The Role of Codon Usage, tRNA Availability, and Cell Proliferation in EBV Latency and (Re)Activation

Abstract

Epstein–Barr nuclear antigen 1 (EBNA1) protein synthesis is inhibited during Epstein–Barr virus (EBV) latency and is resumed in EBV (re)activation. In analyzing the molecular mechanisms underpinning the translation of EBNA1 in the human host, this article deals with two orders of data. First, it shows that the heavily biased codon usage of the EBNA1 open reading frame cannot be translated due to its noncompliance with the human codon usage pattern and the corresponding tRNA pool. The EBNA1 codon bias resides in the sequence composed exclusively of glycine and alanine, i.e., the Gly-Ala repeat (GAR). Removal of the nucleotide sequence coding for GAR results in an EBNA1 codon usage pattern with a lower codon bias, thus conferring translatability to EBNA1. Second, the data bring cell proliferation to the fore as a conditio sine qua non for qualitatively and quantitatively modifying the host's tRNA pool as required by the translational needs of EBNA1, thus enabling viral reactivation. Taken together, the present work provides a biochemical mechanism for the pathogen's shift from latency to (re)activation and confirms the role of human codon usage as a first-line tool of innate immunity in inhibiting pathogens' expression. Immunologically, this study cautions against using codon optimization and proliferation-inducing substances such as glucocorticoids and adjuvants, which can (re)activate the otherwise quiescent, asymptomatic, and innocuous EBV infection. Lastly, the data pose the question whether the causal pathogenic role attributed to EBV should instead be ascribed to the carcinogenesis-associated cellular proliferation.

Keywords

EBNA1 translation - GAR - codon usage - tRNA availability - cell proliferation - adjuvants

Introduction

Epstein–Barr virus (EBV) is associated with infectious mononucleosis, multiple sclerosis, and numerous types of cancer. Epidemiologically, EBV infects 95% of individuals worldwide, and currently, the extent of the pathological burden is highest, with EBV causally linked to 200,000 cases of cancer each year and approximately 1.8% of total cancer-related deaths annually.[1] [2] [3] However, these numbers—although already impressive in themselves—define only a minimal part of the risks that might associate with EBV infection. Indeed, EBV is ubiquitous in the human population, mainly as an asymptomatic, harmless, latent infection, with only occasional reactivation, which is the harbinger of the EBV-related diseases. This means that if EBV reactivation occurred extensively in the human population, the number and the intensity of EBV-related diseases would increase substantially. In light of such a perspective, understanding the mechanism(s) that dictate and regulate EBV latency/reactivation is a fundamental research priority to prevent a possible wave of EBV-related pathologies.

Numerous factors appear to contribute to determine the EBV latency/(re)activation. Some examples include the following:

• Epigenetic machinery such as DNA methylation, host histone chaperones, m6A RNA modification, and nonsense-mediated decay.[4]

• Psychological and cellular stressors resulting in EBV reactivation.[5]

• Reactive oxygen species production in cells.[5]

• Immune escape strategies.[6]

At the molecular level, research mainly focused on Epstein–Barr nuclear antigen 1 (EBNA1), the expression of which characterizes all the four latent forms of EBV infection, i.e., I, IIa, IIb, and III,[7] [8] [9] [10] thus suggesting crucial roles of this protein in EBV latency. Intriguingly, EBNA1 is also essential for EBV reactivation because the viral protein initiates the EBV replication by binding to specific sites in oriP, the plasmid origin.[11] [12] Additionally, EBNA1 is highly antigenic, and so a dominant line of thought has been that EBV escapes from immune surveillance by limiting EBNA1 protein production to minimal levels that cannot evoke immune responses.[13] [14] [15] [16]

Biochemically, such inhibition of EBNA1 protein synthesis resides in a repetitive sequence composed of Gly and Ala residues, i.e., the EBNA1 Gly-Ala repeat (GAR). In fact, in 2003, Yin et al[15] clearly proved that deletion of the repeat leads to a high level of EBNA1 protein in human lung carcinoma cells.

