scholarly journals Rubella Virus Capsid Protein Interacts with Poly(A)-Binding Protein and Inhibits Translation

2008 ◽  
Vol 82 (9) ◽  
pp. 4284-4294 ◽  
Author(s):  
Carolina S. Ilkow ◽  
Valeria Mancinelli ◽  
Martin D. Beatch ◽  
Tom C. Hobman
Keyword(s):  
Capsid Protein ◽  
Rubella Virus ◽  
Virus Assembly ◽  
Binding Protein ◽  
Protein Translation ◽  
Capsid Proteins ◽  
In Vitro Translation ◽  
Cell Protein ◽  
Translational Efficiency ◽  
Dependent Manner

ABSTRACT During virus assembly, the capsid proteins of RNA viruses bind to genomic RNA to form nucleocapsids. However, it is now evident that capsid proteins have additional functions that are unrelated to nucleocapsid formation. Specifically, their interactions with cellular proteins may influence signaling pathways or other events that affect virus replication. Here we report that the rubella virus (RV) capsid protein binds to poly(A)-binding protein (PABP), a host cell protein that enhances translational efficiency by circularizing mRNAs. Infection of cells with RV resulted in marked increases in the levels of PABP, much of which colocalized with capsid in the cytoplasm. Mapping studies revealed that capsid binds to the C-terminal half of PABP, which interestingly is the region that interacts with other translation regulators, including PABP-interacting protein 1 (Paip1) and Paip2. The addition of capsid to in vitro translation reaction mixtures inhibited protein synthesis in a dose-dependent manner; however, the capsid block was alleviated by excess PABP, indicating that inhibition of translation occurs through a stoichiometric mechanism. To our knowledge, this is the first report of a viral protein that inhibits protein translation by sequestration of PABP. We hypothesize that capsid-dependent inhibition of translation may facilitate the switch from viral translation to packaging RNA into nucleocapsids.

scholarly journals Rubella Virus Capsid Associates with Host Cell Protein p32 and Localizes to Mitochondria

2000 ◽  
Vol 74 (12) ◽  
pp. 5569-5576 ◽  
Author(s):  
Martin D. Beatch ◽  
Tom C. Hobman
Keyword(s):  
Host Cell ◽  
Capsid Protein ◽  
Rubella Virus ◽  
Rna Binding ◽  
Virus Assembly ◽  
Cellular Protein ◽  
Cell Protein ◽  
Multiple Copies

ABSTRACT Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA. The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes. The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein. Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system. One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins. The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria. Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region. The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid. Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.

Characterization and purification of lupus antigen La, and RNA-binding protein

1985 ◽  
Vol 5 (3) ◽  
pp. 586-590
Author(s):  
A M Francoeur ◽  
E K Chan ◽  
J I Garrels ◽  
M B Mathews
Keyword(s):  
Hela Cell ◽  
Gel Electrophoresis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Vitro Translation ◽  
Cell Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
La Antigen

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.

scholarly journals Rubella Virus E2 Signal Peptide Is Required for Perinuclear Localization of Capsid Protein and Virus Assembly

2001 ◽  
Vol 75 (4) ◽  
pp. 1978-1983 ◽  
Author(s):  
Lok Man J. Law ◽  
Robert Duncan ◽  
Ali Esmaili ◽  
Hira L. Nakhasi ◽  
Tom C. Hobman
Keyword(s):  
Endoplasmic Reticulum ◽  
Capsid Protein ◽  
Signal Peptide ◽  
Rubella Virus ◽  
Virus Assembly ◽  
Signal Peptidase ◽  
Structural Proteins ◽  
Carboxy Terminus ◽  
Membrane Anchor ◽  
Polyprotein Precursor

ABSTRACT The rubella virus (RV) structural proteins capsid, E2, and E1 are synthesized as a polyprotein precursor. The signal peptide that initiates translocation of E2 into the lumen of the endoplasmic reticulum remains attached to the carboxy terminus of the capsid protein after cleavage by signal peptidase. Among togaviruses, this feature is unique to RV. The E2 signal peptide has previously been shown to function as a membrane anchor for the capsid protein. In the present study, we demonstrate that this domain is required for RV glycoprotein-dependent localization of the capsid protein to the juxtanuclear region and subsequent virus assembly at the Golgi complex.

