scholarly journals The Cellular RNA Helicase DDX1 Interacts with Coronavirus Nonstructural Protein 14 and Enhances Viral Replication

2010 ◽  
Vol 84 (17) ◽  
pp. 8571-8583 ◽  
Author(s):  
Linghui Xu ◽  
Siti Khadijah ◽  
Shouguo Fang ◽  
Li Wang ◽  
Felicia P. L. Tay ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Rna Helicase ◽  
Host Proteins ◽  
Bait Protein ◽  
Infectious Bronchitis ◽  
Infected Cells ◽  
Interfering Rna ◽  
Discontinuous Transcription ◽  
Two Hybrid ◽  
Hybrid Screening

ABSTRACT The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.

scholarly journals Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex

2002 ◽  
Vol 76 (24) ◽  
pp. 12676-12682 ◽  
Author(s):  
Toru Kubota ◽  
Noriko Yokosawa ◽  
Shin-ichi Yokota ◽  
Nobuhiro Fujii
Keyword(s):  
Signal Transduction Pathway ◽  
Adaptor Protein ◽  
Mumps Virus ◽  
Virus Protein ◽  
V Protein ◽  
Long Form ◽  
Infected Cells ◽  
Intracellular Target ◽  
Two Hybrid ◽  
Hybrid Screening

ABSTRACT It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the β subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione S-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor.

scholarly journals Interferon Regulatory Factor 3 Is a Cellular Partner of Rotavirus NSP1

2002 ◽  
Vol 76 (18) ◽  
pp. 9545-9550 ◽  
Author(s):  
Joel W. Graff ◽  
Dana N. Mitzel ◽  
Carla M. Weisend ◽  
Michelle L. Flenniken ◽  
Michele E. Hardy
Keyword(s):  
Nonstructural Protein ◽  
Interferon Regulatory Factor ◽  
Interferon Response ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
C Terminus ◽  
Cellular Partner ◽  
Infected Cells ◽  
Two Hybrid ◽  
Interaction Trap

ABSTRACT The rotavirus nonstructural protein NSP1 is the least conserved protein in the rotavirus genome, and its function in the replication cycle is not known. We employed NSP1 as bait in the yeast two-hybrid interaction trap to identify candidate cellular partners of NSP1 that may provide clues to its function. Interferon regulatory factor 3 (IRF-3) was identified as an NSP1 interactor. NSP1 synthesized in rotavirus-infected cells bound IRF-3 in a glutathione S-transferase pull-down assay, indicating that the interaction was not unique to the two-hybrid system. NSP1 of murine rotavirus strain EW also interacted with IRF-3. NSP1 deletion and point mutants were constructed to map domains important in the interaction between NSP1 and IRF-3. The data suggest that a binding domain resides in the C terminus of NSP1 and that the N-terminal conserved zinc finger is important but not sufficient to mediate binding to IRF-3. We predict that a role for NSP1 in rotavirus-infected cells is to inhibit activation of IRF-3 and diminish the cellular interferon response.

scholarly journals Role for the Src Family Kinase Fyn in Sphingolipid Acquisition by Chlamydiae

2011 ◽  
Vol 79 (11) ◽  
pp. 4559-4568 ◽  
Author(s):  
Jeffrey Mital ◽  
Ted Hackstadt
Keyword(s):  
Host Proteins ◽  
Content Type ◽  
Obligate Intracellular ◽  
Screening Protocol ◽  
Infected Cells ◽  
Protective Environment ◽  
Membrane Bound ◽  
Obligate Intracellular Pathogen ◽  
Interfering Rna ◽  
Chlamydial Inclusion

ABSTRACTThe bacterial obligate intracellular pathogenChlamydia trachomatisreplicates within a membrane-bound vacuole termed the inclusion. From within this protective environment, chlamydiae usurp numerous functions of the host cell to promote chlamydial survival and replication. Here we utilized a small interfering RNA (siRNA)-based screening protocol designed to identify host proteins involved in the trafficking of sphingomyelin to the chlamydial inclusion. Twenty-six host proteins whose deficiency significantly decreased sphingomyelin trafficking to the inclusion and 16 proteins whose deficiency significantly increased sphingomyelin trafficking to the inclusion were identified. The reduced sphingomyelin trafficking caused by downregulation of the Src family tyrosine kinase Fyn was confirmed in more-detailed analyses. Fyn silencing did not alter sphingomyelin synthesis or trafficking in the absence of chlamydial infection but reduced the amount of sphingomyelin trafficked to the inclusion in infected cells, as determined by two independent quantitative assays. Additionally, inhibition of Src family kinases resulted in increased cellular retention of sphingomyelin and significantly decreased incorporation into elementary bodies of bothC. trachomatisandChlamydophila caviae.