However, despite such intense research and notwithstanding numerous hypotheses,[16] [17] [18] [19] [20] [21] [22] [23] three fundamental questions remain unanswered. (1) Why and how EBNA1 protein synthesis is restricted during latency? (2) Why and how EBNA1 protein synthesis is resumed during EBV reactivation? (3) What is the underlying molecular mechanism of the GAR inhibitory effect?

Here, these issues are analyzed starting from the fact that a high level of molecular mimicry exists between human and pathogen proteins,[24] [25] [26] including EBV proteins,[27] [28] [29] with a consequent potential cross-reactivity and autoimmunity.

According to this molecular mimicry–oriented paradigm, the research rationale of the present work is based first on the consideration that, due to the numerous peptide commonalities, inhibition of pathogen protein synthesis during latency may represent a host device to avoid potential destructive autoimmune cross-reactions.[30] In the case in point, constraining the expression of EBNA1 would prevent the host immune response against the EBNA1 protein as well as cross-reactive reactions with the host proteins that share peptide sequences with the virus,[27] [28] [29] thus possibly explaining why EBNA1 protein synthesis is repressed.

Second, this study searches for possible mechanisms underlying the inhibition of EBNA1 protein synthesis on the basis of previous data[30] [31] [32] [33] that suggested that the synthesis of a protein does not occur if the codon usage of the open reading frame (ORF) coding for the protein does not comply with the codon usage of the host. In this regard, data were obtained for the ORFs coding for (re)activation-related proteins from Herpes simplex virus type 1,[30] Toxoplasma gondii,[30] Plasmodium falciparum,[30] Cryptococcus neoformans,[30] Cytomegalovirus (CMV),[31] [32] and severe acute respiratory syndrome coronavirus 2.[33]

Therefore, as a logical extension of such data, the issues of why and how EBNA1 protein synthesis is restricted/resumed during latency and (re)activation, and the role of EBNA1 GAR have been analyzed through the lens of the compliance to the human codon usage.

Materials and Methods

Codon usage analyses were conducted on the ORF of the EBNA1 gene from EBV B95-8 (www.ncbi.nlm.nih.gov/nuccore/V01555.2), a type 1 strain, which is prevalent worldwide.[34] EBNA1 ORF without the GAR sequence was obtained by deleting the genome nucleotide sequence position 108217–108924.

ORF of the human paired box PAX5 gene (NCBI accession: NM_016734.3, https://www.ncbi.nlm.nih.gov/nuccore/NM_016734.3) was used as a control as PAX5 protein is involved in EBNA1-driven transcription.[35]

Codon usage analyses were performed using GeneInfinity program (http://www.geneinfinity.org). Codon usage of the Homo sapiens ORFeome (40,662,582 codons) was obtained from the international DNA sequence database (http://www.kazusa.or.jp/codon/).³⁶ Codon usage for each codon is given as frequency per thousand. Amino acids (AA) are given in one- or three-letter code.

Results and Discussion

Human versus EBNA1 Codon Usage

[Fig. 1A–D] shows the frequency per thousand of the 61 codons in the human ORFeome and in the ORFs coding for the human control PAX5, EBNA1, and EBNA lacking the GAR nucleotide sequence, respectively. Numerically, data illustrated in [Fig. 1] are tabulated in [Supplementary Table S1].

Fig. 1 Codon usage of (A) human ORFeome, (B) human PAX5 ORF, (C) EBNA1 ORF, and (D) EBNA1 ORF without the nucleotide sequence coding for GAR.

