The lytic cycle of Epstein-Barr virus in the nonproducer Raji line can be rescued by the expression of a 135-kilodalton protein encoded by the BALF2 open reading frame

Abstract

In Epstein-Barr virus (EBV)-carrying nonproducer Raji cells, the induction of the viral replicative cycle by chemical treatment is limited to only the early stage and viral DNA synthesis is totally inhibited. We previously showed the absence of two messenger RNAs that are encoded by the BamHI-A fragment of the EBV genome and that correspond to open reading frames BALF2 and BARF1 in chemically induced Raji cells. Since the BALF2 gene encodes a 135-kDa DNA-binding protein which was immunoprecipitated by antibody against ICP8 protein, a key protein in herpes simplex virus replication, we asked whether the lack of productive cycle in Raji cells is due to the absence of expression of the BALF2 gene. We transfected the Raji cell line with the BALF2 gene. After chemical induction, the BALF2-transfected cells expressed not only early antigens but also late antigens. In these cultures, the viral particles were detected by electron microscopy. The expression of late antigens was completely inhibited by arabinofuranosylthymine, which is a specific inhibitor of viral DNA replication. The BALF2 gene might play an essential role in the induction of the EBV-lytic cycle.

Epstein‐Barr Virus Infection of T Cells: Implications for Altered T‐Lymphocyte Activation, Repertoire Development and Autoimmunity

“…Amino acid sequences of all EBV B95-8 open reading frames (Baer et al 1984) were searched for sequences similar to conserved portions of the D35E motif (Doak et al 1994). A D35E-like motif was identified using this strategy in the protein encoded by the BALF-2 ORF (Figure 4), which is a homologue of other conserved herpes virus proteins collectively denoted the herpes major DNA binding proteins (DBP) (Littler et al 1981, Quinn & McGeogh 1985, Decaussin et al 1995. Similar D35E regions were identified in corresponding regions of other herpes virus DBF A D35E-like region in the RAG-2 protein denoted D36D (amino acid residues 202-240 in the RAG-2 consensus from Sadofsky et al 1994) was also identified using a similar search strategy ( Figure 5).…”

“…Characterization of herpes simplex DBP ICP8 dem- onstrates that expression of DBP is required for replication of the virus in association with DNA polymerases and helicases (Ruyechan & Weir 1984, Gustafsson et al 1995, and that DBP associates with DNA in intranuclear 'replication compartments' (deBruyn-Kops & Knipe 1988). DBP expression is temporally correlated with the linear to circular transition of the herpes virus genome to a latent state (de-Bruyn-Kops & Knipe 1988), as well as the reciprocal circular to linear transition of the genome (Decaussin et al 1995). To date the analogy between the DBP and bacterial single-stranded binding protein has been proposed as a function of the DBP (reviewed in Decaussin et al 1995, Gustafsson et al 1995 based upon the nonspecific single-stranded DNA binding properties of tbe protein.…”

“…DBP expression is temporally correlated with the linear to circular transition of the herpes virus genome to a latent state (de-Bruyn-Kops & Knipe 1988), as well as the reciprocal circular to linear transition of the genome (Decaussin et al 1995). To date the analogy between the DBP and bacterial single-stranded binding protein has been proposed as a function of the DBP (reviewed in Decaussin et al 1995, Gustafsson et al 1995 based upon the nonspecific single-stranded DNA binding properties of tbe protein. However, non-specific single-stranded binding properties are also a functional property shared with the catalytic regions of Tc element transposases (van Luenen et al 1994).…”

“…The DBP may also have a role in the reverse transition of the circular genome to the linear state during lytic induction of the virus (Decaussin et al 1995). Since some indirect evidence suggests that the linearization of EBV from the circular episomal form utilizes sequences similar to the sequences recognized during class switch immunoglobulin recombination (Sun et al 1994), cleavage at class switch sequences may also be a DBP function.…”

Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation

“…(i) in Raji cells, EBV's latent phase is very tightly controlled, but, upon induction the majority of the cells readily enter EBV's lytic phase. The lytic phase, however, is incomplete and does not support the amplification the viral DNA because Raji cells carry a defective EBV genome (Polack et al, 1984;Hatfull et al, 1988;Decaussin et al, 1995).Thus, Raji cells (ii) allow studying the very early modifications in cellular chromatin in the pre-replicative phase when 5 the cells transit from viral latency to lytic reactivation but prior to the formation of replication compartments or amplification factories. In EBV-infected cells they become microscopically visible as early as 16 to 24 hours after lytic phase induction and are characterized by the local accumulation of EBV DNA and the occlusion of cellular chromatin constituents such as histones (Chiu et al, 2013).…”