Structural Insight into the Interaction of Sendai Virus C Protein with Alix To Stimulate Viral Budding

2021 ◽  
Author(s):  
Kosuke Oda ◽  
Yasuyuki Matoba ◽  
Masanori Sugiyama ◽  
Takemasa Sakaguchi
Keyword(s):  
Crystal Structure ◽  
Binding Affinity ◽  
Sendai Virus ◽  
Antiviral Drugs ◽  
Accessory Protein ◽  
Type I ◽  
C Protein ◽  
Viral Budding ◽  
C Proteins

The Sendai virus (SeV), belonging to the Respirovirus genus of the family Paramyxoviridae , harbors an accessory protein, named C protein, which facilitates the viral pathogenicity in mice. In addition, the C protein is known to stimulate the budding of virus-like particles through the binding to the host ALG-2 interacting protein X (Alix), a component of the endosomal sorting complexes required for transport (ESCRT) machinery. However, siRNA-mediated gene knockdown studies suggested that neither Alix nor C protein are related to the SeV budding. In the present study, we determined the crystal structure of a complex comprising of the C -terminal half of the C protein (Y3) and the Bro1 domain of Alix at a resolution of 2.2 Å, to investigate the role of the association in the SeV budding. The structure revealed that a novel consensus sequence, LxxW, which is conserved among the Respirovirus C proteins, is important for the Alix-binding. SeV possessing a mutated C protein with a reduced Alix-binding affinity showed impaired virus production, which correlated with the binding affinity. Infectivity analysis showed a 160-fold reduction at 12 h post-infection compared with non-mutated virus, while C protein competes with CHMP4, one subunit of the ESCRT-III complex, on the binding to Alix. Altogether, these results highlight the critical role of C protein in the SeV budding. IMPORTANCE Human parainfluenza virus type I (hPIV1) is a respiratory pathogen affecting in young children, immunocompromised patients, and the elderly, with no available vaccines or antiviral drugs. Sendai virus (SeV), a murine counterpart of hPIV1, has been extensively studied to determine the molecular and biological properties of hPIV1. These viruses possess a multifunctional accessory protein, C protein, which is essential for stimulating the viral reproduction, however, its role in budding remains controversial. In the present study, the crystal structure of the C -terminal half of the SeV C protein associated with the Bro1 domain of Alix, a component of a cell membrane modulating machinery ESCRT, was elucidated. Based on the structure, we designed mutated C proteins with different binding affinity to Alix, and showed that the interaction between C and Alix is vital for the viral budding. These findings provide new insights into the development of a new antiviral drugs against hPIV1.

scholarly journals Insights into substrate binding and catalysis in bacterial type I dehydroquinase

2014 ◽  
Vol 462 (3) ◽  
pp. 415-424 ◽  
Author(s):  
María Maneiro ◽  
Antonio Peón ◽  
Emilio Lence ◽  
José M. Otero ◽  
Mark J. Van Raaij ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Binding ◽  
Detailed Knowledge ◽  
Type I ◽  
Conserved Residues ◽  
Binding Process ◽  
Bacterial Type

The crystal structure of S. typhi type I dehydroquinase in complex with (2R)-3-methyl-3-dehydroquinic acid is described. A previously unknown key role of several conserved residues and a detailed knowledge of the substrate binding process is detailed.

scholarly journals Swine Acute Diarrhea Syndrome Coronavirus Nucleocapsid Protein Antagonizes Interferon-β Production via Blocking the Interaction Between TRAF3 and TBK1

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhihai Zhou ◽  
Yuan Sun ◽  
Jingya Xu ◽  
Xiaoyu Tang ◽  
Ling Zhou ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Acute Diarrhea ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Type I ◽  
Innate Immune Responses ◽  
N Protein ◽  
Antiviral Innate Immunity ◽  
Diarrhea Syndrome

Swine acute diarrhea syndrome coronavirus (SADS-CoV), first discovered in 2017, is a porcine enteric coronavirus that can cause acute diarrhea syndrome (SADS) in piglets. Here, we studied the role of SADS-CoV nucleocapsid (N) protein in innate immunity. Our results showed that SADS-CoV N protein could inhibit type I interferon (IFN) production mediated by Sendai virus (Sev) and could block the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3). Simultaneously, the IFN-β promoter activity mediated by TANK binding kinase 1 (TBK1) or its upstream molecules in the RLRs signal pathway was inhibited by SADS-CoV N protein. Further investigations revealed that SADS-CoV N protein could counteract interaction between TNF receptor-associated factor 3 (TRAF3) and TBK1, which led to reduced TBK1 activation and IFN-β production. Our study is the first report of the interaction between SADS-CoV N protein and the host antiviral innate immune responses, and the mechanism utilized by SADS-CoV N protein provides a new insight of coronaviruses evading host antiviral innate immunity.

scholarly journals Structural Basis of the Inhibition of STAT1 Activity by Sendai Virus C Protein

