Perspective Chapter: Modulation of Latent to Lytic Cycle Infection Switch and Its Implication in EBV Mediated Tumorigenicity

Abstract

Epstein-Barr virus (EBV) is a lymphotropic herpesvirus termed human herpesvirus 4 (HHV4). It was initially identified in biopsies of Burkitt’s lymphoma, arising in the jaw and other site of the body in childhood or early adolescent individuals in sub-Saharan region. Subsequently, its tight association with other type of lymphomas has been described, and the tightest association has been seen in nasopharyngeal carcinoma (NPC), endemic with southeast Asia and southern part of China. The malignant transforming potential of EBV has been identified in immune compromised individuals; in the context all viral genomic products are expressed among which oncogenic proteins or non-coding RNAs are expressed. The interactions between cellular and viral oncoprotein as well as host gene expression regulation by the viral genetic products have been investigated in human tumors. The switch from latent form of infection to lytic phase has been studied in EBV-associated human tumors, and the modulation by intracellular signaling pathways has been known to be of importance in EBV-mediated carcinogenesis.

1. Introduction

Epstein-Barr virus (EBV) is a lymphotropic DNA virus belonging to herpesviridae family. The human herpesviruses fall into a family with eight members; systematically, EBV is termed human herpesvirus type 4 (HHV-4); it was initially identified more than 60 years from the biopsies of Burkitt’s lymphoma (BL), a well-differentiated tumor arising in the jaw of individuals at childhood and early adolescence in sub-Saharan region of Africa [1]. It was the first virus documented to be of direct association with human cancers, leading to nasopharyngeal carcinoma (NPC) [2], endemic of southern China, Southeast Asia, North Africa, and Greenland. Biologically, EBV adopts two distinct phases in its life cycle when replicating on entering the human host; that is a lytic form of infection during which the virus replicates, and mature particles are assembled with the production of new virions with infection potentially to enter new host cells on releasing from the previous host cell having established parasitism.

EBV also established a latent form of infection when the full-length viral DNA is integrated into the genome in the invaded cells persisting in dormancy throughout the lifetime of the host [for a review, see Ref. [3]]. The infection of EBV in B cells has been intensively studied. It has been shown that the viral is capable of entering a cell type suitable for long-term latency and periodic reactivation, as manifested by replication ending with rupture of the host cells and release of mature viral particles [4].

EBV assesses to the site in the host B cell to initiate its life cycle of latent infection mimics the natural differentiation triggered by antigen exposure in the same type of cells. The presence of EBV at different developmental stages of B cells, and its ability to infect a range of cells of epithelial origin, also contributes to the pathogenesis, including the genesis of diverse lymphomas and carcinomas [5, 6, 7], exemplified by non-Hodgkin’s lymphoma, lymphomas and lymphoproliferative diseases in the immunocompromised, post-transplantation lymphoma, and NPC- and EBV-associated gastric cancer.

High titers of anti-EBV antibodies against viral capsid antigen (VCA) expressed on the surface of viral particles, and early diffuse (EAd/BMRF1) in patients with undifferentiated NPC seen in the endemic region with high incidence, suggest a tight association of EBV infection with occurrence of the cancer arising in the coating epithelium of nasopharynx [8, 9], and also, the frequent entering to lytic cycle of EBV harbored in the lesion of NPC, replicating in the tumor cells. The detection of the antiserum against the two EBV antigens whose coding gene cluster scheme is indicated in Figure 1 is diagnostic of NPC in combination of other factors like genetic predisposition.

Figure 1.

Genetic mapping of EAd coding gene BMRF1. The gene ID is 3,783,718, with sequence spanning the nucleotides 67,552–69,916 on genome DNA of EBV (NC_007605.1) with full length of 171 kb. Retrieved from https://www.ncbi.nlm.nih.gov/gene/3783718 on 4th July, 2023.

EBV is not identified in other forms of head and neck cancers, with exception of salivary gland tumors [10]. The replication site in the lesion is constituted with stratified squamous epithelium with differentiating properties; the microenvironment is speculated to be favorable for a program of lytic replication adopted by invading EBV. It is believed that the intracellular niche of less differentiated is suitable for EBV to establish infection; EBV genome is identified in poorly or undifferentiated but not well-differentiated cases of cancer of nasopharynx.

