EBER EBV: Intracellular Pathways and Roles in Viral Pathogenesis

Epstein-Barr virus (EBV) is a widespread human herpesvirus linked to various malignancies and immune disorders. Among its key non-coding RNAs, Epstein-Barr-encoded RNAs (EBERs) play crucial roles in viral persistence and pathogenesis despite not encoding proteins. Their interactions with host cellular pathways significantly contribute to EBV-associated diseases.

EBER Transcription Mechanisms

EBERs, consisting of EBER1 and EBER2, are transcribed by RNA polymerase III, which synthesizes small non-coding RNAs. Their transcription is driven by internal promoter elements, specifically an A-box and B-box, which recruit transcription factors such as TFIIIC and TFIIIB. These factors facilitate RNA polymerase III binding to the EBER loci, ensuring robust transcription even in the absence of viral replication. Unlike many viral RNAs, EBERs are constitutively expressed in latently infected cells, reaching intracellular concentrations exceeding 10⁶ copies per cell, indicating strong regulatory mechanisms for stability and persistence.

Post-transcriptional modifications enhance EBER stability. Their secondary structures, with extensive stem-loop formations, resist exonuclease degradation. Additionally, EBERs associate with La protein, an RNA chaperone that stabilizes RNA polymerase III transcripts, further ensuring their longevity. Initially retained in the nucleus, EBERs are rapidly exported to the cytoplasm through a CRM1-dependent pathway, a common route for structured non-coding RNAs.

EBER transcription is influenced by viral and cellular factors. EBV-encoded nuclear proteins, such as EBNA1, modulate their expression, though precise mechanisms remain under investigation. Cellular stress conditions, including oxidative stress and hypoxia, upregulate EBER transcription, suggesting EBV exploits host stress responses. Epigenetic modifications, such as histone acetylation and DNA methylation, fine-tune transcriptional activity, ensuring consistent EBER production in latently infected cells.

Intracellular Distribution

EBERs exhibit dynamic intracellular localization that influences their function. Initially retained in the nucleus, they accumulate in nucleoplasmic regions rather than associating with chromatin or nucleoli. This transient nuclear residency suggests brief interactions with nuclear factors before export to the cytoplasm, where their functional roles expand.

Their export relies on the CRM1-dependent pathway, used by structured RNAs and viral transcripts. CRM1, also known as exportin 1, recognizes nuclear export signals and facilitates transport through the nuclear pore complex. EBERs achieve this by associating with La protein, which stabilizes them and aids in nuclear export. Their structured nature prevents passive diffusion, necessitating active transport.

In the cytoplasm, EBERs interact with ribonucleoprotein complexes and modulate cellular processes. Despite their cytoplasmic presence, they do not engage in translation, reinforcing their role as regulatory non-coding RNAs. Their accumulation in stress granules and processing bodies suggests involvement in RNA metabolism and post-transcriptional regulation, potentially influencing RNA stability and activity.

Molecular Interactions With Host Factors

EBERs interact with host cellular components, influencing RNA-binding proteins, immune pathways, and signaling networks, allowing EBV to manipulate host cell functions and promote viral persistence.

RNA-Binding Proteins

EBERs form stable complexes with RNA-binding proteins, most notably La protein, which stabilizes them by binding to their 3′ ends and shielding them from degradation. This interaction suggests EBV co-opts host machinery to maintain EBER function.

Beyond La, EBERs associate with hnRNP-K and PKR. Their binding to PKR inhibits its function, preventing eIF2α phosphorylation and circumventing host antiviral responses. This inhibition allows EBV-infected cells to evade translational repression, supporting viral latency. Additionally, EBERs interact with ribosomal proteins, potentially influencing ribosome assembly and RNA metabolism.

Immune Pathways

EBERs modulate immune signaling by engaging with components of the innate immune system. They bind to RIG-I, a cytoplasmic RNA sensor, triggering type I interferon production. However, in EBV infection, this response may contribute to chronic immune activation rather than viral clearance.

