Virus-host Interactome Research Articles

Identification of phytochemicals in Qingfei Paidu decoction for the treatment of coronavirus disease 2019 by targeting the virus-host interactome

Qingfei Paidu decoction (QFPDD) has been clinically proven to be effective in the treatment of coronavirus disease 2019 (COVID-19). However, the bioactive components and therapeutic mechanisms remain unclear. This study aimed to explore the effective components and underlying mechanisms of QFPDD in the treatment of COVID-19 by targeting the virus-host interactome and verifying the antiviral activities of its active components in vitro. Key active components and targets were identified by analysing the topological features of a compound-target-pathway-disease regulatory network of QFPDD for the treatment of COVID-19. The antiviral activity of the active components was determined by a live virus infection assay, and possible mechanisms were analysed by pseudotyped virus infection and molecular docking assays. The inhibitory effects of the components tested on the virus-induced release of IL-6, IL-1β and CXCL-10 were detected by ELISA. Three components of QFPDD, oroxylin A, hesperetin and scutellarin, exhibited potent antiviral activities against live SARS-CoV-2 virus and HCoV-OC43 virus with IC50 values ranging from 18.68 to 63.27 μM. Oroxylin A inhibited the entry of SARS-CoV-2 pseudovirus into target cells and inhibited SARS-CoV-2 S protein-mediated cell-cell fusion by binding with the ACE2 receptor. The active components of QFPDD obviously inhibited the IL-6, IL-1β and CXCL-10 release induced by the SARS-CoV-2 S protein. This study supports the clinical application of QFPDD and provides an effective analysis method for the in-depth study of the mechanisms of traditional Chinese medicine (TCM) in the prevention and treatment of COVID-19.

Interaction of HDAC2 with SARS-CoV-2 NSP5 and IRF3 Is Not Required for NSP5-Mediated Inhibition of Type I Interferon Signaling Pathway.

ABSTRACTOver the last 2 years, several global virus-host interactome studies have been published with SARS-CoV-2 proteins with the purpose of better understanding how specific viral proteins can subvert or utilize different cellular processes to promote viral infection and pathogenesis. However, most of the virus–host protein interactions have not yet been confirmed experimentally, and their biological significance is largely unknown. The goal of this study was to verify the interaction of NSP5, the main protease of SARS-CoV-2, with the host epigenetic factor histone deacetylase 2 (HDAC2) and test if HDAC2 is required for NSP5-mediated inhibition of the type I interferon signaling pathway. Our results show that NSP5 can significantly reduce the expression of a subset of immune response genes such as IL-6, IL-1β, and IFNβ, which requires NSP5’s protease activity. We also found that NSP5 can inhibit Sendai virus-, RNA sensor-, and DNA sensor-mediated induction of IFNβ promoter, block the IFN response pathway, and reduce the expression of IFN-stimulated genes. We also provide evidence for HDAC2 interacting with IRF3, and NSP5 can abrogate their interaction by binding to both IRF3 and HDAC2. In addition, we found that HDAC2 plays an inhibitory role in the regulation of IFNβ and IFN-induced promoters, but our results indicate that HDAC2 is not involved in NSP5-mediated inhibition of IFNβ gene expression. Taken together, our data show that NSP5 interacts with HDAC2 but NSP5 inhibits the IFNβ gene expression and interferon-signaling pathway in an HDAC2-independent manner.IMPORTANCE SARS-CoV-2 has developed multiple strategies to antagonize the host antiviral response, such as blocking the IFN signaling pathway, which favors the replication and spreading of the virus. A recent SARS-CoV-2 protein interaction mapping revealed that the main viral protease NSP5 interacts with the host epigenetic factor HDAC2, but the interaction was not confirmed experimentally and its biological importance remains unclear. Here, we not only verified the interaction of HDAC2 with NSP5, but we also found that HDAC2 also binds to IRF3, and NSP5 can disrupt the IRF3-HDAC2 complex. Furthermore, our results show that NSP5 can efficiently repress the IFN signaling pathway regardless of whether viral infections, RNA, or DNA sensors activated it. However, our data indicate that HDAC2 is not involved in NSP5-mediated inhibition of IFNβ promoter induction and IFNβ gene expression.

