The Influence of Toll-Like Receptor Stimulation on Expression of EBV Lytic Genes

Abstract

Epstein-Barr virus (EBV) establishes latency in the resting memory B-cell compartment. It has been recently suggested that maintenance of chronic infection is dependent on periodic reactivation. Although the stimuli for EBV reactivation in vivo during natural infections are largely unknown, there is evidence indicating that heterologous infections could trigger herpesviruses reactivation. The purpose of this work was to identify the influence of Toll-like receptors stimulation on EBV replication in EBV latently infected Burkitt lymphoma cells (P3HR-1, Raji and Namalwa). The cells were stimulated with Pam3CSK4 (synthetic triacylated lipoprotein), PolyI:C (synthetic analog of dsRNA), LPS (lipopolysaccharide from E.coli), measles virus (MeV) and PMA (phorbol myristate acetate). Non-stimulated cells (NS) served as control. EBV expression was investigated at mRNA level for three viral lytic genes: BZLF1 (immediate early, ZEBRA), BALF2 (early, EA) and BcLF1 (late, VCA). Additionally, the effect of stimulation on NF-kBp65 and inflammatory cytokines (IL-lb, IL-6, IL-8, IL-10, IL-12p70, and TNF) was investigated. Stimulation of TLRs led to limited changes in EBV expression manifesting as increase of ZEBRA at mRNA level in cells treated with PolyI:C and Pam3CSK4. Stimulation with PolyI:C, Pam3CSK4 and LPS also lead to considerable increase of NF-kBp65, while increased levels of inflammatory cytokines were observed for IL-8, TNF and IL-6 in cells treated with PMA and MeV. In conclusion, the results of our experiments support the suggestion that TLRs stimulation with microbial ligands influences EBV virus replication.

Anti-viral and pro-inflammatory functions of Toll-like receptors during gamma-herpesvirus infections

“… Artificial stimulation triggers reactivation? 1/2 Cell surface; Diacyl lipopeptides (Pam3CSK4) Activated – EBV [ 21 , 26 ] No – KSHV[ 35 ] Yes – EBV [ 44 , 45 ] Yes – KSHV [ 62 ] Yes – MHV68 in vivo [ 15 ] No – EBV [ 31 ] No – EBV [ 39 ] Yes – EBV [ 67 , 68 ] Minor effect – MHV68 in cells [ 34 ] Yes – MHV68 in cells [ 42 ] 2/6 Cell surface; Triacyl lipopeptides(FSL-1) Activated – EBV [ 21 , 26 ] No – KSHV[ 35 ] Yes – EBV [ 44 , 45 ] Yes – KSHV [ 62 ] Yes – MHV68 in vivo [ 15 ] Minor effect – MHV68 in cells [ 34 ] No – EBV [ 39 ] Yes – EBV [ 67 , 68 ] Yes – MHV68 in cells [ 42 ] No – higher—MHV68 in vivo [ 15 ] 3 Endosome; dsRNA (polyI:C) …”

“…In the case of EBV, virus reactivation and EBV-linked hepatitis were reported in a patient with infection with the syphilis pathogen Treponema pallidum, a known inducer of TLR2 [ 67 ]. In addition, stimulation of multiple TLRs can induce expression of the lytic regulator ZEBRA in EBV latently infected cell lines [ 68 ]. Collectively, these studies underscore the multiplicity of functions that TLR signaling can have during γ-herpesvirus infection, including a role in promoting lytic replication.…”

Impaired surface marker expression in stimulated Epstein-Barr virus transformed lymphoblasts from Barth Syndrome patients

“…However, studies have also indicated that Epstein-Barr virus transformation of human B lymphocytes may hinder host immune function 12 . B lymphoblasts express Toll-like receptors (TLRs) including TLR4 which can be stimulated with lipopolysaccharide (LPS) 13 . In addition, cytosine linked to a guanine by a phosphate bond deoxyribonucleic acid (CpG DNA) activates human B lymphocytes through TLR9 regardless of whether the DNA is in the form of genomic bacterial DNA or in the form of a synthetic oligodeoxynucleotide 14 .…”