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Identification and comparative analysis of hepatitis C virus-host cell protein interactions.

ABSTRACT: Hepatitis C virus (HCV) alters the global behavior of the host cell to create an environment conducive to its own replication, but much remains unknown about how HCV proteins elicit these changes. Thus, a better understanding of the interface between the virus and host cell is required. Here we report the results of a large-scale yeast two-hybrid screen to identify protein-protein interactions between HCV genotype 2a (strain JFH1) and cellular factors. Our study identified 112 unique interactions between 7 HCV and 94 human proteins, over 40% of which have been linked to HCV infection by other studies. These interactions develop a more complete picture of HCV infection, providing insight into HCV manipulation of pathways, such as lipid and cholesterol metabolism, that were previously linked to HCV infection and implicating novel targets within microtubule-organizing centers, the complement system and cell cycle regulatory machinery. In an effort to understand the relationship between HCV and related viruses, we compared the HCV 2a interactome to those of other HCV genotypes and to the related dengue virus. Greater overlap was observed between HCV and dengue virus targets than between HCV genotypes, demonstrating the value of parallel screening approaches when comparing virus-host cell interactomes. Using siRNAs to inhibit expression of cellular proteins, we found that five of the ten shared targets tested (CUL7, PCM1, RILPL2, RNASET2, and TCF7L2) were required for replication of both HCV and dengue virus. These shared interactions provide insight into common features of the viral life cycles of the family Flaviviridae.

SUBMITTER: Dolan PT

PROVIDER: S-EPMC4171131 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Identification and comparative analysis of hepatitis C virus-host cell protein interactions.

Dolan Patrick T PT Zhang Chaoying C Khadka Sudip S Arumugaswami Vaithilingaraja V Vangeloff Abbey D AD Heaton Nicholas S NS Sahasrabudhe Sudhir S Randall Glenn G Sun Ren R LaCount Douglas J DJ

Molecular bioSystems 20131018 12

Hepatitis C virus (HCV) alters the global behavior of the host cell to create an environment conducive to its own replication, but much remains unknown about how HCV proteins elicit these changes. Thus, a better understanding of the interface between the virus and host cell is required. Here we report the results of a large-scale yeast two-hybrid screen to identify protein-protein interactions between HCV genotype 2a (strain JFH1) and cellular factors. Our study identified 112 unique interaction ...[more]

PMID: 24136289

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Project description:Arthropod-borne viruses, such as the members of the genus Alphavirus, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish and complete their life cycles. Despite considerable efforts to define the host-pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA-protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduce a robust viral RNA-protein discovery method to elucidate the Sindbis virus (SINV) RNA-protein host interface. Cross-link-assisted mRNP purification (CLAMP) assessment revealed an extensive array of host-pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of protein-vRNA interaction by a UV cross-linking and immunoprecipitation sequencing (CLIP-seq) approach and assessed the consequences of the RNA-protein binding event of hnRNP K, hnRNP I, and hnRNP M in regard to viral infection. Here, we demonstrate that mutation of the prioritized hnRNP-vRNA interaction sites effectively disrupts hnRNP-vRNA interaction. Correlating with disrupted hnRNP-vRNA binding, SINV growth kinetics were reduced relative to wild-type parental viral infections in vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics was found to be dysregulated structural-gene expression. Collectively, this study further defines the scope and importance of the alphavirus host-pathogen vRNA-protein interactions.IMPORTANCE Members of the genus Alphavirus are widely recognized for their potential to cause severe disease. Despite this recognition, there are no antiviral therapeutics, or safe and effective vaccines, currently available to treat alphaviral infection. Alphaviruses utilize the host cell machinery to efficiently establish and complete their life cycle. However, the extent and importance of host-pathogen RNA-protein interactions are woefully undercharacterized. The efforts detailed in this study fill this critical gap, and the significance of this research is 3-fold. First, the data presented here fundamentally expand the scope and understanding of alphavirus host-pathogen interactions. Second, this study identifies the sites of interaction for several prioritized interactions and defines the contribution of the RNA-protein interaction at the molecular level. Finally, these studies build a strategy by which the importance of the given host-pathogen interactions may be assessed in the future, using a mouse model of infection.