EBERs also influence Toll-like receptor 3 (TLR3) signaling, inducing pro-inflammatory cytokine secretion, including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). This may contribute to the tumor microenvironment in EBV-associated malignancies. Additionally, EBERs suppress apoptosis by modulating Bcl-2 family proteins, promoting infected cell survival.

Signaling Networks

EBERs influence intracellular signaling pathways, favoring viral persistence. They activate the PI3K/Akt pathway, which regulates cell survival and proliferation, promoting resistance to apoptosis. This activation is particularly relevant in EBV-associated cancers, where sustained PI3K/Akt signaling contributes to tumor progression.

EBERs also enhance JAK/STAT signaling, upregulating genes involved in cell survival and immune modulation. Persistent STAT activation may contribute to inflammation in EBV-associated diseases. Additionally, EBERs sustain NF-κB activity, reinforcing a pro-survival and pro-inflammatory state that supports EBV latency and pathogenesis.

EBER Roles In Viral Pathogenesis

EBERs enable conditions that support viral persistence and disease progression. Their expression across all EBV latency types suggests a fundamental role in maintaining infected cell viability, particularly in malignancies such as Burkitt’s lymphoma, nasopharyngeal carcinoma, and Hodgkin’s lymphoma. Unlike viral proteins targeted by the immune system, EBERs evade direct immune recognition, allowing persistence in infected cells.

EBERs correlate with increased tumor cell proliferation and resistance to apoptosis. They enhance the expression of genes linked to cell cycle progression, including those regulated by c-Myc, an oncogene frequently dysregulated in EBV-associated cancers. Additionally, their impact on epigenetic regulation suggests they influence gene expression patterns favoring long-term viral persistence.

EBERs In Chronic Infection States

EBERs contribute to EBV persistence in chronic infections by sustaining infected cell survival. Unlike lytic-phase viral genes, which are tightly regulated, EBERs remain abundant in latently infected cells. Their presence has been detected in conditions such as post-transplant lymphoproliferative disorders (PTLD), chronic active EBV infection (CAEBV), and autoimmune diseases like systemic lupus erythematosus (SLE).

EBERs influence cellular senescence and proliferation dynamics, altering the balance between pro-apoptotic and anti-apoptotic signaling. This resistance to programmed cell death enables EBV-infected cells to accumulate, increasing the likelihood of oncogenic transformation. Additionally, EBERs are found in exosomes released by infected cells, suggesting a role in intercellular communication. These extracellular vesicles may facilitate viral influence on uninfected cells, modulating gene expression and contributing to systemic effects in chronic EBV-associated diseases.

Techniques For EBER Detection

Detecting EBERs is essential for diagnosing EBV-associated diseases and understanding viral pathogenesis. Their high transcription levels make them reliable biomarkers for EBV latency, particularly in malignancies such as Hodgkin’s lymphoma and nasopharyngeal carcinoma.

In situ hybridization (ISH) remains the gold standard for detecting EBERs in tissue samples. This technique employs complementary RNA or DNA probes labeled with fluorescent or chromogenic markers, allowing pathologists to visualize EBER transcripts within individual cells. EBER-ISH is widely used in clinical diagnostics to confirm EBV-associated lymphomas.

Quantitative polymerase chain reaction (qPCR) provides a rapid and sensitive method for detecting EBER transcripts in blood, saliva, and tissue samples, allowing monitoring of EBV activity in acute and chronic infections.

Emerging technologies such as digital droplet PCR and RNA sequencing have further refined EBER detection. Digital droplet PCR enables absolute quantification of EBER copies, useful for tracking viral load in immunocompromised patients. RNA sequencing allows simultaneous analysis of EBERs and host transcriptomic changes, providing insights into their influence on cellular gene expression. Integrating these advanced techniques into clinical workflows continues to improve EBV diagnostics and understanding of EBER-mediated pathogenesis.