcription factors NF-B and interferon (IFN) regulatory factors (IRFs) to translocate to the nucleus, bind to specific promoter sequences, and induce the transcription of type I IFN mRNA. After IFN is synthesized and secreted, it binds to IFN receptors to signal to the same or neighboring cells that an infection has occurred, triggering the production of IFN-stimulated genes (ISGs). ISGs have direct antiviral activities and thus are the effectors of the IFN response. Because IFN initiates a strong antiviral response in the infected host, viruses usually encode one or more ways to prevent induction of IFN or to interfere with the signaling cascade downstream of IFN receptors. HOW DOES ROTAVIRUS AVOID DETECTION BY THE HOST PATHOGEN RECOGNITION MACHINERY?Rotavirus is a member of the Reoviridae family. Although there are eight different species of rotaviruses (species A to H), Rotavirus A is responsible for the majority of life-threatening diarrhea in children (1, 2). The viral particle is nonenveloped and consists of three protein layers that surround the 11 segments of genomic doublestranded RNA (dsRNA). While synthetic dsRNA and Reoviridae genomes are commonly used to experimentally stimulate the IFN response, rotavirus takes many precautionary measures to protect itself from the innate host defenses encountered during infection.In its natural host, rotavirus replicates in mature enterocytes at the tips of the small intestinal villi. Upon entering a cell, the virus sheds only the outermost layer of its protein shell, leaving a transcriptionally active double-layered particle to synthesize and extrude (ϩ) RNAs for translation by host ribosomes. The virus encodes and packages its own RNA-dependent RNA polymerase (viral protein VP1) and capping enzyme (VP3) to synthesize (ϩ) RNAs that have a 5= cap structure equivalent to that of the host mRNA but that lack a polyadenylated tail. As viral proteins accumulate, they are concentrated in nonenveloped inclusions called viroplasms, which form in the cytoplasm and serve as the centers of viral replication. Within viroplasms, (ϩ) RNAs can be used by newly forming viral particle...

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Rotavirus NSP1 Mediates Degradation of Interferon Regulatory Factors through Targeting of the Dimerization Domain

Cited by 60 publications

References 65 publications

Rotavirus Disease Mechanisms Diarrhea, Vomiting and Inflammation : How and Why

Rotavirus Disease Mechanisms Diarrhea, Vomiting and Inflammation : How and Why

Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

The Rotavirus Interferon Antagonist NSP1: Many Targets, Many Questions

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