Conquering the Nuclear Envelope Barriers by EBV Lytic Replication

Abstract

The nuclear envelope (NE) of eukaryotic cells has a highly structural architecture, comprising double lipid-bilayer membranes, nuclear pore complexes, and an underlying nuclear lamina network. The NE structure is held in place through the membrane-bound LINC (linker of nucleoskeleton and cytoskeleton) complex, spanning the inner and outer nuclear membranes. The NE functions as a barrier between the nucleus and cytoplasm and as a transverse scaffold for various cellular processes. Epstein-Barr virus (EBV) is a human pathogen that infects most of the world's population and is associated with several well-known malignancies. Within the nucleus, the replicated viral DNA is packaged into capsids, which subsequently egress from the nucleus into the cytoplasm for tegumentation and final envelopment. There is increasing evidence that viral lytic gene expression or replication contributes to the pathogenesis of EBV. Various EBV lytic proteins regulate and modulate the nuclear envelope structure in different ways, especially the viral BGLF4 kinase and the nuclear egress complex BFRF1/BFRF2. From the aspects of nuclear membrane structure, viral components, and fundamental nucleocytoplasmic transport controls, this review summarizes our findings and recently updated information on NE structure modification and NE-related cellular processes mediated by EBV.

Keywords: BFRF1; BGLF4 kinase; Epstein–Barr virus; nuclear egress; nuclear envelope modulation.

Figure 1

A diagram of EBV virion replication, production, and maturation. (A) An overview of the nuclear envelope (NE) structure with selected NE-associated proteins and nuclear pore complexes. The NE is composed of two lipid bilayers, inner and outer nuclear membranes (INM and ONM), and the nuclear lamina formed by lamin filaments. The nuclear pore complexes (NPCs) not only serve as gates for material transport, but also structurally hold the membranes and lamina together. The ONM, which is continuous with the ER, is characterized by cytoskeleton-associated nesprin proteins tethered by SUN1 and SUN2 in the INM. Emerin is an INM integrated protein that anchors on the nuclear lamina. (B) After EBV infects cells, the viral genome is injected into the nucleus through nuclear pore complexes. The linear genome is circularized into the episomal form through the terminal repeats and maintained as an episomal form in the latently infected cells. With chemical stimulations or reactivation signals, the viral transactivator Zta or Rta activates replication-related viral proteins and lytic viral DNA replication. Along with the production and assembly of viral capsid proteins, the viral genome is encapsidated into the preformed procapsid to form the mature nucleocapsid. Simultaneously, the viral BGLF4 protein kinase mediates the phosphorylation and partial disassembly of nuclear membrane-underlying nuclear lamina, allowing the access of nucleocapsids to the nuclear membrane. In our observations, the nucleocapsids then traverse through the nuclear envelope with the coordination of the viral nuclear egress complex BFRF1/BFLF2, cellular ESCRT machinery, and Nedd4-like ubiquitin ligases. The cytoplasm-distributed nucleocapsids may subsequently transport into the juxtanuclear “viral assembly compartment”. This specialized compartment, containing highly reorganized membrane structures and organelles, may provide a site for efficient tegumentation and secondary envelopment of the nucleocapsids. Finally, the mature virion is transported close to the cell margins and released from the infected cells by exocytosis. NPC, nuclear pore complex.

Figure 2

The regulatory functions and the conserved kinase motifs of EBV BGLF4 kinase. (A) BGLF4 is a virion-associated protein kinase. After virus infection or reactivation, the mainly nucleus-distributed BGLF4 regulates the cellular environment in many respects. The BGLF4-mediated phosphorylation of IRF3 and UXT is known to suppress the IRF3- and NFκB-mediated cellular suppression of virus replication. BGLF4 regulates the cellular chromatin or chromosome structure by phosphorylating TIP60, condensin, and topoisomerase. The expression of BGLF4 delays cell cycle progression and results in cell growth retardation. These regulations can provide an optimal cellular environment for efficient virus replication. Additionally, BGLF4 plays a crucial role in regulating virion production and maturation. BGLF4 phosphorylates the nuclear lamin A/C and the components of the nuclear pore complex (NPC). The phosphorylation of these structural components attenuates the nuclear envelope barriers and facilitates the transport of viral late proteins and subsequently the nuclear egress of nucleocapsids. In our recent observation, reorganization of the cytoskeleton may also facilitate the maturation and production of the cytoplasm-located nucleocapsids. (B) Conserved kinase motifs of BGLF4 were identified by alignment with eukaryotic protein kinases. There are 11 conserved motifs defined in eukaryotic protein kinases [63]. Only motifs I, II, VIb, VII, IX, and XI can be identified in the kinase domain (gray region) of herpesviral kinases [64]. I, ATP binding; II and VIb, catalysis; VII, Mg²⁺ chelating; IX, catalytic loop maintaining. SUMO interaction motifs (SIMs) at a.a. 37–40 and 344–350 and the NES at a.a. 342–359 were identified in a recent study [65]. Amino acids 386–393 were predicted to comprise a putative NLS [66]. However, our study showed that the alpha-helical structure, rather than the positive charges of these amino acids, is critical for BGLF4 nuclear targeting [43]. The putative secondary structure of BGLF4 was analyzed by the GOR secondary structure prediction program (http://www.expasy.ch/tools/ (accessed on 23 May 2012)). Random coils, alpha helices, and extended strands are indicated as thin lines, boxes, and arrows, respectively. The red boxes indicate the BGLF4 unique regions crucial for nucleoporin interaction and nuclear targeting. The hatched boxes underneath the predicted secondary structure indicate the critical regions (a.a. 290–313, 386–393, and 410–419) for BGLF4 nuclear targeting. The critical helical regions for BGLF4 nuclear targeting were also predicted by Protein Structure Prediction Server (PS)² (version 2; http://ps2v2.life.nctu.edu.tw/ (accessed on 23 May 2012)) using casein kinase 2 alpha 1 polypeptide (PDB accession number 3bqcA) as the template and shown in enlarged 3D images for a.a. 296–312 and a.a. 380–406. This figure is adapted and reorganized from our previous study published in Journal of Virology 2012, 86:8072-85 [43].

