**2.4.3 Immune evasion**

90 Bacterial Artificial Chromosomes

progeny with high levels of gH/gL/gp42 complexes, facilitating B-cell infection. B-cells in turn, generate virus progeny with low levels of gH/gL/gp42 complexes which efficiently infect epithelial cells, but not B cells. To some extent, this relative switch of cell tropism after alternate replication in epithelial and B-cells directs infection from one cell type to the other.

There are three recombinant wild-type EBVs that have been generated so far (Delecluse, Hilsendegen et al. 1998; Kanda, Yajima et al. 2004; Chen, Divisconte et al. 2005). They were generated by the insertion of the prokaryotic F-plasmid (F-factor) in two B95.8 or one Akata strains. Although the insertion sites differ in these three EBV BACs, at the site of the B95.8 deletion (Delecluse, Hilsendegen et al. 1998), or at the major internal repeat region of the B95.5 strain (Chen, Divisconte et al. 2005), or at BXLF1 open reading frame (ORF) in Akata strain (Kanda, Yajima et al. 2004), the insertion site of the F-plasmid does not affect the

EBV is tightly related to the development of many human cancers. Chen et al (Chen, Divisconte et al. 2005) has developed a BAC-GFP-EBV (containing 172-kb of the EBV genome) system to monitor early cellular and viral events associated with EBV infection. BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line (Halder, Murakami et al. 2009). Then the progeny virus produced by a chemical was used to immortalize human primary B-cell which can be easily monitored by green fluorescence and proliferation. The results showed a dramatic increase in Ki-67, CD40, and CD23 signals. The viral genes express a pattern of an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that the resulting progeny virus is capable of infecting new primary B-cells (Halder, Murakami et al. 2009). This process may be critical for establishment of latency prior to cellular transformation

EBV is associated with a number of human malignancies. There is increasing research interest in the molecular functions of these EBV gene products in transformation and evasion from host immune surveillance systems (Izumi 2001). BAC technology made the study on the molecular function of EBV transforming genes feasible because some latent genes such as EBNA1 cannot be maintained in latently infected B cells using traditional cosmid technology (Izumi 2001; Feederle, Bartlett et al. 2010). EBNA1 was found to function as a transactivator of other latent proteins, and was required for replication of the viral genome (Altmann, Pich et al. 2006). When 71kb of EBV DNA genome was amplified in *E.coli* and transfected into primary B-lymphocyes, Altmann et al (Altmann, Pich et al. 2006) identified that EBV DNA is sufficient to immortalize primary human B lymphocytes. Kempkes et al (Kempkes, Pich et al. 1995; Izumi 2001) also identified EBNA3a as a transforming gene, which contributes primarily to the initiation of cell proliferation (Kempkes, Pich et al. 1995; Izumi 2001). Two genes BALF1 and BHRF1 which encode homologous cellular antiapoptotic viral Bcl-2 proteins (vBcl-2), were suggested to interfere with the cell apoptosis program to counteract cell death, which protects the virus from apoptosis in its host cell during virus synthesis (Altmann and Hammerschmidt 2005).

**2.4 Study of the immune response against the EBV using EBV BACs** 

phenotype of the virus.

(Halder, Murakami et al. 2009).

**2.4.2 EBV transformation** 

**2.4.1 EBV infection** 

Several viral proteins have been found to block immune recognition of viral proteins as antigens during lytic replication, such as BGLF5, BZLF2, BILF1 and BNLF2a (Ressing, van Leeuwen et al. 2005; Rowe, Glaunsinger et al. 2007; Zuo, Thomas et al. 2008; Croft, Shannon-Lowe et al. 2009; Zuo, Currin et al. 2009; Zuo, Quinn et al. 2011). The direct contribution of BNFL2a in immune evasion was evidenced using an EBV BAC which initially disrupted the BNLF2a gene of the B95.8 strain by insertional mutagenesis (Croft, Shannon-Lowe et al. 2009). BNLF2 inhibits transporter associated with antigen (TAP). It encodes a 60 amino acid protein which prevents both peptide- and ATP-binding to TAP complex (Hislop, Ressing et al. 2007). Consequently, when co-expressed with targetantigens, cells expressing BNLF2a show decreased levels of surface human leukocyte antigen (HLA)-class I and are resistant to CD8+ cytotoxic T cell killing (Hislop, Ressing et al. 2007). Croft et al (Croft, Shannon-Lowe et al. 2009) created a targeting plasmid with BNLF2a gene which was replaced by tetracycline resistant cassette. This plasmid was then flanked by FLP recombinase target (FRT) sites. This vector was homologously recombined with the EBV BAC, and designated as ∆DBNLF2a, which had the tetracycline gene removed by FLP recombinase. ∆DBNLF2a BACs were then stably transduced into 293 cells, virus replication induced by transfection of a plasmid encoding the EBV lytic switch protein BZLF1 (Feederle, Kost et al. 2000). Compared to wild-type EBV BAC, this recombinant virus induces a strong MHC I T cell response against viral lytic genes than the wild type viruses (Feederle, Bartlett et al. 2010). Overall, these results indicate that BNLF2 prevents the immediate early and early proteins from being efficiently processed and presented to CD8 + T cells during lytic cycle replication. Contrary to BNLF2a in early evasion mechanism in the lytic cycle of EBV, other mechanism seems to operate later during immune evasion (Croft, Shannon-Lowe et al. 2009). Such stage-specific expression of immune evasion genes are a feature of several herpesviruses, such as CMV (Croft, Shannon-Lowe et al. 2009). Taken together, BNLF2a acts in concert with other immuneevasion genes encoded by EBV T-cell surveillance (Croft, Shannon-Lowe et al. 2009).