Three main points stand out from [Fig. 1]:

• The codon usage of the human PAX5 ORF ([Fig. 1B]) complies with the codon usage of the human ORFeome ([Fig. 1A]), thus following the fundamental principle according to which each gene in a genome tends to conform to its species' codon usage pattern.[37] [38]

• In contrast, the codon usage of the ORF coding for EBNA1 is markedly different from that of the human ORFeome and is characterized by a heavily biased codon usage pattern ([Fig. 1C] vs [Fig. 1A]). In practice, coding of Gly and Ala in the long nucleotide sequence corresponding to EBNA1 GAR (i.e., position 108217–108924 in the EBV genome) is mostly delegated to three codons, i.e., GGG (Gly), GGA (Gly), and GCA (Ala), in front of the possible eight synonymous codons—four for each—that code for Gly and Ala.

• Deletion of the EBNA1 nucleotide sequence coding for GAR decreases the bias degree of EBNA1 codon usage pattern ([Fig. 1D] vs [Fig. 1C]).

Furthermore, it is of note that the codon usage of EBNA2, EBNA3, EBNA4, EBNA5, and EBNA6 showed, although to a lesser extent than that present in the EBNA1 ORF, a certain degree of codon bias compared with the human codon usage (see [Supplementary Fig. S1]).

Codon Usage Bias and tRNA Availability: Translational Regulation of EBNA1

The data displayed in [Fig. 1] provide a key for understanding the biochemical mechanism by which EBNA1 and its repeat allow the long-term persistence of the EBV genome. Indeed, according to a basic notion known since the 1980s,[37] [38] the degree of biased codon usage is proportional to the production levels of individual genes, with highly expressed genes using only a small subset of codons, i.e., exhibiting greater codon bias compared with poorly expressed genes. Therefore, theoretically, the highly biased EBNA1 codon usage ([Fig. 1C]) is apt to ensure an abundant production of EBNA1 protein. On the other hand, such EBNA1 codon optimization has no effect on the translational efficiency in the human host. Actually, Ikemura[39] and Ikemura and Ozeki[40] demonstrated that genes characterized by biased codon usage can be efficiently translated only in the presence of a specularly biased tRNA population. That is, codon usage and tRNA availability are functionally coadapted to each other in determining gene translation to protein.

In the case at issue, the profile of the tRNA pool quantitatively and qualitatively matches the pattern of the human codon usage, but not the highly biased codon usage of the viral EBNA1. In the human host, the foreign EBNA1 ORF has no chance to be translated by being unavailable the biased tRNA profile corresponding to the biased EBNA1 codon usage.

On this subject, it is worth recalling that viral ORFs characterized by suboptimal codon usage, i.e., populated by codons rarely used in the human codon usage, are likewise expected to remain untranslated in the host. This is the case of human CMV latency, which is characterized by restriction of viral protein synthesis.[32] In fact, in analyzing the molecular factors that hinder CMV expression in the human host, it was previously showed that the CMV genes frequently use six codons that are rarely used in the human host and that, in some instances, the rare host codons are clustered in viral nucleotide sequences coding for single AA repeats, thus posing extra translational constraints to CMV expression.[32]

Modifying the tRNA Pool: Cell Proliferation as a Primary Factor

In light of the above, the fundamental question of what is the mechanism that leads to resume EBNA1 protein synthesis and, consequently, determines EBV (re)activation becomes the following one: what is the mechanism able to change the host tRNA pool according to the EBNA1 translational needs? To this author's knowledge,[41] [42] [43] [44] [45] [46] [47] a primary process capable of removing the inhibition of EBNA1 translation is represented by cell proliferation.

Indeed, the tRNA profile sharply changes in the human host during cell proliferation induced, for example, by partial hepatectomy or cancer[41] [42] [43] [44] [45] [46] [47] so that different tRNA populations characterize quiescence and proliferation, with quantitative increases in minor tRNAs scarcely expressed during quiescence and, vice versa, decreases in tRNAs abundantly expressed during the quiescent phase.