2. The switch from latency to lytic cycle as a characteristic process in the life cycle of herpesviruses

The life cycle of EBV, the first human tumorigenic virus ever being identified, has been defined as latency and lytic or productive infection by the early seminal work by George and Eva Klein. Up to now, at least 80 proteins encoded by the viral genome have been discovered, and a number of them still remain to be definitively identified [11]. During latent infection of EBV, the integrated viral genome synthesizes six nuclear antigens, termed EBV determined nuclear antigens (EBNAs), and they are alternatively called EBNA1-6 or EBNA 1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA-leader protein, together with three membrane integral proteins, namely latent membrane protein 1, 2A, and 2B (LMP1, 2A, 2B) [11]. Some EBNAs are expressed in lymphocytes with similar phenotype of lymphoblastoid cell lines (LCL) and of antigen activated in immunocompromised individuals, manifested as lymphoproliferative disorders; their transforming potential may play a role of genesis of malignancies. A latent EBV antigen, EBNA3C, alternatively called EBNA6 is mainly expressed in immunocompromised hosts due to its high immunogenicity. It has been described to be required for in vitro transformation of B cells. EBNA3C interacts with various cellular and viral factors to act as a transcriptional coregulatory. It has been revealed that EBNA-3C primarily targets two important cellular pathways—cell cycle and apoptosis. During EBV latency, EBNA-3C promotes B-cell lymphomagenesis by seizing cellular pathways [12].

One of the integral proteins encoded by EBV, LMP1, behaves as a constitutively active surface receptor to stimulate proliferation through the engagement of intracellular signaling pathways, notably of transcription factor NF-κB [13, 14], and JNK [15], its C-terminal activation regions (CTARs) of LMP1 selectively activate STAT family proteins, such as STAT3, STAT5, and STAT1 [16, 17]. LMP1 is therefore classified as a viral oncoprotein. It has been reported that LMP1 is detected in considerable amount of NPC cases, which are clinically immunocompetent [18, 19].

The latent infection of EBV is switched to lytic phase, which is activated by some chemicals in vitro, like sodium butyrate (NaB). The switch of latent infection to lytic cycle in EBV and other human herpesviruses has been studied in cultured B cell lines; it has been shown that the activation of lytic cycle in EBV is controlled by two EBV-encoded transcription factors, ZEBRA and Rta (reviewed in Ref. [20]). Distinctive genomic products are expressed in two phases of the EBV life cycle, latent and lytic cycle, contributing to the initiation and maintenance of the cycle [20]. The main products of EBV-encoded proteins and non-coding RNAs are listed in Table 1.

Time phase/gene exp. profile	Latent	Lytic cycle
		IE	E	L
Proteins	EBNA 1,; EBNA 2,; EBNA 3s,; EBNA LP; LMP1, 2A, 2B	BZLF1; BRLF1	BMRF1; BALF2; BMLF2/3	VCA etc.
Non-coding RNAs	EBER1, 2; BARTs

Table 1.

The life cycle of EBV and expression profile of its genomic product.

The life cycle is divided into latent and lytic cycle; the latter is comprised of three phases, immediate early (IE), early (E), and late (L). During the three time courses IE, E, and L, the genomic products expressed are categorized as transactivators, replication factors, and structural proteins respectively.

The mapping of the genomic products expressed in EBV lytic cycle, contributing to its initiation and maintenance [20] on the linear structure of EBV genome is indicated in Figure 1. The two EBV-derived transcriptional activators are essential for the viral replication; strains with deletion of BZLF1 or BRLF1 in the genome are not competent for replication of viral DNA or production of mature virions [21]. At the initial step of lytic cycle, early genes-encoded proteins are upregulated.

Replication of EBV during lytic cycle is regulated by two immediate-early genes, BZLF1 and BRLF1 (Figure 2). They code for viral transcriptional activators Z and R, termed Zta and Rta, respectively [22]. EBV-encoded BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt’s lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes [22].

Figure 2.

The genetic cluster of the immediate early lytic genes BRLF1 and BZLF1. The gene ID for the two genes are 3,783,727 and 3,783,744, spanning nucleotides 89,838–93,925(complement) and 89,838–90,943(complement). The number on the scale above the figure indicates the position of nucleotides within the genomic sequence. They code for lytic proteins Rta and Zta, respectively. Retrieved from https://www.ncbi.nlm.nih.gov/gene/3783727 and https://www.ncbi.nlm.nih.gov/gene/3783744 on 4th July 2023.

Late genes coding for structural proteins is expressed after replication of EBV DNA. The lytic genes of EBV respond to Zta and Rta at varying extent [23]. They are activated by either Rta or Zta primarily [24].

