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Dengue-2 structural proteins associate with human proteins to produce a coagulation and innate immune response biased interactome.

Folly BB, Weffort-Santos AM, Fathman CG, Soares LR - BMC Infect. Dis. (2011)

Bottom Line: A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network.In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection.Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Federal University of Paraná, Pharmaceutical Sciences Post-graduation Program, Av. Pref. Lothário Meissner 632, CEP 80210-170, Curitiba-PR, Brazil.

ABSTRACT

Background: Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem.

Methods: A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology.

Results: Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis.

Conclusions: Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue proteins with a proposed human protein interaction network produces a modified biological outcome that may be behind the hallmark pathologies of dengue infection.

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Related in: MedlinePlus

Graphical representation of the DENV2 structural protein interactome. (A) Primary network formed by the D2 structural proteins and its targets, obtained experimentally in the two-hybrid assay. (B) Topological representation of the network in (A) overlayed on a human-human interaction network. Red nodes and red edges represent the nodes and edges seen in "A" and blue nodes and edges the remainder of the human-human network.
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Figure 1: Graphical representation of the DENV2 structural protein interactome. (A) Primary network formed by the D2 structural proteins and its targets, obtained experimentally in the two-hybrid assay. (B) Topological representation of the network in (A) overlayed on a human-human interaction network. Red nodes and red edges represent the nodes and edges seen in "A" and blue nodes and edges the remainder of the human-human network.

Mentions: To study dengue-human protein-protein interactions, we individually cloned the Env, PrM and Cap genes from a DENV2 [1] isolate into the bacterial two-hybrid vector pBT. Interactions were identified in the bacterial two-hybrid screen by assaying the individual baits against a whole brain, and a liver cDNA library, both cloned into pTRG vectors. In total, 106 clones were screened in each library. Carbenicillin resistant beta-Gal positive colonies were retested at higher stringency conditions by gradually increasing the dose of carbenicillin. Surviving colonies were re-assayed for beta-Gal activity, and colonies staining for the highest intensity beta-Gal expression (blue coloration) were selected and sequenced. Forty-seven, and 30 in-frame sequences were obtained for the brain and liver screens, respectively, representing 31 unique proteins, presented here in Cytoscape (Figure 1A and Table 1). A few human proteins in our screen interacted with more than one viral protein, a feature also found in a recent HCV interactome study, and attributed to a biological "essentiality" of these host proteins for the virus life cycle.


Dengue-2 structural proteins associate with human proteins to produce a coagulation and innate immune response biased interactome.

Folly BB, Weffort-Santos AM, Fathman CG, Soares LR - BMC Infect. Dis. (2011)

Graphical representation of the DENV2 structural protein interactome. (A) Primary network formed by the D2 structural proteins and its targets, obtained experimentally in the two-hybrid assay. (B) Topological representation of the network in (A) overlayed on a human-human interaction network. Red nodes and red edges represent the nodes and edges seen in "A" and blue nodes and edges the remainder of the human-human network.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037883&req=5

Figure 1: Graphical representation of the DENV2 structural protein interactome. (A) Primary network formed by the D2 structural proteins and its targets, obtained experimentally in the two-hybrid assay. (B) Topological representation of the network in (A) overlayed on a human-human interaction network. Red nodes and red edges represent the nodes and edges seen in "A" and blue nodes and edges the remainder of the human-human network.
Mentions: To study dengue-human protein-protein interactions, we individually cloned the Env, PrM and Cap genes from a DENV2 [1] isolate into the bacterial two-hybrid vector pBT. Interactions were identified in the bacterial two-hybrid screen by assaying the individual baits against a whole brain, and a liver cDNA library, both cloned into pTRG vectors. In total, 106 clones were screened in each library. Carbenicillin resistant beta-Gal positive colonies were retested at higher stringency conditions by gradually increasing the dose of carbenicillin. Surviving colonies were re-assayed for beta-Gal activity, and colonies staining for the highest intensity beta-Gal expression (blue coloration) were selected and sequenced. Forty-seven, and 30 in-frame sequences were obtained for the brain and liver screens, respectively, representing 31 unique proteins, presented here in Cytoscape (Figure 1A and Table 1). A few human proteins in our screen interacted with more than one viral protein, a feature also found in a recent HCV interactome study, and attributed to a biological "essentiality" of these host proteins for the virus life cycle.

Bottom Line: A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network.In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection.Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Federal University of Paraná, Pharmaceutical Sciences Post-graduation Program, Av. Pref. Lothário Meissner 632, CEP 80210-170, Curitiba-PR, Brazil.

ABSTRACT

Background: Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem.

Methods: A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology.

Results: Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis.

Conclusions: Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue proteins with a proposed human protein interaction network produces a modified biological outcome that may be behind the hallmark pathologies of dengue infection.

Show MeSH
Related in: MedlinePlus