Unraveling the Mechanism of Action of a Novel Anti-Epstein-Barr Virus Compound Extracted from Soil-Dwelling Bacteria

Abstract

Introduction: Epstein-Barr Virus (EBV) is a member of the herpesvirus family that is also known as Human herpes virus 4 (HHV-4). EBV infects around 90% of the world population. It establishes latency, and hence indefinite infection, in memory B cells. Despite the innumerable approaches to develop potential vaccines and antiviral drugs, there are currently no available efficient ones to combat EBV. Therefore, our group investigated potential anti-viral natural products from soil-dwelling bacteria. We detected that QSB-12 C, a secondary metabolite produced by the QSB-12 bacterial strain, displayed effective anti-EBV activity with minimal cytotoxic effects. This was tested against an EBV-harboring virus producer cell line. The aim of the study at hand was therefore to establish de novo EBV infection in EBV negative cells to determine the mechanism of action of the novel anti-viral compound. We also aimed to scale up production of the compound for further testing. Methods: We determined the Multiplicity of Infection (MOI) ratio, crucial for establishing de novo viral infection without compromising cellular viability, in the EBV- negative BC-3 cells cultured at varying EBV loads. Subsequently, the dynamics of EBV replication and latency were assessed through the temporal profiling of BZLF and LMP2A relative expression, the lytic and latency phase marker genes respectively. This was followed by a Time-of-Addition Assay (TOA) to elucidate the mechanism of action of QSB-12 C. Moreover, a scale-up fermentation process was employed to increase the yield of the active antiviral compound, with subsequent bio-guided fractionation through High-Performance Liquid Chromatography (HPLC) to isolate potent compounds. Genomic characterization through whole-genome sequencing (WGS) was conducted to identify biosynthetic gene clusters associated with antiviral activity. Finally, the antiviral screening assay involved testing fractions obtained after column chromatography (CC) for their efficacy against EBV in the EBV-harboring P3HR-1 cells, coupled with cytotoxicity assessment using the trypan blue exclusion assay. Results: An MOI of 3.5 successfully established de novo EBV infection in BC-3 cells, yielding substantial viral replication while preserving cellular viability. Following infection, BZLF relative expression exhibited a gradual increase during the initial incubation hours, peaking notably at hour 5, coinciding with the highest observed levels of EBV genome copies in the cells and supernatants. This was followed by a progressive decrease beyond hour 6 in BZLF and EBV genome copy levels which was concomitant with a steady persistent increase in the LMP2A relative expression, indicative of a shift 2 from active replication towards viral latency within the infected BC-3 cells. This enabled employment of the TOA demonstrating that QSB-12 C primarily targets post-entry stages, particularly impeding the genomic replication and biosynthesis processes. The efficacy of this antiviral activity surpassed that of acyclovir by approximately 4 folds with minimal cytotoxicity. WGS allowed identification of the QSB-12 isolate as a Microbacterium species with 99% similarity, closely related to Microbacterium foliorum and belonging to the Actinomycetes phylum harboring unique biosynthetic gene clusters. Scaled-up fermentation from this isolate yielded fractions with significant antiviral activity, notably the chloroform fraction with a 6-fold decrease in the EBV DNA copies compared to the PMA-induced DMSO-treated control with negligible cytotoxicity. The HPLC chromatogram displayed 12 pure compounds, prompting additional sub fractionation via CC. Subsequent testing identified 3 subfractions, B, C and D with significant EBV inhibition without inducing cytotoxicity. Conclusion: Altogether, the results underscore the potential of QSB-12 C as an effective antiviral agent against EBV, offering a promising avenue for further exploration in therapeutic development.