scholarly journals Combating viral contaminants in CHO cells by engineering STAT1 mediated innate immunity

2018 ◽  
Author(s):  
Austin W.T. Chiang ◽  
Shangzhong Li ◽  
Benjamin P. Kellman ◽  
Gouri Chattopadhyay ◽  
Yaqin Zhang ◽  
...  
Keyword(s):  
Cho Cells ◽  
Rna Virus ◽  
Mammalian Cell Culture ◽  
Encephalomyocarditis Virus ◽  
Poly I:C ◽  
Type I ◽  
Dependent Manner ◽  
Viral Contaminants ◽  
Vesicular Stomatitis ◽  
Biopharmaceutical Manufacturing

AbstractViral contamination in biopharmaceutical manufacturing can lead to shortages in the supply of critical therapeutics. To facilitate the protection of bioprocesses, we explored the basis for the susceptibility of CHO cells, the most commonly used cell line in biomanufacturing, to RNA virus infection. Upon infection with certain ssRNA and dsRNA viruses, CHO cells fail to generate a significant interferon (IFN) response. Nonetheless, the downstream machinery for generating IFN responses and its antiviral activity is intact in these cells: treatment of cells with exogenously-added type I IFN or poly I:C prior to infection limited the cytopathic effect from Vesicular stomatitis virus (VSV), Encephalomyocarditis virus (EMCV), and Reovirus-3 virus (Reo-3) in a STAT1-dependent manner. To harness the intrinsic antiviral mechanism, we used RNA-Seq to identify two upstream repressors of STAT1: Gfi1 and Trim24. By knocking out these genes, the engineered CHO cells exhibited increased resistance to the prototype RNA viruses tested. Thus, omics-guided engineering of mammalian cell culture can be deployed to increase safety in biotherapeutic protein production among many other biomedical applications.

scholarly journals JMJD6 negatively regulates cytosolic RNA induced antiviral signaling by recruiting RNF5 to promote activated IRF3 K48 ubiquitination

2021 ◽  
Vol 17 (3) ◽  
pp. e1009366
Author(s):  
Wei Zhang ◽  
Qi Wang ◽  
Fan Yang ◽  
Zixiang Zhu ◽  
Yueyue Duan ◽  
...  
Keyword(s):  
Immune Responses ◽  
Type I Interferon ◽  
Sendai Virus ◽  
Rna Virus ◽  
Negative Regulator ◽  
Viral Rna ◽  
Poly I:C ◽  
Type I ◽  
Dependent Manner ◽  
Antiviral Signaling

The negative regulation of antiviral immune responses is essential for the host to maintain homeostasis. Jumonji domain-containing protein 6 (JMJD6) was previously identified with a number of functions during RNA virus infection. Upon viral RNA recognition, retinoic acid-inducible gene-I-like receptors (RLRs) physically interact with the mitochondrial antiviral signaling protein (MAVS) and activate TANK-binding kinase 1 (TBK1) to induce type-I interferon (IFN-I) production. Here, JMJD6 was demonstrated to reduce type-I interferon (IFN-I) production in response to cytosolic poly (I:C) and RNA virus infections, including Sendai virus (SeV) and Vesicular stomatitis virus (VSV). Genetic inactivation of JMJD6 enhanced IFN-I production and impaired viral replication. Our unbiased proteomic screen demonstrated JMJD6 contributes to IRF3 K48 ubiquitination degradation in an RNF5-dependent manner. Mice with gene deletion of JMJD6 through piggyBac transposon-mediated gene transfer showed increased VSV-triggered IFN-I production and reduced susceptibility to the virus. These findings classify JMJD6 as a negative regulator of the host’s innate immune responses to cytosolic viral RNA.

scholarly journals N6-methyladenosine RNA modification suppresses antiviral innate sensing pathways via reshaping double-stranded RNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weinan Qiu ◽  
Qingyang Zhang ◽  
Rui Zhang ◽  
Yangxu Lu ◽  
Xin Wang ◽  
...  
Keyword(s):  
Rna Virus ◽  
Negative Regulator ◽  
Rna Modification ◽  
Type I ◽  
Viral Immunity ◽  
Potential Therapeutic Target ◽  
Viral Dsrna ◽  
Double Stranded Rna ◽  
Genetic Ablation ◽  
Vesicular Stomatitis