An example of the tight relationship between proliferation and changes of the tRNA profile is illustrated in [Fig. 2] where a visual representation is given of the different tRNA patterns that characterize human gastric and colorectal carcinomas ([Fig. 2B,C]) compared with noncancer nonproliferating control tissue ([Fig. 2A]).[45] Of note, data from [Fig. 2] assume a relevant significance regarding the EBNA1 translation given that tRNAs^Ala and tRNAs^Gly represent the main tRNAs involved in the qualitative and quantitative changes of the tRNA profile in human gastric and colorectal tumors, as detailed by Kanduc et al.[45]

Fig. 2 HPLC profile of tRNA pools in gastric and colorectal tumors. tRNAs from: (A) control colon tissue from obese resected patient and (B) gastric cancer tissue and (C) colorectal cancer tissue from cancer patients. (Reproduced with permission from Kanduc et al.[45])

Then, [Fig. 2] might mechanistically explain the EBNA1 protein synthesis obtained by Yin et al[15] using human lung carcinoma cells and deleting EBNA1 GAR. That is, EBNA1 protein synthesis became possible since human lung carcinoma cells supplied the oncoproliferative cellular context for inducing tRNAs not available in the human quiescent host, while deletion of the nucleotide sequence coding for GAR modified the highly biased EBNA1 codon usage to a less biased and potentially more translatable pattern.

Hence, [Fig. 2] also poses the issue of the causal link between EBV infection and cancer. That is, since EBV and gastric cancer represent the most common form of EBV-associated neoplasm[48] and EBV is significantly associated with colorectal cancer,[49] from a logical point of view it is justified to hypothesize that the causal pathogenic role—currently attributed to EBV—should be ascribed to the carcinogenesis-associated proliferation. Indeed, by inducing tRNA patterns able to favor EBV translation, oncoproliferation might cause, in the following order: EBV protein expression, host's anti-EBV immune responses, cross-reactivity with host's proteins sharing peptide sequences with EBV,[27] [28] [29] and, as a logical final consequence, numerous diseases from lymphomas to lupus and multiple sclerosis.[28]

The fundamental role of cell proliferation in EBV (re)activation is also supported by the facts that: (1) EBV latency and lytic gene expression may be modulated by epigenetic mechanisms[4] such as DNA hypomethylation, a ubiquitous feature of cellular (onco)proliferation,[50] [51] [52] and (2) cellular and viral DNA hypomethylation are known to induce EBV lytic cycle,[53] and indeed azacytidine, which is a DNA methyltransferase inhibitor, rapidly activates the EBV lytic cycle.[54]

Conclusions

This study analyzes the factors that may underlie the inhibition of EBNA1 protein synthesis during latency and highlights the fundamental role of cell proliferation for adapting qualitatively and quantitatively the human tRNA pool to the translational needs of EBNA1.

Indeed, the requirement of functional coadaptation between the pattern of a gene codon usage and the abundance of tRNA species is so tight[39] [40] that, from an evolutionary point of view, it substantiates the concept that the codon usage pattern of the various (micro)organisms, tissues, and cells has been selected to be specifically adapted to the tRNA profiles of the (micro)organisms, tissues, and cells, instead of the tRNA pool having been adapted as a function of the codon usage profiles.[55] [56]

Clinically, the data exposed here might help understand the issue of EBV reactivation during pregnancy and in fetuses and newborns,[57] [58] [59] i.e., in rapidly proliferating organisms, as well as in subjects treated with immunosuppressive drugs, for example, after organ transplant. De facto, it is well known that glucocorticoids promote cell proliferation[60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] and, consequently, can induce tRNA changes favoring EBV (re)activation.

Immunologically, it is noteworthy that the mechanism of the adjuvants in active immunization consists in stimulating powerful B- and T-cell proliferation.[72] [73] [74] This means that adjuvant-induced proliferation might lead to changes in the cellular tRNA pools, thus opening the door to reactivation of latent and inherently harmless infections with consequent pathologic sequelae such as autoimmune cross-reactivity. This risk appears even more menacing considering that latent EBV infection is present in 95% of the human population. Then, according to the data discussed, prophylactic/therapeutic campaigns of anti-EBV vaccination would be possible only if based on the concept of peptide uniqueness, i.e., on peptides unique to the viral plural: proteins and absent in the human host.[75]

Conflict of Interest

None declared.