The Landscape of Virus-Host Protein-Protein Interaction Databases.

Knowledge of virus-host interactomes has advanced exponentially in the last decade by the use of high-throughput screening technologies to obtain a more comprehensive landscape of virus-host protein–protein interactions. In this article, we present a systematic review of the available virus-host protein–protein interaction database resources. The resources covered in this review are both generic virus-host protein–protein interaction databases and databases of protein–protein interactions for a specific virus or for those viruses that infect a particular host. The databases are reviewed on the basis of the specificity for a particular virus or host, the number of virus-host protein–protein interactions included, and the functionality in terms of browse, search, visualization, and download. Further, we also analyze the overlap of the databases, that is, the number of virus-host protein–protein interactions shared by the various databases, as well as the structure of the virus-host protein–protein interaction network, across viruses and hosts.

The Intricacy of the Viral-Human Protein Interaction Networks: Resources, Data, and Analyses.

Viral infections are one of the major causes of human diseases that cause yearly millions of deaths and seriously threaten global health, as we have experienced with the COVID-19 pandemic. Numerous approaches have been adopted to understand viral diseases and develop pharmacological treatments. Among them, the study of virus-host protein-protein interactions is a powerful strategy to comprehend the molecular mechanisms employed by the virus to infect the host cells and to interact with their components. Experimental protein-protein interactions described in the scientific literature have been systematically captured into several molecular interaction databases. These data are organized in structured formats and can be easily downloaded by users to perform further bioinformatic and network studies. Network analysis of available virus-host interactomes allow us to understand how the host interactome is perturbed upon viral infection and what are the key host proteins targeted by the virus and the main cellular pathways that are subverted. In this review, we give an overview of publicly available viral-human protein-protein interactions resources and the community standards, curation rules and adopted ontologies. A description of the main virus-human interactome available is provided, together with the main network analyses that have been performed. We finally discuss the main limitations and future challenges to assess the quality and reliability of protein-protein interaction datasets and resources.

A BioID-Derived Proximity Interactome for SARS-CoV-2 Proteins.

The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here, the cellular impact of expressing SARS-CoV-2 viral proteins was studied by global proteomic analysis, and proximity biotinylation (BioID) was used to map the SARS-CoV-2 virus–host interactome in human lung cancer-derived cells. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are associated with SARS-CoV-2 proteins. We have established a website to host the proteomic data to allow for public access and continued analysis of host–viral protein associations and whole-cell proteomes of cells expressing the viral–BioID fusion proteins. Furthermore, we identified 66 high-confidence interactions by comparing this study with previous reports, providing a strong foundation for future follow-up studies. Finally, we cross-referenced candidate interactors with the CLUE drug library to identify potential therapeutics for drug-repurposing efforts. Collectively, these studies provide a valuable resource to uncover novel SARS-CoV-2 biology and inform development of antivirals.

Unraveling Protein Interactions between the Temperate Virus Bam35 and Its Bacillus Host Using an Integrative Yeast Two Hybrid–High Throughput Sequencing Approach

Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. Interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, which are known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein–protein interactions (PPIs) network for a tectivirus–host system by studying the Bam35–Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS). We generated and thoroughly analyzed a genomic library of Bam35′s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait–prey couples. Initial analysis of the raw data enabled the identification of over 4000 candidate interactions, which were sequentially filtered to produce 182 high-confidence interactions that were defined as part of the core virus–host interactome. Overall, host metabolism proteins and peptidases were particularly enriched within the detected interactions, distinguishing this host–phage system from the other reported host–phage PPIs. Our approach also suggested biological roles for several Bam35 proteins of unknown function, including the membrane structural protein P25, which may be a viral hub with a role in host membrane modification during viral particle morphogenesis. This work resulted in a better understanding of the Bam35–B. thuringiensis interaction at the molecular level and holds great potential for the generalization of the Y2H-HTS approach for other virus–host models.

