Dynamic Epstein-Barr virus gene expression on the path to B-cell transformation

Abstract

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus in the γ-herpesvirinae subfamily that contains a 170-180kb double-stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B-cell compartment of the peripheral blood. EBV can be reactivated from its latent state, leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome and structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady-state viral gene expression within EBV-immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection, EBV only expressed the well-characterized latency-associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation and delayed responses in the known latency genes. This chapter summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, the inhibition of apoptosis, and innate and adaptive immune responses.

Keywords: EBNA; Epstein–Barr virus; Herpesvirus; LMP; Latency; Lytic; Viral gene expression; Viral transformation.

Figure 1. Latency III gene expression in a Lymphoblastoid Cell Line

Schematic diagram of latency proteins and RNAs expressed at steady-state in EBV-transformed LCLs. The nucleus is depicted by the inner, gray-shaded dotted circle. The latent membrane proteins (LMPs) are depicted in the plasma membrane as monomers, but likely exist as multimers and signal from multiple cellular membranes. The EBNA proteins are all shown as nuclear, but may have functions in the cytoplasm as well (e.g. EBNA-LP).

Figure 2. Schematic of the Epstein-Barr Virus genome

Letters on the inner edge of the circular genome denote BamHI digestion fragments. Cis-acting elements within the genome, such as the origin of plasmid replication (oriP), the two origins of lytic replication (oriLyt) and the terminal repeats formed when the linear genome is circularized are denoted in blue squares. Lytic genes that appear to be active early after infection in the pre-latent phase are shown in orange boxes. Coding exons for the latency genes are shown in green boxes. EBV latent mRNAs can be initiated from different promoters depending on latency type and time after infection: the W promoter (Wp), the C Promoter (Cp), the Q promoter (Qp, only in Latency I), and the LMP promoters are labeled. The unspliced pre-mRNAs driven from these promoters is shown as a dotted line. EBV encoded noncoding RNAs, such as the miR-BHRF1 cluster, the miR-BART cluster, and the EBERs are shown as red triangles.

Figure 3. EBV latency mRNAs are expressed as alternative isoforms and distinct transcripts

At the top is a schematized EBV linear genome showing the positions of latency gene exons in black boxes and BamHI fragment names listed below (not to scale). Also shown on the genome are the terminal repeats (open boxes), the C promoter (Cp, green boxes), the W promoter (Wp, yellow box), the Q promoter (Qp, blue box), the bi-directional Latent Membrane Protein promoter (LMPp, purple boxes), the LMP2A-specific promoter (purple box), and the canonical EBNA poly-adenylation sites (pA, arrow). The ORF-containing exon of the lytic gene BHRF1 is shown as an orange box. All coding exons are shown as full height boxes while non-coding exons are half-height. Early after infection latency transcripts are initiated primarily from the W promoter, as shown in yellow. The special instance of alternative splicing between the upstream Wp or Cp splice donor and the W1 or W1′ exon that leads to inclusion of an ATG start codon and EBNA-LP protein production is shown (Inset). After EBNA2 and EBNA-LP production reach a significant level early after infection, the C promoter is activated and transcribes the rest of the EBNAs, as shown in green. Later in infection, the LMP promoters are active and LMP1, LMP2A, and LMP2B and transcribed and spliced as shown in purple. In Latency I only the Q promoter is active to transcribe EBNA1.

Figure 4. Timing of latent and lytic gene expression after infection by EBV

Relative expression levels of the RNA species are shown as shaded bars. Dark shading is indicative of the relative maximum amount of expression of the given RNA over the course of B cell growth transformation by EBV. Lytic genes expressed during the pre-latent phase are shown in orange, latency genes are shown in green, and noncoding RNAs are shown in red.