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Epstein-Barr Virus (EBV)
persistence & latency

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.

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.

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:

• The presence of the viral genome in epithelial cells of undifferentiated nasopharyngeal carcinoma (NPC);
• Early studies searching for the viral genome in normal tissue by in situ hybridization;
• The analogy to other lymphotropic herpesviruses;
• Replication of EBV in the lateral tongue epithelium of acquired immune deficiency syndrome (AIDS) patients with oral hairy leucoplakia.

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.

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.

Latent EBV gene expression programs

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Principal EBV latent proteins and their function

References

• Fox CP, Shannon-Lowe C, Rowe M. Deciphering the role of Epstein-Barr virus in the pathogenesis of T and NK cell lymphoproliferations. Herpesviridae. 2011 Sep 7;2:8.

  

• Ning S. Innate immune modulation in EBV infection. Herpesviridae. 2011 Jan 5;2(1):1.

  

  Elicitation of innate immune response in EBV infection. EBV dUTPase and GP350 act as ligands for TLR2. EBV EBERs can mount innate immune responses via both TLR3 and RIG-I signaling pathways. Infection of EBV also activates TLR9 signaling leading to IFNα production in pDCs. In latency, EBV manipulates the TLR7/IRF5 signaling pathway, which promotes cell proliferation. EBV products are indicated in red fonts.

  

  Evasion of immune responses by individual EBV products. Individual EBV products, including proteins, miRNAs, and EBERs, are shown to evade immune responses in both latent (upper) and lytic (lower) infections. For innate immune evasion, three main strategies are employed: (1) manipulating type I and II IFN Jak-STAT pathway; (2) regulating expression and activity of IRFs, and (3) repressing apoptosis signaling. EBV products are indicated in red fonts. PRD: positive regulatory domain; VRE: virus responsive element; ISRE: IFN-stimulated response element.