Affinity Purification Combined with Mass Spectrometry to Identify Herpes Simplex Virus Protein–Protein Interactions

2014 ◽  
pp. 209-222 ◽  
Author(s):  
David G. Meckes
Keyword(s):  
Mass Spectrometry ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Virus Protein ◽  
Protein Protein Interactions ◽  
Simplex Virus

scholarly journals Functional and Physical Interactions of the Herpes Simplex Virus Type 1 UL20 Membrane Protein with Glycoprotein K

2008 ◽  
Vol 82 (13) ◽  
pp. 6310-6323 ◽  
Author(s):  
Timothy P. Foster ◽  
Vladimir N. Chouljenko ◽  
K. G. Kousoulas
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Herpes Simplex Virus Type ◽  
Protein Protein Interactions ◽  
Glycoprotein K ◽  
Er Retention ◽  
Simplex Virus

ABSTRACT Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.

scholarly journals Identification of structural protein–protein interactions of herpes simplex virus type 1

Virology ◽  
2008 ◽  
Vol 378 (2) ◽  
pp. 347-354 ◽  
Author(s):  
Jin H. Lee ◽  
Valerio Vittone ◽  
Eve Diefenbach ◽  
Anthony L. Cunningham ◽  
Russell J. Diefenbach
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Protein Interactions ◽  
Herpes Simplex Virus Type ◽  
Structural Protein ◽  
Protein Protein Interactions ◽  
Simplex Virus

scholarly journals HVint: A Strategy for Identifying Novel Protein-Protein Interactions in Herpes Simplex Virus Type 1

2016 ◽  
Vol 15 (9) ◽  
pp. 2939-2953 ◽  
Author(s):  
Paul Ashford ◽  
Anna Hernandez ◽  
Todd Michael Greco ◽  
Anna Buch ◽  
Beate Sodeik ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Protein Interactions ◽  
Herpes Simplex Virus Type ◽  
Protein Protein Interactions ◽  
Simplex Virus ◽  
Novel Protein

scholarly journals Determination of Interactions between Tegument Proteins of Herpes Simplex Virus Type 1

2005 ◽  
Vol 79 (15) ◽  
pp. 9566-9571 ◽  
Author(s):  
Valerio Vittone ◽  
Eve Diefenbach ◽  
Damian Triffett ◽  
Mark W. Douglas ◽  
Anthony L. Cunningham ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Interactions ◽  
Herpes Simplex Virus Type ◽  
Protein Protein Interactions ◽  
Tegument Proteins ◽  
Simplex Virus ◽  
Two Hybrid ◽  
Hsv 1

ABSTRACT The aim of this study was to elucidate protein-protein interactions between tegument proteins of herpes simplex virus type 1 (HSV-1). To do so, we have cloned and expressed in the LexA yeast (Saccharomyces cerevisiae) two-hybrid system, 13 of the 21 currently known tegument proteins of HSV-1. These included the tegument proteins essential for replication in cell lines, UL17, UL36, UL37, UL48, and UL49, and the nonessential tegument proteins US11, UL11, UL14, UL16, UL21, UL41, UL46, and UL47. A total of 104 combinations were screened in the yeast two-hybrid assay, with 9 interactions identified. These included: UL11-UL16, UL36-UL37, UL36-UL48, UL46-UL48, UL47-UL48, and UL48-UL49. The remaining interactions consisted of self-associations that were observed for US11, UL37, and UL49. The interactions UL36-UL37, UL36-UL48, UL37-UL37, UL46-UL48, and UL47-UL48 have not been previously reported for HSV-1. The interaction of UL46-UL48 was verified using an in vitro pull-down assay. The interactions of UL36-UL37 and UL37-UL37 were verified with a coimmunoprecipitation assay. Knowledge of HSV-1 tegument protein-protein interactions will provide insights into the pathways of tegument assembly, and the identified interactions are potential targets for new antiviral drugs.

