Epstein-Barr virus (EBV), a human herpesvirus causing infectious mononucleosis, is highly immunogenic having a more than 95% seropositive ratio in the world community and is associated with Burkitt lymphoma and nasopharyngeal carcinoma in etiology [18,19]. It contributes to 200,000 cancers per year around the world [20], and after the establishment of latent infection in B lymphocytes, EBV can persist in the human host for life [21]. EB virus transformation is a standard method to immortalize isolated human B cells [22]. EBV can transform isolated mononuclear leukocytes, which contain human peripheral blood B lymphocytes, into immortal human lymphoblastoid cell lines (LCLs) in an appropriate in vitro environment, and their biochemical and molecular biological characteristics remain unchanged [23,24]. The EBV genome is a linear double strand of about 172 kb containing more than 85 genes, but only a few are expressed in EBV-infected B lymphocytes, called latent genes [24,25]. Latent proteins can activate the interaction between cell growth factor and its receptor, and change the life cycle of B lymphocytes, thus immortalizing the cells.

Six latent virus genes, including Epstein-Barr nuclear antigen LP (EBNA-LP), nuclear antigen 3A (EBNA3A), nuclear antigen 3C (EBNA3C), nuclear antigen 1 (EBNA1), nuclear antigen 2 (EBNA2), and latent membrane protein 1 (LMP1) [26], are mainly expressed during the transformation of B cells into LCLs by EB virus [27]. EBNA1 and EBNA2 are the keys to inducing the immortalization of B cells, while EBNA3A, EBNA3C, and LMP1 are also involved in the in vitro transformation of B cells. EBNA1 is a homologous dimer DNA-binding protein that can bind to multiple sites in the host genome [28]. EBNA1′s expression in the latent and release periods of viruses mediates replication and segregation of the respective viral genomes [29,30], which is crucial for maintaining replication termination at OriP and viral episome maintenance [31]. EBNA2 is a primary viral transcription activator and can up-regulate the expression of host genes and other EBNA genes [32]. It activates the expression of viral oncoprotein LMP1 during the initiation and maintenance of B cell immortalization [33], which accelerates the immortalization of infected B cells into LCLs, enabling them to proliferate indefinitely during culture. Thus, the LMP1 oncoprotein is essential for the continued growth and survival of LCLs [34]. It simulates a pro-survival tumor necrosis factor receptor, which constitutively sends out signals through the NF-KB pathway to promote the proliferation of transformed cells and inhibit apoptosis [35]. Inhibition of the activated NF-KB pathway downstream of LMP1 resulted in the apoptosis of LCLs. However, studies have shown that B cells still proliferate rapidly with LMP1 expression and low NF-KB activation levels in the early post-infection period [35,36]. In early infected B cells, the expression of EBNA3A protein can induce the resistance to apoptosis of B lymphocytes and primary B cells by down-regulating the Bim gene [37], thus reducing the “starting power” of infected cells to apoptosis. So, these early infected cells showed no apparent apoptosis, although the cell DNA damage response was strongly activated [38]. In addition, the EBN3A family epigenetically down-regulates other human genes involved in cell cycle regulation [39]. For example, the expression of two host proteins, MCL-1 and BFL-1, is controlled by EBNA3A. Meanwhile, EBNA3C and EBNA3A inhibit the expression of tumor suppressor p16 in combination to maintain the continuous proliferation of B cells and transform B cells into immortalized LCLs [40,41]. In summary, the EBV latent gene mimics the critical factors of natural B cell growth, promotes its proliferation to form immortalized LCLs, and inhibits host innate tumor suppressor responses to uncontrollable proliferation (Figure 1).

Simian virus 40 (SV40) was first identified in rhesus monkey kidney cell cultures used to produce the polio vaccine in 1960 [55]. It is named for the effect on infected cells that produce a number of abnormal vacuoles, as SV40 may promote tumor growth in animal models and induce the transformation of primary cultured human cells [56,57]. SV40 infects rodent or mammalian cells to improve cell immortalization rate and is widely used in experimental models of mammalian cell replication and gene expression [58,59]. SV40 is a double-stranded DNA virus whose genome encodes seven proteins, three structural proteins, and four functional proteins in an overlapping reading frame [58,60]. Structural proteins are VP1, VP2, and VP3, and functional proteins include large T (LT) antigens and small T (ST) antigens essential to the virus life cycle and two small proteins whose functions are unknown [59]. SV40 LT antigen, a multifunctional regulatory protein [61], is classified as a member of the helicase superfamily and has the property of releasing double-stranded DNA and RNA, which play a decisive role in the initiation of virus-transformed cells [62].

There are two main mechanisms by which the SV40 LT antigen immortalizes cells (Figure 2) [63]. One is the activation of E2F-mediated transcription by binding to the Rb-E2F complex. The other is to inhibit p53 activity by blocking p53-dependent transcriptional activation and p53-independent growth arrest. LT antigen binds to the Rb protein to release the inhibition of cell cycle regulation by the Rb-E2F complex, leading to the entry of the S phase with E2F-dependent gene transcription from the growth arrest pathway and resulting in continuous cell proliferation. During the cell transformation by SV40, the LT antigen interacts with the tumor suppressor p53, a transcriptional activator that mediates apoptosis under adverse conditions such as DNA damage, nucleotide deficiency, and abnormal inhibition of Rb protein, thereby transforming cells and extending their lifespan [64,65]. Moreover, the binding of LT to cellular factors, p300/CBP, also prevents apoptosis and leads to the survival of cells independent of direct interaction between LT and p53 [66]. However, ST antigens can enhance cell transformation but are not necessary. In brief, LT antigens and ST antigens maintain cell transformation phenotypes together [67].