Epstein-Barr Virus Stimulates Torque Teno Virus Replication: A Possible Relationship to Multiple Sclerosis

Borkosky, Silvia S.; Whitley, Corinna; Kopp-Schneider, Annette; zur Hausen, Harald; deVilliers, Ethel-Michele

We have transfected the full-length genomes of two TT viruses isolated from brain tissue from an MS patient in order to ascertain first, whether viral replication takes place in different B lymphocytic and Burkitt's lymphoma cell lines and, secondly, to determine whether EBV acts as a helper virus for the replication of TTV. The results presented here demonstrate replication of both TTV-HD14b and TTV-HD14c up to day 21 post-transfection. Overall, we observed that the replication pattern of both TT viruses is cell-type dependent with higher levels of replication measured in the EBV positive cell lines, but showing variability in the DDCt at certain time points (Figure 2) . A number of studies have demonstrated the ability of herpesviruses to induce amplification of other persisting viral DNA [80] . Several lines of evidence may indicate the mechanism through which EBV exerts its helper-function for TTV replication. Our previous study [67] demonstrated a requirement for a helper-function in the replication and propagation of a series of TTV isolates in the 293TT cell line which had been constructed to express high levels of SV40 large T-antigen [81] . The putative origin of replication in TT viruses harbours a number of pentanucleotide motifs very similar to binding sites for SV40 large T-antigen. EBNA-1 is required for the episomal maintenance of EBV DNA and binds to the origin of replication in a sequence-specific manner [82] . Cellular transcription and gene expression is also regulated by EBNA-1 through binding to cellular promoters. Analyses of a large number of these promoters indicated differences in EBNA-1 binding motifs between those found in the EBV genome and cellular genome [83] , indicating a notion of additional, yet unidentified, binding sites for EBNA-1. Cellular promoters devoid of EBNA-1 binding sites may even be upregulated by the mere presence of EBNA-1 in the cell [84] . Enhancement of HCV replication by EBNA-1 via a transactivating function has been demonstrated [73] . Additional investigation is needed to clarify the mechanism through which EBNA-1 may act to stimulate the TTV genome. We selected several EBV-positive Burkitt's lymphomaand B cell lines in order to evaluate the TTV-HD14b and TTV-HD14c replication. All these EBV-positive cell lines contained the EBNA-1 as evidenced by the quantitative measurement of the envelope glycoprotein gp350/220 mRNA as marker for EBV lytic activity ( Figure 3 ). The TTV origin of replication is present in both TTV-HD14 isolates used in the present study and was not influenced by other differences between these 2 viral genomes [this study, 67]. A role for virus infections in the etiology of multiple sclerosis has repeatedly been investigated [4, 36] . A possible role for TTV infection in autoimmune disease including multiple sclerosis, has been reported previously [43, 52, [61] [62] [63] . The role of EBV infections in MS has however been controversial ranging from failure to demonstrate any presence [38] [39] [40] 85 ] to demonstration of an increased risk for a previous EBV infection [9, 86] and elevated EBV antibodies, more specifically EBNA-1 antibodies in MS patients [4, [87] [88] [89] [90] [91] .