Figure 3

Hypothetical model of the EBV BGLF4-induced structural and functional changes of the nuclear pore complex. (Left part) Under physiological conditions, the nuclear import of NLS-containing proteins is mediated by an importin α-importin β complex and is dependent on RanGTP hydrolysis. (Right part) In the presence of BGLF4, BGLF4 is transported into the nucleus through direct interaction and phosphorylation of FG-NUPs, including NUP62 and NUP153, to dilate the nuclear pores. Simultaneously, BGLF4 also induces reorganization of microtubules and causes changes in the nuclear shape. The nuclear envelope becomes irregular, and the nuclear envelope-associated proteins, such as SUN1 and SUN2, are redistributed. Non-NLS-containing viral proteins can be transported into the nucleus at the same time through at least three different mechanisms: (1) dilated nuclear pores, (2) a microtubule reorganization-dependent mechanism, and (3) direct interaction with or phosphorylation by BGLF4. (4) At the same time, the nuclear import of canonical NLS-containing proteins is partly inhibited. This model is adapted from our previous paper published in Journal of Virology 2015, 89:1703-18 [44].

Figure 4

A hypothetical model of the interaction among BFRF1, cellular ESCRT components, and Nedd4-like ligases in vesicle formation and modulation of the nuclear membrane. (A) After EBV reactivation, membrane anchoring BFRF1 targets to the nucleus-associated membranes. BFRF1 potentially regulates the nuclear membrane through protein oligomerization. It then recruits Alix, Nedd4-like ubiquitin ligases, and, sequentially, other ESCRT components to generate vesicles derived from the double layer nuclear envelope (I), outer nuclear membrane (ONM), or from the inner nuclear membrane (INM) (II). These nuclear membrane-derived vesicles may contain some nucleoporins and other INM proteins, such as lamin A/C or emerin. Cooperating with other viral factors, such as BFLF2, the BFRF1-mediated vesicles may further recruit and pack some nucleocapsids for the subsequent nuclear egress. In addition, the expression of BFRF1 induces the formation of multilayered cisternal nuclear membranes (III), which may provide a more membranous structure for viral budding. ER, endoplasmic reticulum. NPC, nuclear pore complex. This model represents a refined version of BFRF1-ESCRT component interactions as analogously published in PLOS Pathogens 2012, e1002904 [99]. (B) Prediction of the 3D structural BFRF1, based on the HCMV 5DOB chain B. The EBV-specific region (ESR), consisting of amino acids 180–313, could not be predicted because of a lack of sequence similarity. The LD1 domain is shown in green, the LD2 domain is shown in blue, and the ID domain is shown in pink. The regions involved in BFRF1 interaction are indicated in the BFRF1 functional domains and in the 3D modeling. This figure is adapted and reorganized from a previous figure published in Journal of Virology 2020, 94: e01498-19 [103].

Figure 5

The hypothetical model for the cellular and viral regulation of EBV BFRF1-induced nuclear envelope (NE) modulation. The NE plays a pivotal role in cellular signaling and homeostasis. During EBV reactivation, the viral NE protein BFRF1 can modulate NE structure and form cytoplasmic vesicles by recruiting cellular ESCRT components and Itch ubiquitin ligase [99,119]. The ER/NE-derived vesicles provide nucleocytoplasmic transport for the nuclear egress of nucleocapsids (upper part). The presence of BFRF1 may mediate the blockage of late autophagic proteolysis for transported nucleocapsids in the cytoplasm [140]. Previously, we showed that BFRF1-induced vesicles potentially mediate the delivery and clearance of nuclear protein aggregates through autophagy proteolysis in the cytoplasm [120] (lower part). This information obtained may provide insights into the fundamental functions of the NE and knowledge of NE-related and protein aggregation diseases.