• References

• 1 Shannon-Lowe C, Rickinson A. The global landscape of EBV-associated tumors. Front Oncol 2019; 9: 713
• 2 Becnel D, Abdelghani R, Nanbo A. et al. Pathogenic role of Epstein-Barr virus in lung cancers. Viruses 2021; 13 (05) 877
• 3 Ayee R, Ofori MEO, Wright E, Quaye O. Epstein Barr Virus associated lymphomas and epithelia cancers in humans. J Cancer 2020; 11 (07) 1737-1750
• 4 Guo R, Gewurz BE. Epigenetic control of the Epstein-Barr lifecycle. Curr Opin Virol 2022; 52: 78-88
• 5 Sausen DG, Bhutta MS, Gallo ES, Dahari H, Borenstein R. Stress-induced Epstein-Barr virus reactivation. Biomolecules 2021; 11 (09) 1380
• 6 Bauer M, Jasinski-Bergner S, Mandelboim O, Wickenhauser C, Seliger B. Epstein-Barr virus-associated malignancies and immune escape: the role of the tumor microenvironment and tumor cell evasion strategies. Cancers (Basel) 2021; 13 (20) 5189
• 7 Kempkes B, Robertson ES. Epstein-Barr virus latency: current and future perspectives. Curr Opin Virol 2015; 14: 138-144
• 8 Price AM, Luftig MA. To be or not IIb: a multi-step process for Epstein-Barr virus latency establishment and consequences for B cell tumorigenesis. PLoS Pathog 2015; 11 (03) e1004656
• 9 Thorley-Lawson DA. EBV persistence—introducing the virus. Curr Top Microbiol Immunol 2015; 390 (Pt 1): 151-209
• 10 Murata T, Sugimoto A, Inagaki T. et al. Molecular basis of Epstein-Barr virus latency establishment and lytic reactivation. Viruses 2021; 13 (12) 2344
• 11 Frappier L. EBNA1. Curr Top Microbiol Immunol 2015; 391: 3-34
• 12 Yates JL, Camiolo SM, Bashaw JM. The minimal replicator of Epstein-Barr virus oriP. J Virol 2000; 74 (10) 4512-4522
• 13 Blake N, Lee S, Redchenko I. et al. Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing. Immunity 1997; 7 (06) 791-802
• 14 Levitskaya J, Coram M, Levitsky V. et al. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature 1995; 375 (6533): 685-688
• 15 Yin Y, Manoury B, Fåhraeus R. Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1. Science 2003; 301 (5638): 1371-1374
• 16 Apcher S, Komarova A, Daskalogianni C, Yin Y, Malbert-Colas L, Fåhraeus R. mRNA translation regulation by the Gly-Ala repeat of Epstein-Barr virus nuclear antigen 1. J Virol 2009; 83 (03) 1289-1298
• 17 Daskalogianni C, Apcher S, Candeias MM, Naski N, Calvo F, Fåhraeus R. Gly-Ala repeats induce position- and substrate-specific regulation of 26 S proteasome-dependent partial processing. J Biol Chem 2008; 283 (44) 30090-30100
• 18 Tellam JT, Lekieffre L, Zhong J, Lynn DJ, Khanna R. Messenger RNA sequence rather than protein sequence determines the level of self-synthesis and antigen presentation of the EBV-encoded antigen, EBNA1. PLoS Pathog 2012; 8 (12) e1003112
• 19 Coppotelli G, Mughal N, Masucci MG. The Gly-Ala repeat modulates the interaction of Epstein-Barr virus nuclear antigen-1 with cellular chromatin. Biochem Biophys Res Commun 2013; 431 (04) 706-711
• 20 Tellam JT, Zhong J, Lekieffre L. et al. mRNA structural constraints on EBNA1 synthesis impact on in vivo antigen presentation and early priming of CD8+ T cells. PLoS Pathog 2014; 10 (10) e1004423
