Cellular Factors Involved in the Hepatitis D Virus Life Cycle

Abstract

Hepatitis D virus (HDV) is a defective RNA virus with a negative-strand RNA genome encompassing less than 1700 nucleotides. The HDV genome encodes only for one protein, the hepatitis delta antigen (HDAg), which exists in two forms acting as nucleoproteins. HDV depends on the envelope proteins of the hepatitis B virus as a helper virus for packaging its ribonucleoprotein complex (RNP). HDV is considered the causative agent for the most severe form of viral hepatitis leading to liver fibrosis/cirrhosis and hepatocellular carcinoma. Many steps of the life cycle of HDV are still enigmatic. This review gives an overview of the complete life cycle of HDV and identifies gaps in knowledge. The focus is on the description of cellular factors being involved in the life cycle of HDV and the deregulation of cellular pathways by HDV with respect to their relevance for viral replication, morphogenesis and HDV-associated pathogenesis. Moreover, recent progress in antiviral strategies targeting cellular structures is summarized in this article.

Keywords: HBV; HDV; MVB; NTCP; RNA genome; morphogenesis; replication; surface protein.

Figure 1

Illustration of the viral particles of Hepatitis D and Hepatitis B virus. (A) Sizes of the respective particles are indicated by the bars shown. (B) S- and L-HDAg with their respective functional domains indicated by red boxes. Sites for methylation or farnesylation are indicated by red circles. RBD: RNA-binding domain; NLS: Nuclear localization sequence; NES: Nuclear export sequence; ARM: Arginine-rich motif.

Figure 2

Interactions with the host during entry and egress. (1) Initially, low-affinity interaction of S-HBs with HSPG is followed by the interaction of the preS1 domain and NTCP. Subsequent internalization of bound virus and NTCP with EGFR as cofactor occurs most likely in a clathrin-mediated manner. (2) The viral nucleocapsid is released from the endosome and transported into the nucleus where viral replication takes place (3) (see Section 7). Upon assembly of the viral ribonucleoprotein (RNP) around the gRNA of HDV, the RNP is exported from the nucleus and enveloped by budding into the ER lumen (4). The enveloped RNP is subsequently believed to be released through the secretory pathway, following the spherical SVPs of HBV (5). Pregenomic RNA of HBV is encapsidated in the cytosol by the core protein and subsequently enveloped by either budding into the ER lumen or directed towards MVBs through the interaction with a-taxilin and tsg101 (7). Similarly, nucleocapsids of HBV enveloped by budding into the ER lumen can be transported to MVBs and ultimately be released inside of exosomes (8). MVB-dependent or exosomal release of HDV RNPs is a possibility but remains to be investigated (7).

Figure 3

Interaction of HDV with the host during replication. Upon release of the viral RNP from the endosome, the RNP complex is transported into the nucleus through the nuclear pore complex (NPC) via interaction with karyopherin 2a (1). In the nucleus, S-HDAg recruits and modulates RNAP-II to facilitate mRNA transcription. Transcription is promoted by various host cell factors (2). The replication intermediate of HDV is generated upon shuttling of gRNA into the nucleolus, possibly via interaction with B23 and NCL (4), where RNAP-I is recruited and carries out the rolling circle replication with help of GAPDH (5). Next, auto-catalytic cleavage by the HDV ribozyme (6) and host-provided ligase generates quasi-double-stranded agRNA molecules (7), which either serve as template for the production of gRNA (8, 11) or as target for adenosine deaminase acting on RNA (ADAR1) (9) to generate the template for the L-HDAg mRNA (10, 12, 13). Synthesized S- and L-HDAg mRNA are likely exported through interaction with Aly/REF, the nuclear RNA export factor (NXF1) and other components of the Transcription export (TREX) complex (3). Translation of S- and L-HDAg in the cytosol might be assisted by eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) and recruitment of tRNA (14). Subsequent re-import of S- and L-HDAg into the nucleus and phosphorylation facilitated by different kinases as well as other post-translational modifications (PTMs) are responsible for the regulation and different functions of the HDAg isoforms (16). Farnesylation of L-HDAg is carried out by farnesyltransferase (FTase) and is essential for the interaction with HBsAg and subsequent release (15). S- and L-HDAg associate with the gRNA to form novel HDV RNPs (17).