The expression of either Zta or Rta induces other lytic proteins and disrupts viral latency [25, 26]; activation of Rta and Zta has been found in vitro that their promoters are responsive to cross-linking of B-cell receptor (BCR), chemicals such as phorbol esters, and inophores, in addition to NaB.

Rta in EBV is homologous to open-reading frame 50 (ORF 50) within the genome of Kaposi Sarcoma-associated herpesvirus (KSHV), known as human herpesvirus 8 (HHV8). It activates lytic cycle through two distinct phases: The upstream events control the expression of lytic cycle activator genes encoded by the viral genome, and the downstream events drive the viral lytic cycle to express and hence initiate the replication of viral DNA.

The lytic cycle of EBV, and also of KSHV, is activated by a panel of chemicals like NaB. It is a classic inhibitor of histone deacetylase, which prevents removing acetyl group from modified amino acid. It has been shown that NaB disrupts latency of EBV, to allow the virus to enter lytic cycle. In case of EBV, protein synthesis is required when lytic cycle is activated by EBV [27].

An EBV immediate-early gene BZLF1 codes for a protein ZEBRA, which drives the lytic cycle, and this is the major downstream mediator of EBV lytic cycle activation. It acts both as a transcription activator and an essential replication protein. It has been shown that these two functions are separated by phosphorylation of ZEBRA at its casein kinase 2 (CK2) site. The activation of lytic replication by ZEBRA but not through induction of expression of early lytic cycle genes requires phosphorylation by CK (Figure 2) [28].

Zp, the promoter of the EBV immediate-early gene BZLF1, contains several phorbol ester response elements. Study on the role of PKC pathway in lytic cycle suggests that in lytic cycle induction by histone deacetylase (HDAC) inhibitors involves protein kinase C (PKC) [28], which is a group of enzymes that regulates the activation of activator protein-1, NF-кB and cAMP responsive element binding protein to stimulate the expression of a panel of genes. When activated by the phorbol ester TPA, B95–8 cells were activated into the lytic cycle a PKC-dependent mechanism but not by HDAC inhibitors such as n-butyrate and trichostatin A (TSA). In cells in which the immediate-early promoters Zp and Rp were simultaneously activated by the HDAC inhibitors, TPA by itself failed to activate lytic gene expression. Inhibition of PKC activity by bisindolylmaleimide I did not block lytic cycle activation in the EBV-positive Burkitt lymphoma cells by n-butyrate or TSA.

In an extensive exploration of the mechanism underlying the different responses, the variable role of the PKC pathway found in different cell lines could not be accounted for by significant polymorphisms in the promoters of the immediate-early genes, or by differences in the nucleosomal organization of EBV DNA in the region of Zp or Rp [29].

3. The regulation of latency-to-lytic phase by viral and cellular machinery: the implication of transcription factors

The transcriptional activators Z (Zta) and R (Rta) are encoded by two immediate-early genes, BZLF1 and BRLF1 of EBV, to regulate EBV replication. Zta is a basic zipper (bZIP) protein with DNA binding capacity; it binds and activates promoters containing AP1 sites (TGASTCA) and related sequences called Zta-responsive elements [30]. The EBV-encoded transcriptional activator, Rta has a length of 605-amino-acid (aa) residues; it is an acidic transactivator protein; its homolog has not been found in any DNA-binding proteins in the cell. Its homologs, however, are present in all gamma-herpesviruses, and the greatest homology exists in their N-terminal DNA binding domains (DBDs) on the amino(N)-terminus with up to 40% similarity between EBV and KSHV [30].

Another EBV-encoded transcriptional activator, Rta binds to a particular sequence, Rta response elements (RREs), conforming to the consensus GNCCN9GGNG, to activate various promoters through a direct mechanism [31, 32, 33, 34]. It also activates other promoters lacking the sequence of RREs through an indirect mechanism(s). Such target sequences include their own promoter (Rp). Rta activates the sequences through Sp1/Sp3 binding sites [35].

The Zta promoter (Zp) activates many promoters via the ZII cyclic AMP(cAMP) response element [36]; and the BALF5 DNA polymerase gene, a lytic EBV gene, through USF and E2F binding sites [37, 38]. For the promoter Rp, it has been proposed that Rta is targeted indirectly to Sp1/Sp3 sites through an interaction with MCAF1, an Sp1-associated factor [39]. This mechanism closely mirrors that of KSHV Rta, which activates promoters indirectly via an interaction with the DNA-binding protein RBP-Jκ [40, 41, 42].