• 21 Lista MJ, Martins RP, Billant O. et al. Nucleolin directly mediates Epstein-Barr virus immune evasion through binding to G-quadruplexes of EBNA1 mRNA. Nat Commun 2017; 8: 16043
• 22 Wilson JB, Manet E, Gruffat H, Busson P, Blondel M, Fahraeus R. EBNA1: oncogenic activity, immune evasion and biochemical functions provide targets for novel therapeutic strategies against Epstein-Barr virus-associated cancers. Cancers (Basel) 2018; 10 (04) 109
• 23 Deakyne JS, Malecka KA, Messick TE, Lieberman PM. Structural and functional basis for an EBNA1 hexameric ring in Epstein-Barr virus episome maintenance. J Virol 2017; 91 (19) e01046-e17
• 24 Lucchese G, Kanduc D. Cytomegalovirus infection: the neurodevelopmental peptide signatures. Curr Drug Discov Technol 2018; 15 (03) 251-262
• 25 Kanduc D. From hepatitis C virus immunoproteomics to rheumatology via cross-reactivity in one table. Curr Opin Rheumatol 2019; 31 (05) 488-492
• 26 Kanduc D. From anti-SARS-CoV-2 immune responses to COVID-19 via molecular mimicry. Antibodies (Basel) 2020; 9 (03) 33
• 27 Capone G, Calabrò M, Lucchese G. et al. Peptide matching between Epstein-Barr virus and human proteins. Pathog Dis 2013; 69 (03) 205-212
• 28 Kanduc D, Shoenfeld Y. From anti-EBV immune responses to the EBV diseasome via cross-reactivity. Glob Med Genet 2020; 7 (02) 51-63
• 29 Kanduc D. Proteome-wide Epstein-Barr virus analysis of peptide sharing with human systemic lupus erythematosus autoantigens. Isr Med Assoc J 2019; 21 (07) 444-448
• 30 Kanduc D. Human codon usage: the genetic basis of pathogen latency. Glob Med Genet 2021; 8 (03) 109-115
• 31 Kanduc D. Role of codon usage and tRNA changes in rat cytomegalovirus latency and (re)activation. J Basic Microbiol 2016; 56 (06) 617-626
• 32 Kanduc D. Rare human codons and HCMV translational regulation. J Mol Microbiol Biotechnol 2017; 27 (04) 213-216
• 33 Kanduc D. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): codon usage and replicative fitness. Glob Med Genet 2020; 7 (03) 92-94
• 34 Kanda T, Yajima M, Ikuta K. Epstein-Barr virus strain variation and cancer. Cancer Sci 2019; 110 (04) 1132-1139
• 35 Liu CD, Lee HL, Peng CW. B cell-specific transcription activator PAX5 recruits p300 to support EBNA1-driven transcription. J Virol 2020; 94 (07) e02028-e19
• 36 Nakamura Y, Gojobori T, Ikemura T. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res 2000; 28 (01) 292
• 37 Grantham R, Gautier C, Gouy M, Mercier R, Pavé A. Codon catalog usage and the genome hypothesis. Nucleic Acids Res 1980; 8 (01) r49-r62
• 38 de Boer HA, Kastelein RA. Biased codon usage: an exploration of its role in optimization of translation. In: Reznikoff W, Gold L. eds. Maximizing Gene Expression. Montvale, MA: Butterworth Publishers; 1986: 225-286
• 39 Ikemura T. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol 1981; 151 (03) 389-409
• 40 Ikemura T, Ozeki H. Codon usage and transfer RNA contents: organism-specific codon-choice patterns in reference to the isoacceptor contents. Cold Spring Harb Symp Quant Biol 1983; 47 (Pt 2): 1087-1097
