The Human Cytomegalovirus pUL145 Isoforms Act as Viral DDB1-Cullin-Associated Factors to Instruct Host Protein Degradation to Impede Innate Immunity

ABSTRACTControl of human cytomegalovirus (HCMV) requires a continuous immune surveillance, thus HCMV is the most important viral pathogen in severely immunocompromised individuals. Both innate and adaptive immunity contribute to the control of HCMV. Here, we report that peripheral blood natural killer cells (PBNKs) from HCMV-seropositive donors showed an enhanced activity toward HCMV-infected autologous macrophages. However, this enhanced response was abolished when purified NK cells were applied as effectors. We demonstrate that this enhanced PBNK activity was dependent on the interleukin-2 (IL-2) secretion of CD4+T cells when reexposed to the virus. Purified T cells enhanced the activity of purified NK cells in response to HCMV-infected macrophages. This effect could be suppressed by IL-2 blocking. Our findings not only extend the knowledge on the immune surveillance in HCMV—namely, that NK cell-mediated innate immunity can be enhanced by a preexisting T cell antiviral immunity—but also indicate a potential clinical implication for patients at risk for severe HCMV manifestations due to immunosuppressive drugs, which mainly suppress IL-2 production and T cell responsiveness.IMPORTANCEHuman cytomegalovirus (HCMV) is never cleared by the host after primary infection but instead establishes a lifelong latent infection with possible reactivations when the host′s immunity becomes suppressed. Both innate immunity and adaptive immunity are important for the control of viral infections. Natural killer (NK) cells are main innate effectors providing a rapid response to virus-infected cells. Virus-specific T cells are the main adaptive effectors that are critical for the control of the latent infection and limitation of reinfection. In this study, we found that IL-2 secreted by adaptive CD4+T cells after reexposure to HCMV enhances the activity of NK cells in response to HCMV-infected target cells. This is the first direct evidence that the adaptive T cells can help NK cells to act against HCMV infection.

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Toxoplasma GRA15 Activates the NF-κB Pathway through Interactions with TNF Receptor-Associated Factors

mBio ◽

10.1128/mbio.00808-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 9

Author(s):

Lamba Omar Sangaré ◽

Ninghan Yang ◽

Eleni K. Konstantinou ◽

Diana Lu ◽

Debanjan Mukhopadhyay ◽

...

Keyword(s):

Innate Immunity ◽

Cell Death ◽

Transcription Factor ◽

Host Cell ◽

Binding Sites ◽

Associated Factors ◽

Nuclear Factor Kappa B ◽

Adaptor Proteins ◽

Nuclear Factor ◽

Tnf Receptor

ABSTRACT The protozoan parasite Toxoplasma gondii secretes proteins from specialized organelles, the rhoptries, and dense granules, which are involved in the modulation of host cell processes. Dense granule protein GRA15 activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. Exactly how GRA15 activates the NF-κB pathway is unknown. Here we show that GRA15 interacts with tumor necrosis factor receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. We identified several TRAF binding sites in the GRA15 amino acid sequence and showed that these are involved in NF-κB activation. Furthermore, a TRAF2 knockout cell line has impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation. IMPORTANCE The parasite Toxoplasma can cause birth defects and severe disease in immunosuppressed patients. Strain differences in pathogenicity exist, and these differences are due to polymorphic effector proteins that Toxoplasma secretes into the host cell to coopt host cell functions. The effector protein GRA15 of some Toxoplasma strains activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. We show that GRA15 interacts with TNF receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. Deletion of TRAF-binding sites in GRA15 greatly reduces its ability to activate the NF-κB pathway, and TRAF2 knockout cells have impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.

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Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein Degradation

Journal of Virology ◽

10.1128/jvi.01690-17 ◽

2017 ◽

Vol 92 (1) ◽

Cited By ~ 11

Author(s):

Dylan A. Frabutt ◽

Bin Wang ◽

Sana Riaz ◽

Richard C. Schwartz ◽

Yong-Hui Zheng

Keyword(s):