Project description:Host-pathogen interactions are crucial for the successful propagation of pathogens inside the host cell. Knowledge of interactions between host proteins and viral proteins or viral RNA may provide clues for developing novel antiviral strategies. Hepatitis E virus (HEV), a water-borne pathogen that causes acute hepatitis in humans, is responsible for epidemics in developing countries. HEV pathology and molecular biology have been poorly explored due to the lack of efficient culture systems. A contemporary approach, to better understand the viral infection cycle at the molecular level, is the use of system biology tools depicting virus-host interactions. To determine the host proteins which participate in the regulation of HEV replication, we indentified liver cell proteins interacting with HEV RNA at its putative promoter region and those interacting with HEV polymerase (RdRp) protein. We employed affinity chromatography followed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) to identify the interacting host proteins. Protein-protein interaction networks (PPI) were plotted and analyzed using web-based tools. Topological analysis of the network revealed that the constructed network is potentially significant and relevant for viral replication. Gene ontology and pathway enrichment analysis revealed that HEV RNA promoter- and polymerase-interacting host proteins belong to different cellular pathways such as RNA splicing, RNA metabolism, protein processing in endoplasmic reticulum, unfolded protein response, innate immune pathways, secretory vesicle pathway, and glucose metabolism. We showed that hnRNPK and hnRNPA2B1 interact with both HEV putative promoters and HEV RdRp, which suggest that they may have crucial roles in HEV replication. We demonstrated in vitro binding of hnRNPK and hnRNPA2B1 proteins with the HEV targets in the study, assuring the authenticity of the interactions obtained through mass spectrometry. Thus, our study highlights the ability of viruses, such as HEV, to maneuver host systems to create favorable cellular environments for virus propagation. Studying the host-virus interactions can facilitate the identification of antiviral therapeutic strategies and novel targets.

Project description:To study virus-host protein interactions, knowledge about viral and host protein architectures and repertoires, their particular evolutionary mechanisms, and information on relevant sources of biological data is essential. The purpose of this review article is to provide a thorough overview about these aspects. Protein domains are basic units defining protein interactions, and the uniqueness of viral domain repertoires, their mode of evolution, and their roles during viral infection make viruses interesting models of study. Mutations at protein interfaces can reduce or increase their binding affinities by changing protein electrostatics and structural properties. During the course of a viral infection, both pathogen and cellular proteins are constantly competing for binding partners. Endogenous interfaces mediating intraspecific interactions-viral-viral or host-host interactions-are constantly targeted and inhibited by exogenous interfaces mediating viral-host interactions. From a biomedical perspective, blocking such interactions is the main mechanism underlying antiviral therapies. Some proteins are able to bind multiple partners, and their modes of interaction define how fast these "hub proteins" evolve. "Party hubs" have multiple interfaces; they establish simultaneous/stable (domain-domain) interactions, and tend to evolve slowly. On the other hand, "date hubs" have few interfaces; they establish transient/weak (domain-motif) interactions by means of short linear peptides (15 or fewer residues), and can evolve faster. Viral infections are mediated by several protein-protein interactions (PPIs), which can be represented as networks (protein interaction networks, PINs), with proteins being depicted as nodes, and their interactions as edges. It has been suggested that viral proteins tend to establish interactions with more central and highly connected host proteins. In an evolutionary arms race, viral and host proteins are constantly changing their interface residues, either to evade or to optimize their binding capabilities. Apart from gaining and losing interactions via rewiring mechanisms, virus-host PINs also evolve via gene duplication (paralogy); conservation (orthology); horizontal gene transfer (HGT) (xenology); and molecular mimicry (convergence). The last sections of this review focus on PPI experimental approaches and their limitations, and provide an overview of sources of biomolecular data for studying virus-host protein interactions.

Dataset Information

Identification and comparative analysis of hepatitis C virus-host cell protein interactions.

Publications

Identification and comparative analysis of hepatitis C virus-host cell protein interactions.

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