Network-Based Identification and Experimental Validation of Drug Candidates Toward SARS-CoV-2 via Targeting Virus-Host Interactome.

Despite that several therapeutic agents have exhibited promising prevention or treatment on Coronavirus disease-2019 (COVID-19), there is no specific drug discovered for this pandemic. Targeting virus–host interactome provides a more effective strategy for antivirus drug discovery compared with targeting virus proteins. In this study, we developed a network-based infrastructure to prioritize promising drug candidates from natural products and approved drugs via targeting host proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). We firstly measured the network distances between drug targets and COVID-19 disease module utilizing the network proximity approach, and identified 229 approved drugs as well as 432 natural products had significant associations with SARS-CoV-2. After searching for previous literature evidence, we found that 60.7% (139/229) of approved drugs and 39.6% (171/432) of natural products were confirmed with antivirus or anti-inflammation. We further integrated our network-based predictions and validated anti-SARS-CoV-2 activities of some compounds. Four drug candidates, including hesperidin, isorhapontigenin, salmeterol, and gallocatechin-7-gallate, have exhibited activity on SARS-COV-2 virus-infected Vero cells. Finally, we showcased the mechanism of actions of isorhapontigenin and salmeterol via network analysis. Overall, this study offers forceful approaches for in silico identification of drug candidates on COVID-19, which may facilitate the discovery of antiviral drug therapies.

SARS-CoV-2 Infection Remodels the Phenotype and Promotes Angiogenesis of Primary Human Lung Endothelial Cells.

SARS-CoV-2-associated acute respiratory distress syndrome (ARDS) and acute lung injury are life-threatening manifestations of severe viral infection. The pathogenic mechanisms that lead to respiratory complications, such as endothelialitis, intussusceptive angiogenesis, and vascular leakage remain unclear. In this study, by using an immunofluorescence assay and in situ RNA-hybridization, we demonstrate the capability of SARS-CoV-2 to infect human primary lung microvascular endothelial cells (HL-mECs) in the absence of cytopathic effects and release of infectious particles. Preliminary data point to the role of integrins in SARS-CoV-2 entry into HL-mECs in the absence of detectable ACE2 expression. Following infection, HL-mECs were found to release a plethora of pro-inflammatory and pro-angiogenic molecules, as assessed by microarray analyses. This conditioned microenvironment stimulated HL-mECs to acquire an angiogenic phenotype. Proteome analysis confirmed a remodeling of SARS-CoV-2-infected HL-mECs to inflammatory and angiogenic responses and highlighted the expression of antiviral molecules as annexin A6 and MX1. These results support the hypothesis of a direct role of SARS-CoV-2-infected HL-mECs in sustaining vascular dysfunction during the early phases of infection. The construction of virus-host interactomes will be instrumental to identify potential therapeutic targets for COVID-19 aimed to inhibit HL-mEC-sustained inflammation and angiogenesis upon SARS-CoV-2 infection.

Network medicine links SARS-CoV-2/COVID-19 infection to brain microvascular injury and neuroinflammation in dementia-like cognitive impairment