Rta can also activate promoters indirectly through the activation of mitogen-activated protein kinase and phosphatidylinositol-3 kinase, resulting in phosphorylation of ATF2 bound to the ZII element and activation of Zta expression [43, 44].

The induction of viral transcriptional activators Zta and Rta contributes to the switch between the latent and the lytic cycle of EBV infection. In EBV-infected B cells, their expression is likely regulated by CD4+ T cell-derived cytokines. It has been shown that during the first 3 days of culture interleukin-21 decreases constitutive expression of Rta and its target EA-D in EBV-infected B cell lines. In some cell lines, this is followed by a strong increase in the expression of Zta, Rta, and EA-D during the prolonged culture. Additionally, there is evidence suggesting that IL-21-mediated JAK/STAT signals regulate increased expression of Zta [45].

It has been observed that high level of STATs notably remains the state of latent infection of EBV [46]. Protein inhibitor of activated STAT(PIAS) proteins has been identified as negative regulators of STAT signaling. The PIAS proteins exert the inhibitory effect through modification activity of SUMO E3 ligase on ubiquitination of protein substrate. Up to more than 60 proteins, most of them being transcription factors, either positively or negatively regulated by members of the PIAS family through multiple mechanisms, have been identified in biochemical studies [47, 48].

A member of this family, PIAS1 is a restriction factor of EBV, acting to inhibit viral and cellular transcription factors. The interactions of PIAS1 with interferon regulatory factor 8 (IRF8) have been demonstrated and PIAS1 has been shown to inhibit lytic gene activation mediated by IRF8 [45, 49]. In the population refractory to lytic cycle induction, Stat3 and Fos transcripts were preferentially upregulated. Expression of both factors was increased folds compared to untreated cells.

In EBV harboring Burkitt lymphoma cells, the regulation of lytic cycle entry is investigated. When latently infected with EBV, the cells express high levels of STAT3 protein, predominantly in unphosphorylated form. When exposed to NaB, a lytic cycle-inducing agent and a prototype inhibitor of HDAC, entry of lytic cycle entry is induced. The cells were treated with IFN-γ determining whether STAT3 is phosphorylated at the tyrosine residue Y705 in this cell line or if this pathway was defective. Since the increase of Stat3 transcript occurs primarily in refractory cells, the levels in STAT3 protein were examined present in this population. Increased level of STAT3 protein in the refractory population is relative to the untreated cells in a manner of time course dependency after treatment with NaB is identified. STAT3 protein, however, is not significantly upregulated in the subpopulation of lytic cells [20, 21].

The exact molecular mechanisms, such as underlying posttranscriptional processes, to control latency of EBV, the reactivation and progression of the lytic cycle remain to be fully elucidated [50, 51]. Master transcriptional regulators of plasma cell differentiation, including Blimp-1/PRDM1, also activate the promoters of EBV genes BZLF1 (Zp) and BRLF1 (Rp) [51, 52]. Transcription factors such as ATFs, Sp1/3, MEF2D, XBPs, family members of cAMP-responsive element-binding protein (CREB), AP1 heterodimers of c-Fos and phosphorylated c-Jun, and HIF1α interact with Zp in response to challenge of antigen or oxidative stress; Zp further contains cis-regulatory elements to exert autoregulation [50, 51, 52, 53, 54, 55]. Repressors of Zp are repressed by molecules like the zinc-finger E-box-binding proteins encoded by ZEB1 and ZEB2 and the polycomb protein Yin Yang 1 (YY1) [50, 51, 56, 57, 58]. Notably, microRNAs (miRNAs) from the miR-200 family (miR-200b and miR-429 expressed in epithelial cells) which post-transcriptionally silence ZEB1/2 expression are capable of enhancing EBV reactivation through increased Zp activity [59, 60].

It has been shown that high levels of host STAT3 curtail the susceptibility of latently infected cells to lytic cycle activation signals. Cellular PCBP2 [poly(C)-binding protein 2], an RNA-binding protein, has been identified as a transcriptional target of STAT3 in refractory cells. It has been demonstrated that single cells expressing high levels of PCBP2 are refractory to lytic activation of EBV, both spontaneous and induced, lytic susceptibility is regulated by STAT3 via cellular PCBP2, and suppression of PCBP2 levels is sufficient to increase EBV lytic cells in number [61]. The findings would guide efforts to improve oncolytic therapy for EBV-associated cancers.