scholarly journals A two-hybrid screening to isolate proteins that interact with the C. elegans germline DEAD box RNA Helicase VBH-1

2007 ◽  
Vol 306 (1) ◽  
pp. 382-383
Author(s):  
Ayde Mendoza-Oliva ◽  
Valeria Hansberg ◽  
Rosa E. Navarro
Keyword(s):  
Rna Helicase ◽  
C Elegans ◽  
Dead Box ◽  
Two Hybrid ◽  
Hybrid Screening

scholarly journals Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast two-hybrid system

2001 ◽  
Vol 82 (3) ◽  
pp. 677-686 ◽  
Author(s):  
Lissett López ◽  
Ana Urzainqui ◽  
Elvira Domínguez ◽  
Juan Antonio García
Keyword(s):  
Hybrid System ◽  
Rna Helicase ◽  
Full Length ◽  
Genome Replication ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Cylindrical Inclusions ◽  
Infected Cells ◽  
Two Hybrid

Potyvirus CI RNA helicase is a protein involved in RNA genome replication and virus movement. The protein aggregates in the cytoplasm of infected cells to form typical cylindrical inclusions. A yeast two-hybrid system was used to analyse interactions of the CI RNA helicase from plum pox potyvirus (PPV) with itself and with other viral proteins. No interactions could be detected of full-length CI protein with itself or with PPV P3/6K1, NIa, NIb or CP proteins. However, positive self-interactions were detected for N-terminal fragments of the CI protein, allowing the mapping of a CI–CI binding domain to the N-terminal 177 aa of the protein. Further deletion analysis suggested that several regions of this domain contribute to the interaction. Moreover, pull-down experiments demonstrate that, at least in vitro, full-length PPV CI protein is able to self-interact in the absence of other virus or plant factors.

scholarly journals Interactions of Rotavirus VP4 Spike Protein with the Endosomal Protein Rab5 and the Prenylated Rab Acceptor PRA1

2003 ◽  
Vol 77 (12) ◽  
pp. 7041-7047 ◽  
Author(s):  
Vincent Enouf ◽  
Serge Chwetzoff ◽  
Germain Trugnan ◽  
Jean Cohen
Keyword(s):  
Amino Acids ◽  
Cell Attachment ◽  
Small Gtpase ◽  
Spike Protein ◽  
Deletion Mutants ◽  
Vesicular Traffic ◽  
Early Endosomes ◽  
Infected Cells ◽  
Two Hybrid ◽  
Hybrid Screening

ABSTRACT Rotavirus spike protein VP4 is implicated in several important functions, such as cell attachment, penetration, hemagglutination, neutralization, virulence, and host range. It is present at the plasma membrane and colocalizes with the cytoskeleton in infected cells. We looked for cellular partners responsible for the localization of VP4 by two-hybrid screening of a monkey CV1 cell cDNA library. In the screen we isolated repeatedly three cDNAs encoding either two isoforms (a and c) of Rab5 protein or the prenylated Rab acceptor (PRA1). The small GTPase Rab5 is a molecule regulating the vesicular traffic and the motility of early endosomes along microtubules. Rab5 interacts with a large number of effectors, in particular with PRA1. Interactions of VP4 with both partners, Rab5 and PRA1, were confirmed by coimmunoprecipitation from infected- or transfected-cell lysates. Interaction of Rab5 and PRA1 was restricted to free VP4, since neither triple-layered particles nor NSP4-VP4-VP7 heterotrimeric complexes could be coprecipitated. Site-directed and deletion mutants of VP4 were used to map a VP4 domain(s) interacting with Rab5 or PRA1. Of the 10 mutants tested, 2 interacted exclusively with a single partner. In contrast, the domain extending from amino acids 560 to 722 of VP4 is essential for both interactions. These results suggest that Rab5 and PRA1 may be involved in the localization and trafficking of VP4 in infected cells.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

scholarly journals Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication

2015 ◽  
Vol 89 (13) ◽  
pp. 6746-6760 ◽  
Author(s):  
Nenavath Gopal Naik ◽  
Huey-Nan Wu
Keyword(s):  
Life Cycle ◽  
Recombinant Protein ◽  
Dengue Virus ◽  
Protein Expression ◽  
Nonstructural Protein ◽  
Recombinant Protein Expression ◽  
Rna Replication ◽  
Viral Production ◽  
Glycosylation Sites ◽  
Infected Cells

ABSTRACTDengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, andtrans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites.In vivoprotein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.IMPORTANCEThis is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

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