2015 ◽  
Vol 89 (22) ◽  
pp. 11487-11499 ◽  
Author(s):  
Kosuke Oda ◽  
Yasuyuki Matoba ◽  
Takashi Irie ◽  
Ryoko Kawabata ◽  
Masaya Fukushi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Signal Transduction ◽  
Molecular Weight ◽  
Innate Immunity ◽  
Sendai Virus ◽  
Signal Transduction Pathways ◽  
C Protein ◽  
Parallel Form ◽  
Ifn Γ ◽  
Alpha Beta

ABSTRACTSendai virus (SeV) C protein inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/β) and IFN-γ by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3 [amino acids 99 to 204]) at a resolution of 2.0 Å. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an antiparallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anticooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr701by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated STAT1 (pY-STAT1) with Y3 associated with the γ-activated sequence, probably as high-molecular-weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN-γ by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-α/β.IMPORTANCESendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/β) and IFN-γ by binding to the transcription factor STAT1. In virus-infected cells, phosphorylation of STAT1 at the Tyr701residue is potently enhanced, although transcription by STAT1 is inert. Here, we determined the crystal structure of the N-terminal domain of STAT1 associated with the C-terminal half of the C protein. Molecular modeling and experiments suggested that the two C proteins bind to and stabilize the parallel form of the STAT1 dimer, which are likely to be phosphorylated at Tyr701, further inducing high-molecular-weight complex formation and inhibition of transcription by IFN-γ. We also discuss the possible mechanism of inhibition of the IFN-α/β pathways by the C protein. This is the first structural report of the C protein, suggesting a mechanism of evasion of the paramyxovirus from innate immunity.

scholarly journals Sendai Virus C Proteins Counteract the Interferon-Mediated Induction of an Antiviral State

1999 ◽  
Vol 73 (8) ◽  
pp. 6559-6565 ◽  
Author(s):  
Dominique Garcin ◽  
Patrizia Latorre ◽  
Daniel Kolakofsky
Keyword(s):  
Sendai Virus ◽  
Antiviral Response ◽  
Single Amino Acid ◽  
Wild Type ◽  
C Protein ◽  
Antiviral State ◽  
Protein Levels ◽  
Vesicular Stomatitis ◽  
C Proteins ◽  
Virus C

ABSTRACT We have studied the relationship between the Sendai virus (SeV) C proteins (a nested set of four proteins initiated at different start codons) and the interferon (IFN)-mediated antiviral response in IFN-competent cells in culture. SeV strains containing wild-type or various mutant C proteins were examined for their ability (i) to induce an antiviral state (i.e., to prevent the growth of vesicular stomatitis virus [VSV] following a period of SeV infection), (ii) to induce the elevation of Stat1 protein levels, and (iii) to prevent IFN added concomitant with the SeV infection from inducing an antiviral state. We find that expression of the wild-type C gene and, specifically, the AUG114-initiated C protein prevents the establishment of an antiviral state: i.e., cells infected with wild-type SeV exhibited little or no increase in Stat1 levels and were permissive for VSV replication, even in the presence of exogenous IFN. In contrast, in cells infected with SeV lacking the AUG114-initiated C protein or containing a single amino acid substitution in the C protein, the level of Stat1 increased and VSV replication was inhibited. The prevention of the cellular IFN-mediated antiviral response appears to be a key determinant of SeV pathogenicity.

Type I Procollagen C-Propeptide Defects: Study of Genotype-Phenotype Correlation and Predictive Role of Crystal Structure

2014 ◽  
pp. n/a-n/a ◽  
Author(s):  
Sofie Symoens ◽  
David J.S. Hulmes ◽  
Jean-Marie Bourhis ◽  
Paul J. Coucke ◽  
Anne De Paepe ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Type I ◽  
Phenotype Correlation ◽  
Type I Procollagen ◽  
Genotype Phenotype Correlation ◽  
Predictive Role

scholarly journals The Amino-Terminal Half of Sendai Virus C Protein Is Not Responsible for either Counteracting the Antiviral Action of Interferons or Down-Regulating Viral RNA Synthesis

2002 ◽  
Vol 76 (14) ◽  
pp. 7114-7124 ◽  
Author(s):  
Atsushi Kato ◽  
Yukano Ohnishi ◽  
Michiko Hishiyama ◽  
Masayoshi Kohase ◽  
Sakura Saito ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Sendai Virus ◽  
Virus Assembly ◽  
Viral Rna ◽  
Antiviral Action ◽  
C Protein ◽  
Amino Terminal ◽  
Inhibitory Activities ◽  
C Proteins ◽  
Virus C

ABSTRACT The Sendai virus C proteins, C′, C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.

scholarly journals Activation of the Beta Interferon Promoter by Unnatural Sendai Virus Infection Requires RIG-I and Is Inhibited by Viral C Proteins