AbstractDouble-stranded RNA (dsRNA) is a virus-encoded signature capable of triggering intracellular Rig-like receptors (RLR) to activate antiviral signaling, but whether intercellular dsRNA structural reshaping mediated by the N6-methyladenosine (m6A) modification modulates this process remains largely unknown. Here, we show that, in response to infection by the RNA virus Vesicular Stomatitis Virus (VSV), the m6A methyltransferase METTL3 translocates into the cytoplasm to increase m6A modification on virus-derived transcripts and decrease viral dsRNA formation, thereby reducing virus-sensing efficacy by RLRs such as RIG-I and MDA5 and dampening antiviral immune signaling. Meanwhile, the genetic ablation of METTL3 in monocyte or hepatocyte causes enhanced type I IFN expression and accelerates VSV clearance. Our findings thus implicate METTL3-mediated m6A RNA modification on viral RNAs as a negative regulator for innate sensing pathways of dsRNA, and also hint METTL3 as a potential therapeutic target for the modulation of anti-viral immunity.

scholarly journals TRIM24 facilitates antiviral immunity through mediating K63-linked TRAF3 ubiquitination

2020 ◽  
Vol 217 (7) ◽  
Author(s):  
Qingchen Zhu ◽  
Tao Yu ◽  
Shucheng Gan ◽  
Yan Wang ◽  
Yifei Pei ◽  
...  
Keyword(s):  
Virus Infection ◽  
Vesicular Stomatitis Virus ◽  
Rna Virus ◽  
Antiviral Immunity ◽  
Type I ◽  
Antiviral Signaling ◽  
Unknown Mechanism ◽  
Positive Regulator ◽  
Transcriptional Suppression ◽  
Vesicular Stomatitis

Ubiquitination is an essential mechanism in the control of antiviral immunity upon virus infection. Here, we identify a series of ubiquitination-modulating enzymes that are modulated by vesicular stomatitis virus (VSV). Notably, TRIM24 is down-regulated through direct transcriptional suppression induced by VSV-activated IRF3. Reducing or ablating TRIM24 compromises type I IFN (IFN-I) induction upon RNA virus infection and thus renders mice more sensitive to VSV infection. Mechanistically, VSV infection induces abundant TRIM24 translocation to mitochondria, where TRIM24 binds with TRAF3 and directly mediates K63-linked TRAF3 ubiquitination at K429/K436. This modification of TRAF3 enables its association with MAVS and TBK1, which consequently activates downstream antiviral signaling. Together, these findings establish TRIM24 as a critical positive regulator in controlling the activation of antiviral signaling and describe a previously unknown mechanism of TRIM24 function.

scholarly journals ARF-like Protein 16 (ARL16) Inhibits RIG-I by Binding with Its C-terminal Domain in a GTP-dependent Manner

2011 ◽  
Vol 286 (12) ◽  
pp. 10568-10580 ◽  
Author(s):  
Yong-Kang Yang ◽  
Hong Qu ◽  
Dong Gao ◽  
Wei Di ◽  
Hai-Wei Chen ◽  
...  
Keyword(s):  
Family Member ◽  
Sendai Virus ◽  
Rna Virus ◽  
Cell Types ◽  
Type I ◽  
Dependent Manner ◽  
Homologous Proteins ◽  
Downstream Signaling ◽  
Terminal Domain ◽  
Inhibitory Function