Innate Immunity ◽

Endoplasmic Reticulum ◽

Enzymatic Activity ◽

Er Stress ◽

Protein Degradation ◽

Influenza A Virus ◽

Influenza A ◽

Critical Role ◽

Viral Glycoproteins ◽

Stress Pathway

ABSTRACT Innate immunity provides an immediate defense against infection after host cells sense danger signals from microbes. Endoplasmic reticulum (ER) stress arises from accumulation of misfolded/unfolded proteins when protein load overwhelms the ER folding capacity, which activates the unfolded protein response (UPR) to restore ER homeostasis. Here, we show that a mechanism for antiviral innate immunity is triggered after the ER stress pathway senses viral glycoproteins. When hemagglutinin (HA) glycoproteins from influenza A virus (IAV) are expressed in cells, ER stress is induced, resulting in rapid HA degradation via proteasomes. The ER-associated protein degradation (ERAD) pathway, an important UPR function for destruction of aberrant proteins, mediates HA degradation. Three class I α-mannosidases were identified to play a critical role in the degradation process, including EDEM1, EDEM2, and ERManI. HA degradation requires either ERManI enzymatic activity or EDEM1/EDEM2 enzymatic activity when ERManI is not expressed, indicating that demannosylation is a critical step for HA degradation. Silencing of EDEM1, EDEM2, and ERManI strongly increases HA expression and promotes IAV replication. Thus, the ER stress pathway senses influenza HA as “nonself” or misfolded protein and sorts HA to ERAD for degradation, resulting in inhibition of IAV replication. IMPORTANCE Viral nucleic acids are recognized as important inducers of innate antiviral immune responses that are sensed by multiple classes of sensors, but other inducers and sensors of viral innate immunity need to be identified and characterized. Here, we used IAV to investigate how host innate immunity is activated. We found that IAV HA glycoproteins induce ER stress, resulting in HA degradation via ERAD and consequent inhibition of IAV replication. In addition, we have identified three class I α-mannosidases, EDEM1, EDEM2, and ERManI, which play a critical role in initiating HA degradation. Knockdown of these proteins substantially increases HA expression and IAV replication. The enzymatic activities and joint actions of these mannosidases are required for this antiviral activity. Our results suggest that viral glycoproteins induce a strong innate antiviral response through activating the ER stress pathway during viral infection.

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Proteomic Analysis Uncovers Measles Virus Protein C Interaction with p65/iASPP/p53 Protein Complex

10.1101/2020.05.08.084418 ◽

2020 ◽

Author(s):

Alice Meignié ◽

Chantal Combredet ◽

Marc Santolini ◽

István A. Kovács ◽

Thibaut Douché ◽

...

Keyword(s):

Innate Immunity ◽

Cell Death ◽

Protein Complex ◽

Measles Virus ◽

P53 Protein ◽

Host Protein ◽

Mass Spectrometry Analysis ◽

C Protein ◽

Cell Death Pathways ◽

Protein Partners

ABSTRACTViruses manipulate central machineries of host cells to their advantage. They prevent host cell antiviral responses to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two non-structural proteins MV-V and MV-C known to counteract the host interferon response and to regulate cell death pathways. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death pathways have been proposed, whereas MV-C host protein partners are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. To determine which host factors are targeted by MV-C, we captured both direct and indirect host protein partners of MV-C protein. For this, we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. From the list of host proteins specifically interacting with MV-C protein in different cell lines we selected the host targets that belong to immunity and cell death pathways for further validation. Direct protein partners of MV-C were determined by applying protein complementation assay (PCA) and the bioluminescence resonance energy transfer (BRET) approach. As a result, we found that MV-C protein specifically interacts with p65/iASPP/p53 protein complex that controls both cell death and innate immunity pathways.

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Phosphorylation by CK2 increases the SUMO-dependent activity of Cytomegalovirus transactivator IE2

10.1101/655282 ◽

2019 ◽

Author(s):

Vasvi Tripathi ◽

Kiran Sankar Chatterjee ◽

Ranabir Das

Keyword(s):

Human Cytomegalovirus ◽

Cross Talk ◽

Viral Genome ◽

Essential Gene ◽

E3 Ligase ◽

Host Protein ◽

Genome Replication ◽

Salt Bridges ◽

Post Translational Modification ◽

Viral Genes

AbstractViral factors manipulate the host post-translational modification (PTM) machinery for replication. Distinctly, phosphorylation and SUMOylation can regulate the activity of human cytomegalovirus (HCMV) protein IE2. However, the molecular mechanism of this process is unknown. Taking a structural, biochemical and cellular approach, we uncover a cross-talk of phosphorylation and SUMOylation exploited by IE2. A scan for the SUMO Interacting Motifs (SIMs) revealed two SIMs in IE2. A real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhanced IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies proved that IE2 is a SUMO cis-E3 ligase. Two putative CK2 sites adjacent to IE2-SIM1 are phosphorylated in-vitro and in cellular conditions. Phosphorylation drastically increased the IE2/SUMO affinity, IE2-SUMOylation and cis-E3 activity of IE2. Additional salt-bridges between the phosphoserines and SUMO account for the higher IE2/SUMO affinity. Phosphorylation also enhances the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism where SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2, works in tandem to enable transcriptional regulation of viral genes.Author summaryThe host protein SUMO is a crucial regulator of cellular processes. Conjugation of other proteins to SUMO by a process called SUMOylation, can change the protein’s function or localization and regulate downstream cellular events. The SUMO pathway is exploited by viruses to transcribe viral genes and replicate the viral genome. IE2 is an essential gene of human Cytomegalovirus (HCMV), which acts as a transactivator and helps to transcribe other viral proteins required for viral genome replication and viral assembly. SUMOylation of IE2 is necessary for its function. Here, we have uncovered that IE2 functions as a cis-SUMO-E3 ligase, where a SUMO-Interacting Motif (SIM) in IE2 enhances its SUMOylation. Interestingly, phosphorylation of the SIM in IE2 augments its cis-E3 activity to further increase SUMOylation. Moreover, SIM phosphorylation also enhances the interaction between IE2 and SUMOylated binding partners. Thus, we uncover an exciting process, where phosphorylation enhances both covalent and non-covalent interaction of a protein (IE2) and SUMO. We also observe that the cross-talk of phosphorylation and SUMOylation has significant effects on the transactivation function of IE2. Overall, we discover how a viral protein IE2 exploits crosstalk between SUMOylation and Phosphorylation to enhance its activity and in turn, ensure efficient viral replication.

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