scholarly journals Heterogeneous Nuclear Ribonucleoprotein (hnRNP) E1 Binds to hnRNP A2 and Inhibits Translation of A2 Response Element mRNAs

2006 ◽  
Vol 17 (8) ◽  
pp. 3521-3533 ◽  
Author(s):  
Linda D. Kosturko ◽  
Michael J. Maggipinto ◽  
George Korza ◽  
Joo Won Lee ◽  
John H. Carson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Vitro Translation ◽  
Dependent Manner ◽  
Response Element ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Hnrnp E1

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.

scholarly journals The Abundant Nuclear Enzyme PARP Participates in the Life Cycle of Simian Virus 40 and Is Stimulated by Minor Capsid Protein VP3

2003 ◽  
Vol 77 (7) ◽  
pp. 4273-4282 ◽  
Author(s):  
Ariela Gordon-Shaag ◽  
Yael Yosef ◽  
Mahmoud Abd El-Latif ◽  
Ariella Oppenheim
Keyword(s):  
Amino Acids ◽  
Life Cycle ◽  
Capsid Protein ◽  
Membrane Integrity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Capsid Proteins ◽  
Dependent Manner ◽  
Minor Capsid Protein ◽  
Stimulation Of

ABSTRACT The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process.

scholarly journals Rubella virus capsid protein structure and its role in virus assembly and infection

2013 ◽  
Vol 110 (50) ◽  
pp. 20105-20110 ◽  
Author(s):  
V. Mangala Prasad ◽  
S. D. Willows ◽  
A. Fokine ◽  
A. J. Battisti ◽  
S. Sun ◽  
...  
Keyword(s):  
Protein Structure ◽  
Capsid Protein ◽  
Rubella Virus ◽  
Virus Assembly ◽  
Virus Capsid ◽  
Virus Capsid Protein

scholarly journals Functional Characterization of the Canine Heme-Regulated eIF2αKinase: Regulation of Protein Synthesis

2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
Kimon C. Kanelakis ◽  
Jayashree Pyati ◽  
Pamela C. Wagaman ◽  
Jui Chang Chuang ◽  
Young Yang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Expression System ◽  
Functional Characterization ◽  
Protein Translation ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Suitable Model ◽  
Dependent Manner

The heme-regulated inhibitor (HRI) negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α(eIF2α) thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

scholarly journals Cytoplasmic Cargo Receptor p62 Inhibits Avibirnavirus Replication by Mediating Autophagic Degradation of Viral Protein VP2

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Yahui Li ◽  
Boli Hu ◽  
Gang Ji ◽  
Yina Zhang ◽  
Chenyang Xu ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Viral Protein ◽  
Virus Assembly ◽  
Virus Production ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Virus Particles ◽  
Selective Autophagy ◽  
Cargo Receptor ◽  
Autophagic Degradation

ABSTRACT Selective autophagy regulates the degradation of cytoplasmic cargos, such as damaged organelles, invading pathogens, and aggregated proteins. Furthermore, autophagy is capable of degrading avibirnavirus, but the mechanism responsible for this process is unclear. Here, we show that autophagy cargo receptor p62 regulates the degradation of the avibirnavirus capsid protein VP2. Binding of p62 to VP2 enhances autophagic induction and promotes autophagic degradation of viral protein VP2. Further study showed that the interaction of p62 with viral protein VP2 is dependent on ubiquitination at the K411 site of VP2 and the ubiquitin-associated domain of p62. Mutation analysis showed that the K411R mutation of viral protein VP2 prohibits its p62-mediated degradation. Consistent with this finding, p62 lacking the ubiquitin-associated domain or the LC3-interacting region no longer promoted the degradation of VP2. Virus production revealed that the knockout of p62 but not the overexpression of p62 promotes the replication of avibirnavirus. Collectively, our findings suggest that p62 mediates selective autophagic degradation of avibirnavirus protein VP2 in a ubiquitin-dependent manner and is an inhibitor of avibirnavirus replication. IMPORTANCE Avibirnavirus causes severe immunosuppression and mortality in young chickens. VP2, the capsid protein of avibirnavirus, is responsible for virus assembly, maturation, and replication. Previous study showed that avibirnavirus particles could be engulfed into the autophagosome and degradation of virus particles took apart. Selective autophagy is a highly specific and regulated degradation pathway for the clearance of damaged or unwanted cytosolic components and superfluous organelles as well as invading microbes. However, whether and how selective autophagy removes avibirnavirus capsids is largely unknown. Here, we have shown that selective autophagy specifically clears ubiquitinated avibirnavirus protein VP2 by p62 recognition and that p62 is an inhibitor of avibirnavirus replication, highlighting the role of p62 as a potential drug target for mediating the removal of ubiquitinated virus components from cells.