Retinoic acid-inducible gene I (RIG-I) recognizes RNA virus-derived nucleic acids, which leads to the production of type I interferon (IFN) in most cell types. Tight regulation of RIG-I activity is important to prevent ultra-immune responses. In this study, we identified an ARF-like (ARL) family member, ARL16, as a protein that interacts with RIG-I. Overexpression of ARL16, but not its homologous proteins ARL1 and ARF1, inhibited RIG-I-mediated downstream signaling and antiviral activity. Knockdown of endogenous ARL16 by RNAi potentiated Sendai virus-induced IFN-β expression and vesicular stomatitis virus replication. ARL16 interacted with the C-terminal domain (CTD) of RIG-I to suppress the association between RIG-I and RNA. ARL16 (T37N) and ARL16Δ45–54, which were restricted to the GTP-disassociated form, did not interact with RIG-I and also lost the inhibitory function. Furthermore, we suggest that endogenous ARL16 changes to GTP binding status upon viral infection and binds with the RIG-I CTD to negatively control its signaling activity. These findings suggested a novel innate immune function for an ARL family member, and a GTP-dependent model in which RIG-I is regulated.

Expression of ROR1 in Chronic Lymphocytic Leukemia Cells Enhances Cells Survival by Wnt5a Signaling.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 340-340
Author(s):  
Liguang Chen ◽  
Li Tang ◽  
Tetsu Fukuda ◽  
Thomas J. Kipps
Keyword(s):  
Tyrosine Kinase ◽  
Human Serum ◽  
Cell Survival ◽  
Cho Cells ◽  
Wnt Signaling Pathway ◽  
Leukemia Cell ◽  
Chinese Hamster ◽  
Type I ◽  
Dependent Manner

Abstract ROR1 is a type I membrane-receptor tyrosine kinase with intracellular tyrosine kinase domains and extracellular Frizzled-like cysteine-rich domains (CRDs) and kringle domains, which are common to receptors of members of the Wnt-family. We found that ROR1 might serve as an orphan receptor for Wnt5a, which can activate NF-κB via a non-canonical Wnt-signaling pathway. In prior studies we found that CLL cells expressed ROR1 and high-levels of Wnt3, Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16, but not Wnt5a, which is found expressed on dendritic cells and other stromal cells that can support leukemia-cell survival in vitro, and presumably in vivo. As such, we examined whether cells could support the survival of CLL cells in a Wnt5a-dependent manner. For this we cultured CLL cells alone or together with Chinese Hamster Ovary (CHO) cells or CHO transduced to express Wnt5a (CHO-Wnt5a) and monitored the viability of CLL cells over time. We found that the viability of CLL cells co-cultured with CHO cells or CHO-Wnt5a cells was greater than that of CLL cells cultured alone. However the viability of CLL cells co-cultured for two days with CHO-Wnt5a cells (71% ± 18% S.D) was significantly greater than that of CLL cells co-cultured with CHO cells (45% ± 17% S.D) (N=10, P<0.01). The capacity of CHO-Wnt5a to enhance the viability of CLL cells relative to that of CHO cells could be neutralized by anti-ROR1 antisera generated in patients who received autologous CLL cells transduced to express the CD40-ligand (CD154), but not by normal human serum or pre-treatment human serum. CHO cells transduced to express ROR1, but not CHO cells transduced with a control vector, could absorb the capacity of the anti-ROR1 antisera to bind ROR1 and removed its capacity to neutralize the capacity of CHO-Wnt5a cells to provide for a protecive advantage over CHO cells for CLL cell survival in vitro. These studies provide the first evidence that the survival of CLL cells can be enhanced by cells expressing Wnt5a, let alone Wnt factors in general, and might account in part for the enhanced survival of CLL cells in tissue microenvironments containing cells that express Wnt5a. Conceivably, the anti-ROR1 antibodies induced by autologous CLL cells transduced to express CD154, or the administration of antibodies or antagonists that can inhibit Wnt5a-ROR1 signaling, could have therapeutic activity in patients with CLL.

scholarly journals Vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer

2012 ◽  
Vol 93 (12) ◽  
pp. 2529-2545 ◽  
Author(s):  
Eric Hastie ◽  
Valery Z. Grdzelishvili
Keyword(s):  
Cancer Cells ◽  
Vesicular Stomatitis Virus ◽  
Reverse Genetics ◽  
Cell Transformation ◽  
Rna Virus ◽  
Type I ◽  
Delivery Methods ◽  
Recombinant Viruses ◽  
Antiviral Responses ◽  
Vesicular Stomatitis