2007 ◽  
Vol 81 (22) ◽  
pp. 12227-12237 ◽  
Author(s):  
Laura Strähle ◽  
Jean-Baptiste Marq ◽  
Albert Brini ◽  
Stéphane Hausmann ◽  
Daniel Kolakofsky ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Sendai Virus ◽  
Viral Gene ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
C Protein ◽  
Double Stranded Rna ◽  
Beta Interferon ◽  
C Proteins ◽  
Green Fluorescent

ABSTRACT As infection with wild-type (wt) Sendai virus (SeV) normally activates beta interferon (IFN-β) very poorly, two unnatural SeV infections were used to study virus-induced IFN-β activation in mouse embryonic fibroblasts: (i) SeV-DI-H4, which is composed mostly of small, copyback defective interfering (DI) genomes and whose infection overproduces short 5′-triphosphorylated trailer RNAs (pppRNAs) and underproduces viral V and C proteins, and (ii) SeV-GFP(+/−), a coinfection that produces wt amounts of viral gene products but that also produces both green fluorescent protein (GFP) mRNA and its complement, which can form double-stranded RNA (dsRNA) with capped 5′ ends. We found that (i) virus-induced signaling to IFN-β depended predominantly on RIG-I (as opposed to mda-5) for both SeV infections, i.e., that RIG-I senses both pppRNAs and dsRNA without 5′-triphosphorylated ends, and (ii) it is the viral C protein (as opposed to V) that is primarily responsible for countering RIG-I-dependent signaling to IFN-β. Nondefective SeV that cannot specifically express C proteins not only cannot prevent the effects of transfected poly(I-C) or pppRNAs on IFN-β activation but also synergistically enhances these effects. SeV-Vminus infection, in contrast, behaves mostly like wt SeV and counteracts the effects of transfected poly(I-C) or pppRNAs.

scholarly journals The YLDL Sequence within Sendai Virus M Protein Is Critical for Budding of Virus-Like Particles and Interacts with Alix/AIP1 Independently of C Protein

2006 ◽  
Vol 81 (5) ◽  
pp. 2263-2273 ◽  
Author(s):  
Takashi Irie ◽  
Yukie Shimazu ◽  
Tetsuya Yoshida ◽  
Takemasa Sakaguchi
Keyword(s):  
Matrix Protein ◽  
Sendai Virus ◽  
Specific Interaction ◽  
M Protein ◽  
Small Interfering Rnas ◽  
C Protein ◽  
Enveloped Viruses ◽  
Virus Like Particles ◽  
Viral Budding ◽  
N Terminus

ABSTRACT For many enveloped viruses, cellular multivesicular body (MVB) sorting machinery has been reported to be utilized for efficient viral budding. Matrix and Gag proteins have been shown to contain one or two L-domain motifs (PPxY, PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular proteins involved in MVB sorting, which are recruited to the viral budding site. However, for many enveloped viruses, L-domain motifs have not yet been identified and the involvement of MVB sorting machinery in viral budding is still unknown. Here we show that both Sendai virus (SeV) matrix protein M and accessory protein C contribute to virus budding by physically interacting with Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and its specific interaction with the N terminus of Alix/AIP1(1-211) was important for the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP budding was inhibited by the overexpression of some deletion mutant forms of Alix/AIP1 and depletion of endogenous Alix/AIP1 with specific small interfering RNAs. The YLDL sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, and even YPxL. C protein was also able to physically interact with the N terminus of Alix/AIP1(212-357) and enhanced M-VLP budding independently of M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may enhance the efficiency of the utilization of cellular MVB sorting machinery for efficient SeV budding.

scholarly journals DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to co-ordinate assembly of signalling complexes downstream from MAVS

2017 ◽  
Vol 474 (4) ◽  
pp. 571-587 ◽  
Author(s):  
Lili Gu ◽  
Anthony Fullam ◽  
Niamh McCormack ◽  
Yvette Höhn ◽  
Martina Schröder
Keyword(s):  
Sendai Virus ◽  
Direct Interaction ◽  
Type I ◽  
Tnf Receptor ◽  
Signalling Molecules ◽  
Adaptor Molecule ◽  
Dead Box ◽  
Iκb Kinase ◽  
Second Wave

The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon (IFN) induction downstream from antiviral pattern recognition receptors. It binds to TANK-binding kinase 1 and IκB-kinase-ε (IKKε), the two key kinases mediating activation of IFN regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKKε activation downstream from RIG-I and then links the activated kinase to IRF3. In the present study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TNF receptor-associated factor 3 (TRAF3) mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai virus (SeV) infection, both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the mitochondrial antiviral signalling (MAVS) adaptor, DDX3, IκB-kinase-ε (IKKε), TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting that it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented the formation of TRAF3–MAVS and TRAF3–IKKε complexes. Based on our data, we propose that early TRAF3 ubiquitination is required for the formation of a stable MAVS–TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that co-ordinates assembly of different TRAF3, IKKε and IRF3-containing signalling complexes downstream from MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling.

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