Functional motifs responsible for human metapneumovirus M2-2-mediated innate immune evasion

Chen, Yu; Deng, Xiaoling; Deng, Jieqiong; Zhou, Jiehua; Ren, Yuping; Liu, Shengxuan; Prusak, Deborah; Wood, Thomas G.; Bao, Xiaoyong

doi:10.1016/j.virol.2016.09.026

Cited by 17 publications

(34 citation statements)

References 52 publications

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“…Also, not obtained were positive results supporting the previous finding reported by Ren et al and Chen et al regarding inhibition of MAVS (IPS-1)-dependent gene transcription by M2-2 ( Fig. 6 and 7B) (35,36). The reasons for these conflicting results are unclear.…”

Section: Discussionmentioning

confidence: 51%

Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

Kitagawa

Sakai

Funayama

et al. 2017

J Virol

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Human metapneumovirus (HMPV) has the ability to inhibit Toll-like receptor 7 (TLR7)- and TLR9-dependent alpha interferon (IFN-α) production by plasmacytoid dendritic cells (pDCs). However, the inhibition mechanism remains largely unknown. To identify viral proteins responsible for this inhibition, we performed a screening of HMPV open reading frames (ORFs) for the ability to block TLR7/9-dependent signaling reconstituted in HEK293T cells by transfection with myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), IKKα, and IFN regulatory factor 7 (IRF7). This screening demonstrated that the M2-2 protein was the most potent inhibitor of TLR7/9-dependent IFN-α induction. A recombinant HMPV in which the M2-2 ORF was silenced indeed induced greater IFN-α production by human pDCs than wild-type HMPV did. Immunoprecipitation experiments showed direct physical association of the M2-2 protein with the inhibitory domain (ID) of IRF7. As a natural consequence of this, transfection of IRF7 lacking the ID, a constitutively active mutant, resulted in activation of the IFN-α promoter even in the presence of M2-2. Bioluminescence resonance energy transfer assays and split luciferase complementation assays revealed that M2-2 inhibited MyD88/TRAF6/IKKα-induced homodimerization of IRF7. In contrast, expression of the M2-2 protein did not result in inhibition of IPS-1-induced homodimerization and resultant activation of IRF7. This indicates that inhibition of MyD88/TRAF6/IKKα-induced IRF7 homodimerization does not result from a steric effect of M2-2 binding. Instead, it was found that M2-2 inhibited MyD88/TRAF6/IKKα-induced phosphorylation of IRF7 on Ser477. These results suggest that M2-2 blocks TLR7/9-dependent IFN-α induction by preventing IRF7 homodimerization, possibly through its effects on the phosphorylation status of IRF7. The family is divided into two subfamilies, the and the Members of the subfamily have the ability to inhibit TLR7/9-dependent IFN-α production, and the underlying inhibition mechanism has been intensively studied. In contrast, little is known about how members of the subfamily regulate IFN-α production by pDCs. We identified the M2-2 protein of HMPV, a member of the subfamily, as a negative regulator of IFN-α production by pDCs and uncovered the underlying mechanism. This study explains in part why the M2-2 knockout recombinant HMPV is attenuated and further suggests that M2-2 is a potential target for HMPV therapy.

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Section: Discussionmentioning

confidence: 51%

Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

Kitagawa

Sakai

Funayama

et al. 2017

J Virol

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“…Reporter gene assays. MEFs cells were transfected in triplicate with luciferase reporter gene plasmids containing multiple copies of NF-B binding sites (NF-B-Luc) or the IRF-3 binding site (PRDIII-I-Luc and IRF-3-Luc) using FuGene 6 (Roche, Indianapolis, IN), as previously described (60,61). At 30 h posttransfection, cells were infected with RSV for 15 h and lysed to measure luciferase reporter activity.…”

Section: Cell Lines Virus and Antibodiesmentioning

confidence: 99%

Exchange Proteins Directly Activated by cAMP and Their Roles in Respiratory Syncytial Virus Infection

Choi

Ren

Chen

et al. 2018

J Virol

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Respiratory syncytial virus (RSV) is the leading cause of respiratory infection in young children and high-risk adults. However, a specific treatment for this viral infection is not currently available. In this study, we discovered that an exchange protein directly activated by cyclic AMP (EPAC) can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, treatment with EPAC inhibitor (ESI-09), but not protein kinase A inhibitor (H89), significantly inhibits RSV replication and proinflammatory cytokine/chemokine induction. In addition, RSV-activated transcriptional factors belonging to the NF-κB and IRF families are also suppressed by ESI-09. Through isoform-specific gene knockdown, we found that EPAC2, but not EPAC1, plays a dominant role in controlling RSV replication and virus-induced host responses. Experiments using both EPAC2 knockout and EPAC2-specific inhibitor support such roles of EPAC2. Therefore, EPAC2 is a promising therapeutic target to regulate RSV replication and associated inflammation. RSV is a serious public health problem, as it is associated with bronchiolitis, pneumonia, and asthma exacerbations. Currently no effective treatment or vaccine is available, and many molecular mechanisms regarding RSV-induced lung disease are still significantly unknown. This project aims to elucidate an important and novel function of a protein, called EPAC2, in RSV replication and innate inflammatory responses. Our results should provide an important insight into the development of new pharmacologic strategies against RSV infection, thereby reducing RSV-associated morbidity and mortality.

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“…Recently, a wild-type recombinant hMPV was approved as a suitable parent virus for the development of live attenuated hMPV vaccine candidates in experimental human infection trials [ 8 ], providing a promising vaccine study direction for hMPV. We and others have developed several attenuated recombinant strains of hMPV by gene deletion or mutations [ 9 , 10 , 11 , 12 , 13 ]. Compared to other vaccines, live attenuated vaccines offer several advantages for the immunization of infants and young children, including no vaccine-associated enhanced viral disease, the induction of both humoral and mucosal immunity, intranasal vaccine delivery, and viral replication in the upper respiratory tract of young infants despite the presence of passively acquired maternally-derived respiratory syncytial virus (RSV) neutralizing antibodies [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

et al. 2017

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Human metapneumovirus (hMPV) is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s) in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF) and mitochondrial antiviral-signaling (MAVS) proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s). Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s). This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

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Functional motifs responsible for human metapneumovirus M2-2-mediated innate immune evasion

Cited by 17 publications

References 52 publications

Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

Exchange Proteins Directly Activated by cAMP and Their Roles in Respiratory Syncytial Virus Infection

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

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