Host miRNAs in the EBV lytic cycle are profiled. Among small RNAs in reactivated Burkitt lymphoma cells, several miRNAs, such as miR-141, are identified. They are induced upon BCR cross-linking. Notably, EBV encodes a viral miRNA, miR-BART9, with sequence homology to miR-141. Their molecular targets and experimentally validated multiple candidates are commonly regulated by both miRNAs. Targets included B cell transcription factors and known regulators of EBV immediate-early genes, leading us to hypothesize that these miRNAs modulate kinetics of the lytic cascade in B cells. Through functional assays, we identified roles for miR-141 and EBV miR-BART9 and one specific target, FOXO3, in progression of the lytic cycle [62, 63].

4. Lytic cycle activation contributes to viral tumorigenicity

Cellular factors like E2F6, E2F1, tumor suppressor gene (TSG) coding product Rb, and enzymes catalyzing removal of acetyl group from histones and non-histone proteins, histone deacetylase 1 (HDAC1), and HDAC2 are associated with EBV-encoded nuclear antigens, to regulate such events like gene expression, based on the next-generation sequencing (NGS) analysis. A complex of E2F-Rb-HDAC exhibits similar distributions located in genomic regions of EBV-positive cells and its long-range regulatory regions are associated with oncogenic super-enhancers [64].

The transforming EBV latent antigens cooperatively hijack this complex, to bind KLFs gene loci on host genomic sequence, to facilitate the expression of KLF14 gene in LCLs. These results demonstrate that EBV latent antigens have been demonstrated to function as master regulators of this multi-subunit repressor complex (E2F-Rb-HDAC) with tumor suppressive potential, to reverse its activities antagonistic and facilitate downstream gene expression to contribute to the viral transforming protein-mediated lymphomagenesis [64].

To date, several cytogenetic lesions have been reported to be of crucial alterations in the occurrence of NPC, an EBV-associated tumor endemic of certain regions in the world, including southern China, Southeast Asia, and Greenland inhabited by Inuit and North Africa [65]. In Asian NPC, loss of homozygosity (LOH) is frequently detected at several chromosomal regions, particularly the locations 3p, 9p, 11q, 13q, and 14q [66, 67, 68, 69, 70, 71]. It has been reported that the anomaly in 3p is among key changes during occurrence of NPC [66]. A high frequency of LOH in 3p was also found in normal nasopharyngeal epithelial cells and precancerous lesions in individuals from endemic areas, suggesting that the inactivation of TSGs in this chromosome might be an early event in the genesis of NPC [66]. Coding genes for p16/CDKN2A and RASSF1A on 9p and 3p respectively have been recognized as main TSGs in NPC [66, 67, 68, 69, 70, 71]. Mutability of RASSF1A has been reported in NPC [67]. CCND1 coding for cyclin D1(cycD1) has been observed to be amplified in NPC epithelium; the role played by cycD1 in EBV-mediated transformation has been documented [72].

Another 3p21-mapped TSG, BLU codes for a zinc finger motif containing protein ZMYND10 with tumor suppressive potential have been identified. Its inactivation due to epigenetic approach has been observed in clinical specimens, as well as passaged cell lines of NPC. Its promoter hypermethylation was detected in the primary tumors of NPC, but not in normal nasopharyngeal epithelium and cells of immortalized normal epithelial cells [73, 74]. The expression of BLU is correlated with a favorable prognosis, prolonged survival of NPC patients [75].

The cytogenetic aberrations suggested that genome instability is closely associated with the development of NPC. Given that both chemicals and the virus have been shown to be co-carcinogens in the development of cancer [76], it would be interesting to examine the interplay between EBV and chemical carcinogens and their effects on the genome instability of NPC.

With regard to the dietary association with the genesis of NPC, traditionally consuming food from high-risk areas of NPC is found to contain EBV inducers and mutagens, as well as N-nitrosamines [77]. Moreover, it has also been shown that various chemicals aforementioned as inducers of EBV lytic cycle, like phorbol esters and n-butyrate present in several herbal medicines and food sources, can induce the EBV lytic cycle and may be involved in the tumorigenesis of NPC [78, 79, 80].