scholarly journals Arginine Methylation of the ICP27 RGG Box Regulates the Functional Interactions of ICP27 with SRPK1 and Aly/REF during Herpes Simplex Virus 1 Infection

2009 ◽  
Vol 83 (17) ◽  
pp. 8970-8975 ◽  
Author(s):  
Stuart K. Souki ◽  
Rozanne M. Sandri-Goldin
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Interactions ◽  
Arginine Methylation ◽  
Protein Protein Interactions ◽  
Herpes Simplex Virus 1 ◽  
Methylation Inhibitor ◽  
Arginine Residues ◽  
Regulate Protein ◽  
Simplex Virus

ABSTRACT The herpes simplex virus 1 protein ICP27 is methylated on arginine residues within an RGG box, and arginine methylation regulates ICP27 export to the cytoplasm. Arginine methylation can regulate protein-protein interactions; therefore, we examined the effect of hypomethylation on ICP27's interactions with cellular proteins SRPK1 and Aly/REF, which bind to ICP27 through the RGG box region. During infections with viral mutants containing lysine substitutions or the methylation inhibitor adenosine dialdehyde, the interaction of ICP27 with SRPK1 and Aly/REF was decreased, as determined by coimmunoprecipitation and colocalization studies, indicating that ICP27 RGG box methylation regulates interaction with these proteins.

scholarly journals PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

2016 ◽  
Vol 2016 ◽  
pp. 1-13
Author(s):  
Stefan Kalkhof ◽  
Stefan Schildbach ◽  
Conny Blumert ◽  
Friedemann Horn ◽  
Martin von Bergen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Isotope Labeling ◽  
Affinity Purification ◽  
Interaction Network ◽  
Mass Spectrometry Data ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Binding Behavior ◽  
Bona Fide

The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6.

scholarly journals Exploring the Human-Nipah Virus Protein-Protein Interactome

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Luis Martinez-Gil ◽  
Natalia M. Vera-Velasco ◽  
Ismael Mingarro
Keyword(s):  
Protein Interactions ◽  
Affinity Purification ◽  
Nipah Virus ◽  
Productive Infection ◽  
Virus Protein ◽  
Protein Protein Interactions ◽  
Data Set ◽  
Wide Range ◽  
Host Virus Interactions ◽  
Virus Interactions

ABSTRACT Nipah virus is an emerging, highly pathogenic, zoonotic virus of the Paramyxoviridae family. Human transmission occurs by close contact with infected animals, the consumption of contaminated food, or, occasionally, via other infected individuals. Currently, we lack therapeutic or prophylactic treatments for Nipah virus. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. This aim led us to perform the present work, in which we identified 101 human-Nipah virus protein-protein interactions (PPIs), most of which (88) are novel. This data set provides a comprehensive view of the host complexes that are manipulated by viral proteins. Host targets include the PRP19 complex and the microRNA (miRNA) processing machinery. Furthermore, we explored the biologic consequences of the interaction with the PRP19 complex and found that the Nipah virus W protein is capable of altering p53 control and gene expression. We anticipate that these data will help in guiding the development of novel interventional strategies to counter this emerging viral threat. IMPORTANCE Nipah virus is a recently discovered virus that infects a wide range of mammals, including humans. Since its discovery there have been yearly outbreaks, and in some of them the mortality rate has reached 100% of the confirmed cases. However, the study of Nipah virus has been largely neglected, and currently we lack treatments for this infection. To develop these agents we must now improve our understanding of the host-virus interactions that underpin a productive infection. In the present work, we identified 101 human-Nipah virus protein-protein interactions using an affinity purification approach coupled with mass spectrometry. Additionally, we explored the cellular consequences of some of these interactions. Globally, this data set offers a comprehensive and detailed view of the host machinery's contribution to the Nipah virus's life cycle. Furthermore, our data present a large number of putative drug targets that could be exploited for the treatment of this infection.

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