BackgroundDementia-like cognitive impairment is an increasingly reported complication of SARS-CoV-2 infection. However, the underlying mechanisms responsible for this complication remain unclear. A better understanding of causative processes by which COVID-19 may lead to cognitive impairment is essential for developing preventive and therapeutic interventions.MethodsIn this study, we conducted a network-based, multimodal omics comparison of COVID-19 and neurologic complications. We constructed the SARS-CoV-2 virus-host interactome from protein-protein interaction assay and CRISPR-Cas9-based genetic assay results and compared network-based relationships therein with those of known neurological manifestations using network proximity measures. We also investigated the transcriptomic profiles (including single-cell/nuclei RNA-sequencing) of Alzheimer’s disease (AD) marker genes from patients infected with COVID-19, as well as the prevalence of SARS-CoV-2 entry factors in the brains of AD patients not infected with SARS-CoV-2.ResultsWe found significant network-based relationships between COVID-19 and neuroinflammation and brain microvascular injury pathways and processes which are implicated in AD. We also detected aberrant expression of AD biomarkers in the cerebrospinal fluid and blood of patients with COVID-19. While transcriptomic analyses showed relatively low expression of SARS-CoV-2 entry factors in human brain, neuroinflammatory changes were pronounced. In addition, single-nucleus transcriptomic analyses showed that expression of SARS-CoV-2 host factors (BSG and FURIN) and antiviral defense genes (LY6E, IFITM2, IFITM3, and IFNAR1) was elevated in brain endothelial cells of AD patients and healthy controls relative to neurons and other cell types, suggesting a possible role for brain microvascular injury in COVID-19-mediated cognitive impairment. Overall, individuals with the AD risk allele APOE E4/E4 displayed reduced expression of antiviral defense genes compared to APOE E3/E3 individuals.ConclusionOur results suggest significant mechanistic overlap between AD and COVID-19, centered on neuroinflammation and microvascular injury. These results help improve our understanding of COVID-19-associated neurological manifestations and provide guidance for future development of preventive or treatment interventions, although causal relationship and mechanistic pathways between COVID-19 and AD need future investigations.

Characterisation of the Semliki Forest Virus-host cell interactome reveals the viral capsid protein as an inhibitor of nonsense-mediated mRNA decay.

The positive-sense, single-stranded RNA alphaviruses pose a potential epidemic threat. Understanding the complex interactions between the viral and the host cell proteins is crucial for elucidating the mechanisms underlying successful virus replication strategies and for developing specific antiviral interventions. Here we present the first comprehensive protein-protein interaction map between the proteins of Semliki Forest Virus (SFV), a mosquito-borne member of the alphaviruses, and host cell proteins. Among the many identified cellular interactors of SFV proteins, the enrichment of factors involved in translation and nonsense-mediated mRNA decay (NMD) was striking, reflecting the virus' hijacking of the translation machinery and indicating viral countermeasures for escaping NMD by inhibiting NMD at later time points during the infectious cycle. In addition to observing a general inhibition of NMD about 4 hours post infection, we also demonstrate that transient expression of the SFV capsid protein is sufficient to inhibit NMD in cells, suggesting that the massive production of capsid protein during the SFV reproduction cycle is responsible for NMD inhibition.

Open Science Resources for the Mass Spectrometry-Based Analysis of SARS-CoV-2.

The SARS-CoV-2 virus is the causative agent of the 2020 pandemic leading to the COVID-19 respiratory disease. With many scientific and humanitarian efforts ongoing to develop diagnostic tests, vaccines, and treatments for COVID-19, and to prevent the spread of SARS-CoV-2, mass spectrometry research, including proteomics, is playing a role in determining the biology of this viral infection. Proteomics studies are starting to lead to an understanding of the roles of viral and host proteins during SARS-CoV-2 infection, their protein-protein interactions, and post-translational modifications. This is beginning to provide insights into potential therapeutic targets or diagnostic strategies that can be used to reduce the long-term burden of the pandemic. However, the extraordinary situation caused by the global pandemic is also highlighting the need to improve mass spectrometry data and workflow sharing. We therefore describe freely available data and computational resources that can facilitate and assist the mass spectrometry-based analysis of SARS-CoV-2. We exemplify this by reanalyzing a virus-host interactome data set to detect protein-protein interactions and identify host proteins that could potentially be used as targets for drug repurposing.