• 41 Kanduc D, Farber E, Ghoshal A, Nagai M. Sequential alterations in tRNA population of 2-acetylaminofluorene-induced hepatocyte nodules. Biochem Biophys Res Commun 1993; 195 (03) 1309-1313
• 42 Kanduc D. Modulation of in vitro kidney cell growth by hepatic transfer RNAs. Biochem Biophys Res Commun 1996; 221 (03) 735-738
• 43 Kanduc D, Pagano G, Farber E. Changes in tRNA pattern in ethionine-induced rat putative preneoplastic hepatocyte nodules. Biochem Mol Biol Int 1996; 38 (06) 1191-1197
• 44 Kanduc D. Changes of tRNA population during compensatory cell proliferation: differential expression of methionine-tRNA species. Arch Biochem Biophys 1997; 342 (01) 1-5
• 45 Kanduc D, Grazia di Corcia M, Lucchese A, Natale C. Enhanced expression of initiator TRNA(Met) in human gastric and colorectal carcinoma. Biochem Mol Biol Int 1997; 43 (06) 1323-1329
• 46 Kanduc D, Basile AM, Nardelli M. Translational regulation: possible significance of differential tRNA expression during the transition from quiescence to proliferation. In: Bannasch P, Kanduc D, Papa S, Tager JM. eds. Cell Growth and Oncogenesis. Basel: Birkhauser Verlag; 1998: 247-256
• 47 De Pasquale C, Kanduc D. Modulation of HPV16 E7 translation by tRNAs in eukaryotic cell-free translation systems. Biochem Mol Biol Int 1998; 45 (05) 1005-1009
• 48 Young LS, Yap LF, Murray PG. Epstein-Barr virus: more than 50 years old and still providing surprises. Nat Rev Cancer 2016; 16 (12) 789-802
• 49 Salyakina D, Tsinoremas NF. Viral expression associated with gastrointestinal adenocarcinomas in TCGA high-throughput sequencing data. Hum Genomics 2013; 7 (01) 23
• 50 Ehrlich M. DNA hypomethylation in cancer cells. Epigenomics 2009; 1 (02) 239-259
• 51 Kanduc D, Ghoshal A, Quagliariello E, Farber E. DNA hypomethylation in ethionine-induced rat preneoplastic hepatocyte nodules. Biochem Biophys Res Commun 1988; 150 (02) 739-744
• 52 Kanduc D, Rossiello MR, Aresta A, Cavazza C, Quagliariello E, Farber E. Transitory DNA hypomethylation during liver cell proliferation induced by a single dose of lead nitrate. Arch Biochem Biophys 1991; 286 (01) 212-216
• 53 Szyf M, Eliasson L, Mann V, Klein G, Razin A. Cellular and viral DNA hypomethylation associated with induction of Epstein-Barr virus lytic cycle. Proc Natl Acad Sci U S A 1985; 82 (23) 8090-8094
• 54 Miller G, El-Guindy A, Countryman J, Ye J, Gradoville L. Lytic cycle switches of oncogenic human gammaherpesviruses. Adv Cancer Res 2007; 97: 81-109
• 55 Eigen M, Winkler-Oswatitsch R. Transfer-RNA, an early gene?. Naturwissenschaften 1981; 68 (06) 282-292
• 56 Eigen M, Winkler-Oswatitsch R. Transfer-RNA: the early adaptor. Naturwissenschaften 1981; 68 (05) 217-228
• 57 Purtilo DT, Sakamoto K. Reactivation of Epstein-Barr virus in pregnant women: social factors, and immune competence as determinants of lymphoproliferative diseases-a hypothesis. Med Hypotheses 1982; 8 (04) 401-408
• 58 Christian LM, Iams JD, Porter K, Glaser R. Epstein-Barr virus reactivation during pregnancy and postpartum: effects of race and racial discrimination. Brain Behav Immun 2012; 26 (08) 1280-1287
