Investigating genetic-and-epigenetic networks, and the cellular mechanisms occurring in Epstein-Barr virus-infected human B lymphocytes via big data mining and genome-wide two-sided NGS data identification

Abstract

Epstein-Barr virus (EBV), also known as human herpesvirus 4, is prevalent in all human populations. EBV mainly infects human B lymphocytes and epithelial cells, and is therefore associated with their various malignancies. To unravel the cellular mechanisms during the infection, we constructed interspecies networks to investigate the molecular cross-talk mechanisms between human B cells and EBV at the first (0-24 hours) and second (8-72 hours) stages of EBV infection. We first constructed a candidate genome-wide interspecies genetic-and-epigenetic network (the candidate GIGEN) by big database mining. We then pruned false positives in the candidate GIGEN to obtain the real GIGENs at the first and second infection stages in the lytic phase by their corresponding next-generation sequencing data through dynamic interaction models, the system identification approach, and the system order detection method. The real GIGENs are very complex and comprise protein-protein interaction networks, gene/microRNA (miRNA)/long non-coding RNA regulation networks, and host-virus cross-talk networks. To understand the molecular cross-talk mechanisms underlying EBV infection, we extracted the core GIGENs including host-virus core networks and host-virus core pathways from the real GIGENs using the principal network projection method. According to the results, we found that the activities of epigenetics-associated human proteins or genes were initially inhibited by viral proteins and miRNAs, and human immune responses were then dysregulated by epigenetic modification. We suggested that EBV exploits viral proteins and miRNAs, such as EBNA1, BPLF1, BALF3, BVRF1 and miR-BART14, to develop its defensive mechanism to defeat multiple immune attacks by the human immune system, promotes virion production, and facilitates the transportation of viral particles by activating the human genes NRP1 and CLIC5. Ultimately, we propose a therapeutic intervention comprising thymoquinone, valpromide, and zebularine to act as inhibitors of EBV-associated malignancies.

Fig 1. Flow chart describing the constructing of the interspecies GIGEN network and HVCP for the multiple drug targets and potential multi-molecule drugs via the systems biology approach.

The blocks filled with gray indicate the input information exploited in this process, obtained by big data mining to establish the candidate GIGEN, NGS data to obtain the gene expression of human and EBV during the lytic phase, genome-wide DNA methylation profiles to verify the epigenetic regulation of DNA methylation of human genomes, and literature information on multi-molecule drugs for multi-molecule drug design based on the predicted drug targets. The blocks with gray frames represent the systems biology approach exploited to provide the identified information in our results; and the white blocks with solid line frames contain the results obtained from these processes.

Fig 2. Changes of gene expression levels of typical lytic genes based on classification from the literature review.

The NGS data of these typical lytic genes were sequenced by the Reads Per Kilobase per Million mapped reads (RPKM) procedure at every time-point, and are classified as immediate–early (IE), early, and late stages during the lytic phase on the basis of the classification from the literature review.

Fig 3

Real interspecies GIGENs of the first (A) and second (B) infection stages in the lytic phase. The nodes with red frames correspond to the EBV proteins/TF/miRNAs; the nodes with blue frames indicate the human receptors/proteins/TFs/miRNAs/lncRNAs; the edges in green denote the PPIs of humans, EBV, and human-EBV; the edges in purple represent the miRNA repressions of miRNAs on intraspecies and interspecies genes; the edges in black represent the transcriptional regulations of TFs on intraspecies and interspecies genes; the edges in orange signify the lncRNA regulations of lncRNAs on human genes.

Fig 4

HVCNs at the first (A) and second (B) infection stages in the lytic phase. The nodes with red frames correspond to the EBV proteins/TF/miRNAs; the nodes with blue frames indicate the human receptors/proteins/TFs/miRNAs/lncRNAs; the edges in green denote the PPIs of humans, EBV, and human-EBV; the edges in purple represent the miRNA repressions of miRNAs on intraspecies and interspecies genes; the edges in black represent the transcriptional regulations of TFs on intraspecies and interspecies genes.

Fig 5. HVCP in B cells infected with EBV at the first infection stage in the lytic phase.

The solid lines indicate the protein–protein interactions; the dotted lines denote the translocations, including protein translations and miRNA transcriptions; the solid lines that end in arrows, bars, or circles stand for positively transcriptional regulations, negatively transcriptional regulations, and miRNA repressions, respectively; the dash-dot lines represent the gene functions that are inhibited; the bold lines mean the gene functions that are promoted; the short arrows beside the gene functions signify susceptibility to repression or enhancement.

Fig 6. HVCP in B cells infected with EBV at the second infection stage of the lytic phase.

The solid lines indicate protein–protein interactions; the dotted lines denote the translocations, including protein translations and miRNA transcriptions; the solid lines that end in arrows, bars, or circles stand for positively transcriptional regulations, negatively transcriptional regulations, and miRNA repressions, respectively; the dash-dot lines represent the gene functions that are inhibited; the bold lines mean the gene functions that are promoted; the short arrows beside the gene functions signify susceptibility to repression or enhancement.

Fig 7. Signaling pathways of the interspecies molecular mechanisms based on the HVCP in Fig 5 at the first infection stage during EBV infection.

(A) The core pathways promoting cell proliferation and the impairment of immune information by the EBNA2-mediated pathway with receptor CD46; (B) the pro-apoptotic human pathway blocked by EBV through ubiquitination and acetylation; (C) the autophagy mechanism blocked by EBV through the involvement of viral BALF4, BDLF4, EBNA3B, miR-BART14, and miR-BART1-3p; (D) the complete progression of lytic production through the impairment of pro-apoptosis and the promotion of viral translocation and anti-apoptosis; (E) the promotion of the integrated production of infectious virions by silencing autophagy and inhibiting the expression of STAT3.

Fig 8. Signaling pathways of the interspecies molecular mechanisms based on the HVCP in Fig 6 at the second infection stage of EBV infection.

(A) The core pathways of the enhancement of anti-apoptosis, immunosuppression, and genetic diversity pathways by EBV for the packaging, assembly, and transport of viral particles; (B) the promotion of virion production, vesicle trafficking, release, and anti-apoptosis pathways as a result of EBNA1-mediated PML disruption; (C) the maintenance of virion transportation by decreasing the repression of the lytic cycle and increasing anti-apoptosis activities.

Fig 9. Overview of molecular mechanisms in EBV lytic infection and the significant network marker for potential multi-molecule drug design.

The red words indicate the potential drug target proteins for multi-molecule drug design; the green words represent the molecular mechanisms being hijacked by EBV; the blue and pink arrows denote the cellular functions being inhibited or promoted, respectively; the yellow arrows represent the progression from the first into the second infection stage in the lytic phase. Upon EBV reactivation into the lytic phase, the human immune system can detect EBV antigens, trigger the human immune responses, and inhibit the progression of EBV lytic replication. However, EBV mediates some defensive mechanisms against human immune response, and thereby protects the complete virion production and transportation from human immune interference at both infection stages. Additionally, the transition stage of EBV is dependent on the activation of late lytic genes and LMP1 function.

Fig 10

(A) Multi-molecule drug design for the predicted drug targets; (B) Protein structures of EBV interleukin-10 and human interleukin-10. Thymoquinone (TQ) inhibits the RNA expression of viral EBNA2, LMP1, and EBNA1; valpromide (VPM) can prevent the gene expression of viral BZLF1; zebularine (Zeb) can decrease the upregulation of viral LMP2A, LMP2B, and EBNA2. These drugs can block reactivation, interrupt the viral production of virions, interfere with the transportation of viral particles, and destroy viral defensive mechanisms.