Pathway enrichment analysis of virus-host interactome and prioritization of novel compounds targeting the spike glycoprotein receptor binding domain–human angiotensin-converting enzyme 2 interface to combat SARS-CoV-2

SARS-CoV-2 has become a pandemic causing a serious global health concern. The absence of effective drugs for treatment of the disease has caused its rapid spread on a global scale. Similarly to the SARS-CoV, the SARS-CoV-2 is also involved in a complex interplay with the host cells. This infection is characterized by a diffused alveolar damage consistent with the Acute Respiratory Disease Syndrome (ARDS). To explore the complex mechanisms of the disease at the system level, we used a network medicine tools approach. The protein-protein interactions (PPIs) between the SARS-CoV and the associated human cell proteins are crucial for the viral pathogenesis. Since the cellular entry of SARS-CoV-2 is accomplished by binding of the spike glycoprotein binding domain (RBD) to the human angiotensin-converting enzyme 2 (hACE2), a molecule that can bind to the spike RDB-hACE2 interface could block the virus entry. Here, we performed a virtual screening of 55 compounds to identify potential molecules that can bind to the spike glycoprotein and spike-ACE2 complex interface. It was found that the compound ethyl 1-{3-[(2,4-dichlorobenzyl) carbamoyl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-7-quinolinyl}-4-piperidine carboxylate (the S54 ligand) and ethyl 1-{3-[(2,4-dichlorobenzyl) carbamoyl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydro-7-quinolinyl}-4 piperazine carboxylate (the S55 ligand) forms hydrophobic interactions with Tyr41A, Tyr505B and Tyr553B, Leu29A, Phe495B, respectively of the spike glycoprotein, the hotspot residues in the spike glycoprotein RBD-hACE2 binding interface. Furthermore, molecular dynamics simulations and free energy calculations using the MM-GBSA method showed that the S54 ligand is a stronger binder than a known SARS-CoV spike inhibitor SSAA09E3 (N-(9,10-dioxo-9, 10-dihydroanthracen-2-yl) benzamide). Communicated by Ramaswamy H. Sarma

Exploring the SARS-CoV-2 virus-host-drug interactome for drug repurposing

Coronavirus Disease-2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. Various studies exist about the molecular mechanisms of viral infection. However, such information is spread across many publications and it is very time-consuming to integrate, and exploit. We develop CoVex, an interactive online platform for SARS-CoV-2 host interactome exploration and drug (target) identification. CoVex integrates virus-human protein interactions, human protein-protein interactions, and drug-target interactions. It allows visual exploration of the virus-host interactome and implements systems medicine algorithms for network-based prediction of drug candidates. Thus, CoVex is a resource to understand molecular mechanisms of pathogenicity and to prioritize candidate therapeutics. We investigate recent hypotheses on a systems biology level to explore mechanistic virus life cycle drivers, and to extract drug repurposing candidates. CoVex renders COVID-19 drug research systems-medicine-ready by giving the scientific community direct access to network medicine algorithms. It is available at https://exbio.wzw.tum.de/covex/.

Potential Drugs Targeting Early Innate Immune Evasion of SARS-Coronavirus 2 via 2'-O-Methylation of Viral RNA.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causing the COVID-19 respiratory disease pandemic utilizes unique 2′-O-methyltransferase (2′-O-MTase) capping machinery to camouflage its RNA from innate immune recognition. The nsp16 catalytic subunit of the 2′-O-MTase is unusual in its requirement for a stimulatory subunit (nsp10) to catalyze the ribose 2′-O-methylation of the viral RNA cap. Here we provide a computational basis for drug repositioning or de novo drug development based on three differential traits of the intermolecular interactions of the SARS-CoV-2-specific nsp16/nsp10 heterodimer, namely: (1) the S-adenosyl-l-methionine-binding pocket of nsp16, (2) the unique “activating surface” between nsp16 and nsp10, and (3) the RNA-binding groove of nsp16. We employed ≈9000 U.S. Food and Drug Administration (FDA)-approved investigational and experimental drugs from the DrugBank repository for docking virtual screening. After molecular dynamics calculations of the stability of the binding modes of high-scoring nsp16/nsp10–drug complexes, we considered their pharmacological overlapping with functional modules of the virus–host interactome that is relevant to the viral lifecycle, and to the clinical features of COVID-19. Some of the predicted drugs (e.g., tegobuvir, sonidegib, siramesine, antrafenine, bemcentinib, itacitinib, or phthalocyanine) might be suitable for repurposing to pharmacologically reactivate innate immune restriction and antagonism of SARS-CoV-2 RNAs lacking 2′-O-methylation.

