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 tRNAsAla and tRNAsGly 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]