Oncolytic virus (OV) therapy is an emerging anti-cancer approach that utilizes viruses to preferentially infect and kill cancer cells, while not harming healthy cells. Vesicular stomatitis virus (VSV) is a prototypic non-segmented, negative-strand RNA virus with inherent OV qualities. Antiviral responses induced by type I interferon pathways are believed to be impaired in most cancer cells, making them more susceptible to VSV than normal cells. Several other factors make VSV a promising OV candidate for clinical use, including its well-studied biology, a small, easily manipulated genome, relative independence of a receptor or cell cycle, cytoplasmic replication without risk of host-cell transformation, and lack of pre-existing immunity in humans. Moreover, various VSV-based recombinant viruses have been engineered via reverse genetics to improve oncoselectivity, safety, oncotoxicity and stimulation of tumour-specific immunity. Alternative delivery methods are also being studied to minimize premature immune clearance of VSV. OV treatment as a monotherapy is being explored, although many studies have employed VSV in combination with radiotherapy, chemotherapy or other OVs. Preclinical studies with various cancers have demonstrated that VSV is a promising OV; as a result, a human clinical trial using VSV is currently in progress.

scholarly journals Fibrinogen Gamma Chain Promotes Aggregation of Vesicular Stomatitis Virus in Saliva

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 282 ◽  
Author(s):  
Valesca Anschau ◽  
Rafael Sanjuán
Keyword(s):  
Quantitative Analysis ◽  
Vesicular Stomatitis Virus ◽  
Rna Virus ◽  
Comparative Proteomics ◽  
Dependent Manner ◽  
Gamma Chain ◽  
Vesicular Stomatitis ◽  
Molecular Components ◽  
Dose Dependent ◽  
Infectious Unit

The spread of viruses among cells and hosts often involves multi-virion structures. For instance, virions can form aggregates that allow for the co-delivery of multiple genome copies to the same cell from a single infectious unit. Previously, we showed that vesicular stomatitis virus (VSV), an enveloped, negative-strand RNA virus, undergoes strong aggregation in the presence of saliva from certain individuals. However, the molecular components responsible for such aggregation remain unknown. Here we show that saliva-driven aggregation is protein dependent, and we use comparative proteomics to analyze the protein content of strongly versus poorly aggregating saliva. Quantitative analysis of over 300 proteins led to the identification of 18 upregulated proteins in strongly aggregating saliva. One of these proteins, the fibrinogen gamma chain, was verified experimentally as a factor promoting VSV aggregation in a dose-dependent manner. This study hence identifies a protein responsible for saliva-driven VSV aggregation. Yet, the possible involvement of additional proteins or factors cannot be discarded.

scholarly journals Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) membrane (M) protein inhibits type I and III interferon production by targeting RIG-I/MDA-5 signaling

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Yi Zheng ◽  
Meng-Wei Zhuang ◽  
Lulu Han ◽  
Jing Zhang ◽  
Mei-Ling Nan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Nuclear Translocation ◽  
Ectopic Expression ◽  
Antiviral Immunity ◽  
Typical Feature ◽  
M Protein ◽  
Poly I:C ◽  
Type I ◽  
Vesicular Stomatitis ◽  
Insight Into

AbstractCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has quickly spread worldwide and has affected more than 10 million individuals. A typical feature of COVID-19 is the suppression of type I and III interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism by which SARS-CoV-2 evades antiviral immunity remains elusive. Here, we reported that the SARS-CoV-2 membrane (M) protein inhibits the production of type I and III IFNs induced by the cytosolic dsRNA-sensing pathway mediated by RIG-I/MDA-5–MAVS signaling. In addition, the SARS-CoV-2 M protein suppresses type I and III IFN induction stimulated by SeV infection or poly (I:C) transfection. Mechanistically, the SARS-CoV-2 M protein interacts with RIG-I, MAVS, and TBK1, thus preventing the formation of the multiprotein complex containing RIG-I, MAVS, TRAF3, and TBK1 and subsequently impeding the phosphorylation, nuclear translocation, and activation of IRF3. Consequently, ectopic expression of the SARS-CoV-2 M protein facilitates the replication of vesicular stomatitis virus. Taken together, these results indicate that the SARS-CoV-2 M protein antagonizes type I and III IFN production by targeting RIG-I/MDA-5 signaling, which subsequently attenuates antiviral immunity and enhances viral replication. This study provides insight into the interpretation of SARS-CoV-2-induced antiviral immune suppression and illuminates the pathogenic mechanism of COVID-19.