• 59 Haeri S, Baker AM, Boggess KA. Prevalence of Epstein-Barr virus reactivation in pregnancy. Am J Perinatol 2010; 27 (09) 715-719
• 60 Ninomiya E, Hattori T, Toyoda M, Umezawa A, Hamazaki T, Shintaku H. Glucocorticoids promote neural progenitor cell proliferation derived from human induced pluripotent stem cells. Springerplus 2014; 3: 527
• 61 Gündisch S, Boeckeler E, Behrends U, Amtmann E, Ehrhardt H, Jeremias I. Glucocorticoids augment survival and proliferation of tumor cells. Anticancer Res 2012; 32 (10) 4251-4261
• 62 Finlay CA, Cristofalo VJ. Glucocorticoid enhancement of cellular proliferation in vitro. In: Boynton AL, Leffert HL. eds. Control of Animal Cell Proliferation. Vol. II. San Diego, CA: Academic Press; 1987: 203-218
• 63 Phillips PD, Cristofalo VJ. Classification system based on the functional equivalency of mitogens that regulate WI-38 cell proliferation. Exp Cell Res 1988; 175 (02) 396-403
• 64 Rosner BA, Cristofalo VJ. Hydrocortisone: a specific modulator of in vitro cell proliferation and aging. Mech Ageing Dev 1979; 9 (5–6): 485-496
• 65 Neuberger TJ, Kalimi O, Regelson W, Kalimi M, De Vries GH. Glucocorticoids enhance the potency of Schwann cell mitogens. J Neurosci Res 1994; 38 (03) 300-313
• 66 Cai J, Zheng T, Lotz M, Zhang Y, Masood R, Gill P. Glucocorticoids induce Kaposi's sarcoma cell proliferation through the regulation of transforming growth factor-beta. Blood 1997; 89 (05) 1491-1500
• 67 Zheng Y, Izumi K, Li Y, Ishiguro H, Miyamoto H. Contrary regulation of bladder cancer cell proliferation and invasion by dexamethasone-mediated glucocorticoid receptor signals. Mol Cancer Ther 2012; 11 (12) 2621-2632
• 68 Rafacho A, Cestari TM, Taboga SR, Boschero AC, Bosqueiro JR. High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets. Am J Physiol Endocrinol Metab 2009; 296 (04) E681-E689
• 69 Atmani H, Chappard D, Basle MF. Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol. J Cell Biochem 2003; 89 (02) 364-372
• 70 Bourcier T, Forgez P, Borderie V, Scheer S, Rostène W, Laroche L. Regulation of human corneal epithelial cell proliferation and apoptosis by dexamethasone. Invest Ophthalmol Vis Sci 2000; 41 (13) 4133-4141
• 71 Kawamura A, Tamaki N, Kokunai T. Effect of dexamethasone on cell proliferation of neuroepithelial tumor cell lines. Neurol Med Chir (Tokyo) 1998; 38 (10) 633-638 , discussion 638–640
• 72 Chauhan N, Tiwari S, Iype T, Jain U. An overview of adjuvants utilized in prophylactic vaccine formulation as immunomodulators. Expert Rev Vaccines 2017; 16 (05) 491-502
• 73 Awate S, Babiuk LA, Mutwiri G. Mechanisms of action of adjuvants. Front Immunol 2013; 4: 114
• 74 Zhang R, Wang C, Guan Y. et al. Manganese salts function as potent adjuvants. Cell Mol Immunol 2021; 18 (05) 1222-1234
• 75 Kanduc D. Peptide cross-reactivity: the original sin of vaccines. Front Biosci (Schol Ed) 2012; 4 (04) 1393-1401

Publication History

Received: 21 April 2022

Accepted: 09 May 2022

Article published online:
15 September 2022