A High-Resolution Map of SBP1 Interactomes in Plasmodium falciparum-infected Erythrocytes.

SummaryThe pathogenesis of malaria parasites depends on host erythrocyte modifications that are facilitated by parasite proteins exported to the host cytoplasm. These exported proteins form a trafficking complex in the host cytoplasm that transports virulence determinants to the erythrocyte surface; this complex is thus essential for malaria virulence. Here, we report a comprehensive interaction network map of this complex. We developed authentic, unbiased, highly sensitive proteomic approaches to determine the proteins that interact with a core component of the complex, SBP1 (skeleton-binding protein 1). SBP1 interactomes revealed numerous exported proteins and potential interactors associated with SBP1 intracellular trafficking. We identified several host-parasite protein interactions and linked the exported protein MAL8P1.4 to Plasmodium falciparum virulence in infected erythrocytes. Our study highlights the complicated interplay between parasite and host proteins in the host cytoplasm and provides an interaction dataset connecting dozens of exported proteins required for P. falciparum virulence.

Characterizing replication kinetics and plaque production of type I feline infectious peritonitis virus in three feline cell lines

Investigating type I feline coronaviruses (FCoVs) in tissue culture is critical for understanding the basic virology, pathogenesis, and virus-host interactome of these important veterinary pathogens. This has been a perennial challenge as type I FCoV strains do not easily adapt to cell culture. Here we characterize replication kinetics and plaque formation of a model type I strain FIPV Black in Fcwf-4 cells established at Cornell University (Fcwf-4 CU). We determined that maximum virus titers (>107 pfu/mL) were recoverable from infected Fcwf-4 CU cell-free supernatant at 20 h post-infection. Type I FIPV Black and both biotypes of type II FCoV formed uniform and enumerable plaques on Fcwf-4 CU cells. Therefore, these cells were employable in a standardized plaque assay. Finally, we determined that the Fcwf-4 CU cells were morphologically distinct from feline bone marrow-derived macrophages and were less sensitive to exogenous type I interferon than were Fcwf-4 cells purchased from ATCC.

Strain-Specific Contribution of Eukaryotic Elongation Factor 1 Gamma to the Translation of Influenza A Virus Proteins.

Influenza A virus exploits multiple host proteins during infection. To define the virus–host interactome, our group conducted a proteomics-based screen and identified 299 genes that contributed to virus replication and 24 genes that were antiviral. Of these genes, we focused on the role during virus replication of eukaryotic elongation factor 1 gamma (eEF1G), which is a subunit of the eukaryotic elongation factor-1 complex and known to be a pro-viral host protein. Using the CRISPR/Cas9 system, we obtained two clones that were defective in eEF1G expression. In both of these clones, A/WSN/33 (H1N1) virus growth and protein expression were significantly suppressed, but viral mRNA, vRNA, and cRNA expression were not reduced. However, the replication and protein expression of A/California/04/2009 (H1N1pdm) virus in both clones were similar to those in parental cells. We found that the PB2 and PA proteins of WSN virus were responsible for the eEF1G-dependent replication. Our data show that eEF1G plays a role in the translation of virus proteins in a strain-specific manner. Additional analyses may be needed to further understand the role of strain-specific host proteins during virus replication.

The TRiC chaperonin controls reovirus replication through outer-capsid folding.