scholarly journals SARS-CoV-2 nsp12 attenuates type I interferon production by inhibiting IRF3 nuclear translocation

2021 ◽  
Author(s):  
Wenjing Wang ◽  
Zhuo Zhou ◽  
Xia Xiao ◽  
Zhongqin Tian ◽  
Xiaojing Dong ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Type I Interferon ◽  
Sendai Virus ◽  
Polymerase Activity ◽  
Poly I:C ◽  
Type I ◽  
Dependent Manner ◽  
Promoter Activation ◽  
Global Pandemic ◽  
Antiviral Innate Immunity

AbstractSARS-CoV-2 is the pathogenic agent of COVID-19, which has evolved into a global pandemic. Compared with some other respiratory RNA viruses, SARS-CoV-2 is a poor inducer of type I interferon (IFN). Here, we report that SARS-CoV-2 nsp12, the viral RNA-dependent RNA polymerase (RdRp), suppresses host antiviral responses. SARS-CoV-2 nsp12 attenuated Sendai virus (SeV)- or poly(I:C)-induced IFN-β promoter activation in a dose-dependent manner. It also inhibited IFN promoter activation triggered by RIG-I, MDA5, MAVS, and IRF3 overexpression. Nsp12 did not impair IRF3 phosphorylation but suppressed the nuclear translocation of IRF3. Mutational analyses suggested that this suppression was not dependent on the polymerase activity of nsp12. Given these findings, our study reveals that SARS-CoV-2 RdRp can antagonize host antiviral innate immunity and thus provides insights into viral pathogenesis.

scholarly journals Feline Infectious Peritonitis Virus Nsp5 Inhibits Type I Interferon Production by Cleaving NEMO at Multiple Sites

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 43 ◽  
Author(s):  
Si Chen ◽  
Jin Tian ◽  
Zhijie Li ◽  
Hongtao Kang ◽  
Jikai Zhang ◽  
...  
Keyword(s):  
Type I Interferon ◽  
Sendai Virus ◽  
Nuclear Factor Κb ◽  
Poly I:C ◽  
Type I ◽  
Dependent Manner ◽  
Type I Ifn ◽  
Feline Infectious Peritonitis Virus ◽  
Feline Infectious Peritonitis ◽  
Polycytidylic Acid

Feline infectious peritonitis (FIP), caused by virulent feline coronavirus, is the leading infectious cause of death in cats. The type I interferon (type I IFN)-mediated immune responses provide host protection from infectious diseases. Several coronaviruses have been reported to evolve diverse strategies to evade host IFN response. However, whether feline infectious peritonitis virus (FIPV) antagonizes the type I IFN signaling remains unclear. In this study, we demonstrated that FIPV strain DF2 infection not only failed to induce interferon-β (IFN-β) and interferon-stimulated gene (ISG) production, but also inhibited Sendai virus (SEV) or polyinosinic-polycytidylic acid (poly(I:C))-induced IFN-β production. Subsequently, we found that one of the non-structural proteins encoded by the FIPV genome, nsp5, interrupted type I IFN signaling in a protease-dependent manner by cleaving the nuclear factor κB (NF-κB) essential modulator (NEMO) at three sites—glutamine132 (Q132), Q205, and Q231. Further investigation revealed that the cleavage products of NEMO lost the ability to activate the IFN-β promoter. Mechanistically, the nsp5-mediated NEMO cleavage disrupted the recruitment of the TRAF family member-associated NF-κB activator (TANK) to NEMO, which reduced the phosphorylation of interferon regulatory factor 3 (IRF3), leading to the inhibition of type I IFN production. Our research provides new insights into the mechanism for FIPV to counteract host innate immune response.

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