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Epstein-Barr Virus (EBV)
persistence & latency

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Introduction

Epstein-Barr virus' persists in the host organism for lifetime of the host. The strategy of maintaining its continuous presence is two-fold: latent infection of long-lived cell type (memory B-cells) during which minimal number (transient latency) or none (true latency) of non-structural proteins are expressed and occasional reactivation followed by virus replication during which infectious virions infect replication-competent cells of the host organism and then, replenish viral reservoirs in latency-competent cells.

Thus, in the most simplistic interpretation, latency refers to an infected state where the cell contains intact epistomal (i.e. circular not linear) viral genome(s) but infectious virus is not produced and reactivation/replication refers to productive infection that results in virions (linear DNA packaged in capsid, tegumen and envelope) being released from the infected cell.

At the site of true latency, the virus is neither pathogenic to the host nor visible to the immune system. For all intents and purposes, the cell carrying the truly latent virus is healthy, as far as the host is concerned. However, transitional latency may contribute to pathogenesis.

Biological model of EBV persistence

The germinal center model of EBV infection

EBV uses normal B-cell biology to establish infection, persist and replicate.

EBV is spread by saliva. It enters the epithelium of Waldeyer's ring (the adenoids and tonsil) where it infects normal, resting, naive B-cells. Here it expresses the first of its transient latency states (growth program), where all of the latent genes are expressed driving activation and proliferation of B-cells giving rise to B cell blasts. In addition to promoting the cell proliferation, EBV is strongly antiapoptotic and some of the infected cells escape from apoptosis and enter follicle where their undergo a germinal center reaction switching to a second state of transient latency (default program) during which the differentiation of the infected B-cells into the undividing memory compartment occurs. The memory B-cells switch to state of true latency with no viral antigen being expressed. At this state the virus is invisible to immune system. However, expressed microRNAs are not ruled out and this is the biggest challenge to the concept of true latency as it is not yet clear how they can regulate otherwise completely normal host cell carrying dormant viral genome.

Virus in the state of true latency is invisible to the host's immune system. Transiently latent virus expresses up to 9 proteins and several non-translated RNAs. The non-translated RNAs are several microRNAs and EBV-encoded RNAs (EBER1 and EBER2), which have been suggested to protect EBV-infected cells from apoptosis. Of the six latent EBV proteins, 6 are nuclear antigens EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-LP) and three are membrane proteins LMP-1 and LMP-2A and LMP-2B. This limited antigen expression is one of the essential immune escape mechanisms of latent EBV infection. In addition to the reduced number of viral protein expression during latent infection, the copy number of the expressed EBV antigens and of the antigenic peptides processing for MHC presentation from these is also kept very low.

Reactivation of EBV from latently infected memory cells is thought to occur in response to normal physiologic signals that induce differentiation of memory cell into plasma cell. The most recent studies suggest that healthy carriers of the virus continually shed large quantities of infectious viral particles over their lifetime that cannot be accounted for by replication in plasma cells alone, suggesting amplification at a secondary site. There is evidence that this site is epithelium surrounding Waldeyer's ring. During lytic cycle EBV expresses more than 80 antigens. In healthy hosts, the replication program has to be transient, rapid, and relatively rare to minimize the chances to be shut down by host immune system. On the other hand, the virus has developed additional strategies to elude the immune response for successful generation of its progeny.

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Role of epithelial cells in viral life cycle

Epithelial cells of the orophatrynx have long been suspected in playing role in productive infection based on the following:

Epithelial cells are not involved in viral latency that in vivo is restricted to the hemopoietic system. It was convincingly demonstrated by the fact that allogenetic bone marrow transplantation from EBV-seronegative donor to EBV-seropositive recipient can wipe out viral latency in vivo. Also, importance of B lymphoid system for the life cycle of the virus is underlined by the fact that patients with X-linked agammaglobulinemia lacking mature B cells cannot be infected with EBV.

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Latency in immunosuppressed patients

There is now compelling evidence that persistent infection is controlled by a population of EBV-specific (largely CD8+) cytotoxic T lymphocytes (CTL). These recognize epitopes frequently derived from EBNA proteins 2, 3A, 3B, and 3C. Despite the sophistication of the antigen downregulation every infected individual develops T and B cells responses to the latent EBV antigens, and these are thought to keep persistent EBV infection in check and avoid EBV-associated malignancies in most infected people. During immunosuppression the number of latently infected cells in peripheral blood is markedly elevated and is accompanied by a concomitant increase in the amount of virus produced in the oropharynx. Cell sorting and PCR analysis of peripheral blood cells revealed that namely EBV-infected undividing memory B-cells (EBV genome only carrying cells), and not proliferating lymphoblasts (LMP-2A expressing cells), accumulate in the peripheral blood of immunosuppressed patients. This fact indicates that a dynamic equilibrium can be maintained that is stable over virus loads differing by several orders of magnitude before the system collapses. This illustrates extraordinary evolutionary adaptation of the virus to its host. A recent study showed that the pattern of LMP-2A (marker of on-going lymphoproliferation) expression appeared to be unchanged up to a virus load of 1,000 copies per 106 lymphocytes.

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Latent EBV gene expression programs

Latency program Genes expressed
0 EBERs
I EBERs, EBNA-1
II EBERs, EBNA-1, LMP-2A, LMP-1
III EBERs, EBNA-1, LMP-2A, LMP-1, EBNA-2, EBNA-3 (A, B, C), EBNA-LP

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Principal EBV latent proteins and their function

Gene products Description Mechanism of action Latency program B cell differentiation stage Malignancy
EBNA-1 Expressed in all proliferating EBV-infected cells.
Required for viral replication.
Increases survivability of B-cells.
Binds to origin of replication in viral genome.
Regulates transcription.
Destabilizes p53.
I, II, III Blast, Germinal center B-cell, Memory B cell Burkitt's lymphoma, pleural effusion lymphoma (PEL), Post-transplant lymphoproliferative disease (PTLD), Hodgkin's lymphoma, T-cell lymphoma, nasopharyngeal carcinoma, gastric carcinoma
EBNA-2 Regulates transcription of cellular and viral genes.
Essential for B-cell transformation.
Interacts with RBP-Jk transcription factor.
Activates transcription of anti-apoptotic genes.
III Blast Post-transplant lymphoproliferative disease (PTLD)
LMP-1 Integral membrane protein.
Classical oncogene.
Acts as constitutively active TNF receptor-like protein.
Activates NFkB, MAP kinase and PL3k anti-apoptotic pathways. II, III Blast, germinal center B cell Hodgkin's lymphoma, Post-transplant lymphoproliferative disease (PTLD), Hodgkin's lymphoma, T-cell lymphoma, nasopharyngeal carcinoma, gastric carcinoma
LMP-2A Integral membrane protein.
Not essential for B-cell transformation.
Mimic B-cell receptor signaling.
Activates MAP kinase and PI3k anti-apoptotic pathways.
II, III Blast Post-transplant lymphoproliferative disease (PTLD), Hodgkin's lymphoma
EBERs Small untranslated RNAs (EBV-encoded RNAs).
Very abundant in almost all EBV-infected cells.
EBER-1 binds to PKR and inhibits proapoptotic activity of this protein kinase.
Induce expression of autocrine growth-promoting cytokines.
0, I, II, III Blast, germinal center B cell, memory B cell Burkitt's lymphoma, pleural effusion lymphoma (PEL), Post-transplant lymphoproliferative disease (PTLD), Hodgkin's lymphoma, T-cell lymphoma, nasopharyngeal carcinoma, gastric carcinoma
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References