Viruses are molecular machines sustained through a life cycle that requires replication within host cells. Throughout the infectious cycle, viral and cellular components interact to advance the multistep process required to produce progeny virions. Despite progress made in understanding the virus-host protein interactome, much remains to be discovered about the cellular factors that function during infection, especially those operating at terminal steps in replication. In an RNA interference screen, we identified the eukaryotic chaperonin TRiC (also called CCT) as a cellular factor required for late events in the replication of mammalian reovirus. We discovered that TRiC functions in reovirus replication through a mechanism that involves the folding of the viral σ3 outer-capsid protein into a form capable of assembling onto virus particles. TRiC also complexes with homologous capsid proteins of closely related viruses. Our data define a critical function for TRiC in the viral assembly process and raise the possibility that this mechanism is conserved in related nonenveloped viruses. These results also provide insight into TRiC protein substrates and establish a rationale for the development of small-molecule inhibitors of TRiC as potential antiviral therapeutics.

Evaluation of a Bead-Free Coimmunoprecipitation Technique for Identification of Virus-Host Protein Interactions Using High-Resolution Mass Spectrometry.

Protein interactions between virus and host are essential for viral propagation and movement, as viruses lack most of the proteins required to thrive on their own. Precision methods aimed at disrupting virus-host interactions represent new approaches to disease management but require in-depth knowledge of the identity and binding specificity of host proteins within these interaction networks. Protein coimmunoprecipitation (co-IP) coupled with mass spectrometry (MS) provides a high-throughput way to characterize virus-host interactomes in a single experiment. Common co-IP methods use antibodies immobilized on agarose or magnetic beads to isolate virus-host complexes in solutions of host tissue homogenate. Although these workflows are well established, they can be fairly laborious and expensive. Therefore, we evaluated the feasibility of using antibody-coated microtiter plates coupled with MS analysis as an easy, less expensive way to identify host proteins that interact with Potato leafroll virus (PLRV), an insect-borne RNA virus that infects potatoes. With the use of the bead-free platform, we were able to detect 36 plant and 1 nonstructural viral protein significantly coimmunoprecipitating with PLRV. Two of these proteins, a 14-3-3 signal transduction protein and malate dehydrogenase 2 (mMDH2), were detected as having a weakened or lost association with a structural mutant of the virus, demonstrating that the bead-free method is sensitive enough to detect quantitative differences that can be used to pin-point domains of interaction. Collectively, our analysis shows that the bead-free platform is a low-cost alternative that can be used by core facilities and other investigators to identify plant and viral proteins interacting with virions and/or the viral structural proteins.

Prediction of GCRV virus-host protein interactome based on structural motif-domain interactions

BackgroundGrass carp hemorrhagic disease, caused by grass carp reovirus (GCRV), is the most fatal causative agent in grass carp aquaculture. Protein-protein interactions between virus and host are one avenue through which GCRV can trigger infection and induce disease. Experimental approaches for the detection of host-virus interactome have many inherent limitations, and studies on protein-protein interactions between GCRV and its host remain rare.ResultsIn this study, based on known motif-domain interaction information, we systematically predicted the GCRV virus-host protein interactome by using motif-domain interaction pair searching strategy. These proteins derived from different domain families and were predicted to interact with different motif patterns in GCRV. JAM-A protein was successfully predicted to interact with motifs of GCRV Sigma1-like protein, and shared the similar binding mode compared with orthoreovirus. Differentially expressed genes during GCRV infection process were extracted and mapped to our predicted interactome, the overlapped genes displayed different tissue expression distributions on the whole, the overall expression level in intestinal is higher than that of other three tissues, which may suggest that the functions of these genes are more active in intestinal. Function annotation and pathway enrichment analysis revealed that the host targets were largely involved in signaling pathway and immune pathway, such as interferon-gamma signaling pathway, VEGF signaling pathway, EGF receptor signaling pathway, B cell activation, and T cell activation.ConclusionsAlthough the predicted PPIs may contain some false positives due to limited data resource and poor research background in non-model species, the computational method still provide reasonable amount of interactions, which can be further validated by high throughput experiments. The findings of this work will contribute to the development of system biology for GCRV infectious diseases, and help guide the identification of novel receptors of GCRV in its host.