Ribonucleoprotein Y-box-binding protein-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) protein expression after serum stimulation through binding to OXPHOS mRNA

2012 ◽  
Vol 443 (2) ◽  
pp. 573-584 ◽  
Author(s):  
Shinya Matsumoto ◽  
Takeshi Uchiumi ◽  
Hiroyuki Tanamachi ◽  
Toshiro Saito ◽  
Mikako Yagi ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Protein Expression ◽  
Nadh Dehydrogenase ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Protein Translation ◽  
Dependent Manner ◽  
Serum Stimulation ◽  
Iron Sulfur

Mitochondria play key roles in essential cellular functions, such as energy production, metabolic pathways and aging. Growth factor-mediated expression of the mitochondrial OXPHOS (oxidative phosphorylation) complex proteins has been proposed to play a fundamental role in metabolic homoeostasis. Although protein translation is affected by general RNA-binding proteins, very little is known about the mechanism involved in mitochondrial OXPHOS protein translation. In the present study, serum stimulation induced nuclear-encoded OXPHOS protein expression, such as NDUFA9 [NADH dehydrogenase (ubiquinone) 1α subcomplex, 9, 39 kDa], NDUFB8 [NADH dehydrogenase (ubiquinone) 1β subcomplex, 8, 19 kDa], SDHB [succinate dehydrogenase complex, subunit B, iron sulfur (Ip)] and UQCRFS1 (ubiquinol-cytochrome c reductase, Rieske iron–sulfur polypeptide 1), and mitochondrial ATP production, in a translation-dependent manner. We also observed that the major ribonucleoprotein YB-1 (Y-box-binding protein-1) preferentially bound to these OXPHOS mRNAs and regulated the recruitment of mRNAs from inactive mRNPs (messenger ribonucleoprotein particles) to active polysomes. YB-1 depletion led to up-regulation of mitochondrial function through induction of OXPHOS protein translation from inactive mRNP release. In contrast, YB-1 overexpression suppressed the translation of these OXPHOS mRNAs through reduced polysome formation, suggesting that YB-1 regulated the translation of mitochondrial OXPHOS mRNAs through mRNA binding. Taken together, our findings suggest that YB-1 is a critical factor for translation that may control OXPHOS activity.

scholarly journals Purification and characterization of recombinant Xenopus poly(A)+-binding protein expressed in a baculovirus system

1992 ◽  
Vol 287 (3) ◽  
pp. 761-766 ◽  
Author(s):  
R A Stambuk ◽  
R T Moon
Keyword(s):  
Developmental Stages ◽  
Binding Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Cell Protein ◽  
Translational Efficiency ◽  
Functional Protein ◽  
Baculovirus System

The poly(A)(+)-binding protein (PABP) is a highly conserved protein that binds to the poly(A)+ tail of mRNAs. PABP has been shown to regulate message stability and translational efficiency, yet the mechanisms remain unknown. To facilitate further dissection of the functions of this protein, we have expressed and purified Xenopus PABP using a baculovirus expression system. At 48 h after infection of insect Spodoptera frugiperda (Sf9) cells with recombinant virus, approx. 3% of cell protein was PABP. Purification of PABP was achieved by affinity chromatography on poly(A)(+)-Sepharose. The purified protein was indistinguishable from Xenopus PABP with respect to its immunoreactivity and electrophoretic mobility. Furthermore, the recombinant PABP was expressed and purified as a functional protein as indicated by its ability to bind to poly(A)(+)-Sepharose and its ability to enhance the translation of adenylated messages in vitro. By comparing protein extracts from various developmental stages of Xenopus embryos with known amounts of purified PABP, we determined the amount of PABP per embryo. This analysis suggested that there is less than one PABP molecule available per PABP-binding site at early stages of development, and only a slight excess of PABP at later stages.

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