The regions of China with a high annual incidence of NPC were colocalized with those where herbal drugs containing phorbol esters are commonly used [81]. These results suggest that chemical carcinogens may contribute to the carcinogenesis of NPC. However, the underlying mechanism has not been extensively studied yet [65]. The synergy of chemicals including EBV lytic cycle inducers 12-O-tetradecanoylphorbol-13-acetate (TPA) and NaB in enhancing EBV reactivation and genome instability for implication of NPC genesis has been investigated with an EBV-negative nasopharyngeal carcinoma cell line and an EBV-positive NPC cell derived from the same line. The results of expression profile analysis indicate that many carcinogenesis-related genes were altered after recurrent EBV reactivation, and several aberrations are observed in correspondence to alterations in NPC. Cooperation between chemical carcinogens enhances the reactivation of EBV leading to alteration of cancer hallmark gene expression with resultant enhancement of tumorigenesis in NPC [82].

5. Perspectives: application in therapeutic strategy for treating virally associated tumors

EBV infection is associated with a number of human tumors of lymphoid and epithelial origin; the tightest association has been seen with NPC, in virtually all cases, and the viral DNA is isolated from the tumor cells. EBV is mainly present in NPC cells with a pattern of latent infection. Inhibition of virus replication, however, is not efficacious in treating EBV-associated malignancies. Instead, activation of EBV replication is potentially therapeutic, because virus replication can directly kill EBV-infected tumor cells, sensitize them to nucleoside analogues, and stimulate immune-mediated killing via increased virus antigen expression in tumor cells [83, 84]. When EBV is triggered to enter the reproductive lytic phase, immunogenic proteins, in addition to only a few viral proteins and non-coding small RNAs expressed at latent stage, are expressed. The expression of lytic antigens provokes a stronger and more effective immune response [85]. Importantly, viral kinases expressed as lytic cycle proteins render tumor cells to sensitive for certain antiviral treatment, like (val)ganciclovir [86].

The intentional induction of the lytic form of EBV infection combined with ganciclovir (GCV) treatment has been proposed as a novel regimen for anti-EBV positive tumor therapy [87]. EBV-encoded BGLF4 with thymidine kinase activity is expressed only during the lytic form of infection. Its catalytic activity converts a nucleoside analogue, GCV into its active, cytotoxic form. Gemcitabine and doxorubicin induce lytic EBV infection in EBV-transformed B cells in vitro and in vivo. The combination of gemcitabine or doxorubicin and GCV has significantly more cytostatic effective for EBV-driven lymphoproliferative disease in SCID mice than chemotherapy alone. The results suggest that the addition of GCV, due to the presence of active enzyme, may enhance the therapeutic efficacy of gemcitabine- or doxorubicin-containing chemotherapy regimens, for treating EBV-driven lymphoproliferative disease in patients.

In addition to EBV-associated BL, specifically target EBV-positive cells for destruction have been tested as a therapeutic approach for tumors of epithelial origin. The efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive such tumors have been examined. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive gastric carcinoma cells in vitro and NPC tumors. The antitumor effect of the BZLF1 and BRLF1 adenovirus vectors was not significantly affected by adding ganciclovir. These results suggest lytic cycle of EBV induction of a potential cancer therapy against EBV-related tumors [88].

Virus-targeted lytic induction treatment in EBV-associated malignancies aims at evoking more potent immune responses and induce susceptibility to achieve a goal in the virally targeting therapy. Clinically, this strategy was initially attempted in EBV-associated lymphoma [89] and has now been studied in subsequent years [90, 91]. Different agents, such as HDAC inhibitors, chemotherapeutics, radiation, phorbol esters, and butyrates, have been tested as inducers of the lytic phase of EBV in associated tumor cell lines [86, 87, 88]. Until now, only a few clinical proof-of-principle studies on viral lytic induction have been performed [88, 92].

6. Conclusion

The infection of several viruses, including two human herpesviruses, EBV and KSHV/HHV-8, has been found to be associated with carcinogenesis. The tightest association has been seen with EBV in case of BL and NPC. EBV adopts a distinctive pattern of life cycle, with latency and lytic cycle. During lytic cycle, the viral replication leads to host cells rupture and release of mature viral particles, to allow establishment of new infection of latency. The viral-encoded products play a role in achievement of malignancy, and understanding of regulation and drug targeting of viral lytic cycle will contribute to improve anticancer therapy.

Acknowledgments

The authors would like to dedicate the manuscript to the late Professor George Klein (1925-2016), a pioneering tumor biologist who made tremendous contribution in the fields of chemical and viral carcinogenesis together with discoveries in cancer cytogenetics and oncogenes; his life time efforts have enabled tumor biology to evolve as an independent science from a branch of cell biology. We are proud of being on his student chain.