Figure 4

Schematic representation of HDV interference mechanisms on HBV replication. HDV suppresses HBV RNA transcription likely through L-HDAg acting on HBV enhancer elements (1) and through inhibition of binding of the transcription factors STAT3/ HNF3 to HBV enhancer elements by S-HDAg (3). Recruitment of HBV cccDNA transcriptional repressors YY1 and Ezh2 by epigenetic modifications induced by HDV can be speculated (2). Interaction of S-HDAg with RNA polymerase II (RNAP II) increases error rate of RNA pol II and subsequently leads to mutations in HBV pregenomic RNA (pgRNA) (4). Interference of HBV HBsAg glycosylation, which is detrimental for HBV replication, by interaction of HDV with N-glycosylating enzymes can be speculated (5). HDV replication activates expression of the interferon-stimulated genes MxA, DDX60, ISG20 and TRIM22 (6). DDX60 degrades cytoplasmic HBV RNAs (7). ISG20 inhibits packaging of HBV pgRNA into HBV capsids (8). MxA, which can also be activated by L-HDAg only, inhibits HBV capsid assembly (9).

Figure 5

Schematic representation of cellular host factors involved in HDV pathogenesis. L-HDAg activates the proto-oncogene c-Jun by direct interaction and the proto-oncogene c-Fos via activating the serum response factor (SFR). C-Jun and c-Fos dimerize to form the transcription factor AP-1, which in turn is supposed to be involved in hepatocyte transformation (1). L-HDAg promotes the TGF-β signaling cascade by interacting with the TGF-β signal transducer SMAD3. Expression of the plasmogen-activator inhibitor type-1 (PAI-1), which promotes fibrosis and cirrhosis (2), and the transcription factor Twist, which might induce epithelial–mesenchymal transition leading to hepatocellular carcinoma (HCC) (3), are activated by this means. L-HDAg also promotes the TNFα signaling cascade, in particular expression of the master regulator of apoptosis NF-κB, which might induce hepatic injury (4). L-HDAg inhibits clathrin-mediated protein transport. For instance, internalization of the major iron transport protein, transferrin (5) and ligand-bound epidermal growth factor receptor (6) is inhibited by L-HDAg, leading to disrupted iron homeostasis (5) and constitutive cell proliferation (6). Both S- and L-HDAg inhibit autophagic flux, leading to autophagosome accumulation (7). L-HDAg activates the cellular host factors Ero1-α, CYP2E1, Nox4 and Nox1, which in turn increase cellular ROS level, leading to oxidative stress. Moreover, S-HDAg inhibits GSTP1 expression, which acts as a detoxifying enzyme, reducing cellular ROS level (8). L-HDAg transactivates DNA-methyltransferase 3b (DNMT3b) via activation of STAT3. DNMT3B in turn hypermethylates the E2F1 gene, which leads to downregulation of E2F1, a crucial mediator of apoptosis, and consequently to apoptosis inhibition (9). Further, S- and L-HDAg activate expression of clusterin, a molecular chaperone that is strongly associated with HCC metastasis upon overexpression. Clusterin activation is probably mediated by interaction of S- and L-HDAg with histone acetyltransferase, which in turn hyperacetylate the clusterin gene (11). L-HDAg interacts with the nuclear RNA export factor 1 (NXF1). Impairment of NXF1 function by the L-HDAg interaction leading to accumulation of cellular mRNAs can be speculated (12). Moreover, HDV replication in general activates signaling cascades, which are strongly associated with genome instability (10).