Hepatitis A Virus Genome Organization and Replication Strategy

Abstract

Hepatitis A virus (HAV) is a positive-strand RNA virus classified in the genus Hepatovirus of the family Picornaviridae It is an ancient virus with a long evolutionary history and multiple features of its capsid structure, genome organization, and replication cycle that distinguish it from other mammalian picornaviruses. HAV proteins are produced by cap-independent translation of a single, long open reading frame under direction of an inefficient, upstream internal ribosome entry site (IRES). Genome replication occurs slowly and is noncytopathic, with transcription likely primed by a uridylated protein primer as in other picornaviruses. Newly produced quasi-enveloped virions (eHAV) are released from cells in a nonlytic fashion in a unique process mediated by interactions of capsid proteins with components of the host cell endosomal sorting complexes required for transport (ESCRT) system.

Figure 1.

Quasi-enveloped and naked, nonenveloped hepatitis A virus (HAV) virions. Electron microscopic images of negatively stained quasi-enveloped virions (eHAV) (panels i–iv) and naked, 27 nm nonenveloped (panel v) virions recovered from supernatant fluids of infected Huh-7.5 cells. (From Feng et al. 2013; adapted, with permission.)

Figure 2.

Organization of the hepatitis A virus (HAV) RNA genome and processing of the polyprotein. (A) Genome organization, showing the 5′ covalently-linked VPg (3B) protein, 5′ untranslated region (5′UTR), the internal ribosome entry site (IRES), cre, 3′UTR, and 3′ poly(A) tail. The open reading frame (ORF) encoding the polyprotein is shown as a box, with cleavage sites leading to production of mature structural (P1 segment, 1A–1D, shaded) and nonstructural (P23 segment, 2B–3D, open) proteins. (B) Polyprotein processing. The primary cleavage between VP1pX (1D) and 2B is likely to occur cotranslationally. Processing of the nonstructural proteins results in multiple processing intermediates. The kinetics are not known in detail, but cleavages between 2B and 2C, and 3C and 3D appear to be favored (Martin and Lemon 2002). Most scission events are mediated by the 3C^pro cysteine proteinase (“*”). Exceptions are the VP4-VP2 cleavage, which is autocatalytic (“A”) and activated by packaging of the RNA genome, and excision of the pX domain from VP1pX after loss of the quasi-enveloped virion (eHAV) membrane, which is mediated by an unknown host protease (“H”).

Figure 3.

Secondary RNA structure within the positive-strand hepatitis A virus (HAV) RNA. (A) Secondary structure within the 5′ untranslated region (5′UTR) has been established by analysis of covariant substitutions in sequences of different hepatoviruses and nuclease mapping coupled with Mfold predictions (Brown et al. 1991a). Functional assays show that internal ribosome entry site (IRES) activity is dependent upon stem-loops III, IV, and V (Brown et al. 1991a). Nuclease mapping suggests the polypryimidine pY1 track is ordered, and biophysical studies suggest it may form several short stem-loops stabilized by noncanonical U-U and C-C base pairs (Hardin et al. 1999). The two initiator AUG codons are highlighted. (B) Secondary structure of the HAV cre, which is located in the 3D-coding region. It is essential for RNA replication and functions in a position-independent fashion (Yang et al. 2008). Adenines in the apical loop (circled bases) likely template slide-back uridylation of VPg. (C) Mfold prediction of secondary RNA structure in the 3′UTR (ΔG = −37.6). The UGA terminator codon is highlighted. Additional stem-loop structures are predicted to exist within the 3D^pol-coding sequence.

Figure 4.

RNA replication. Picornaviral replication is nonconservative, with more positive-strand RNA (+, solid line) than minus-strand RNA (–, dashed line) synthesized. The process begins after delivery of the (+)-strand genome to the cytosol and internal ribosome entry site (IRES)-directed translation has produced the nonstructural proteins required for replicase assembly. The incoming (+)-strand serves as template for initial synthesis of a (–)-strand, resulting in a full-length duplex double-stranded RNA (dsRNA). 3D^pol catalyzes RNA synthesis, but other viral and host proteins (HPs) are likely to be required for transcription. In a second step, the 5′ end of the (+)-strand dissociates from the (-)-strand in the duplex, allowing priming for synthesis of a new (+)-strand, a process that then repeats. Little if any single-stranded (–)-strand RNA is produced. Uridylated VPg-pUpU serves as the protein primer for both (–)-strand and (+)-strand synthesis in tightly coupled reactions, annealing with terminal adenines at the 3′ ends of both (+)-strand (see inset) and (–)-strand RNA. New (+)-strand RNA molecules are either packaged into nascent capsids, or recycled as a template for more (–)-strand synthesis.

Figure 5.

Confocal fluorescence microscopy showing viral RNA and capsid proteins in hepatitis A virus (HAV)-infected cells. Huh-7.5 cells were fixed on chamber slides at 14-days posttransfection of infectious HM175/p16 synthetic RNA. Double-stranded RNA (dsRNA) (red) was visualized by staining with a specific monoclonal antibody J2 (Scicons), and conformational HAV capsid antigen (green) by staining with JC serum (Feng et al. 2013). Most cells show foci where the two signals colocalize, but there is extensive segregation of dsRNA (RNA synthesis) and capsid antigen (assembled capsids). Nuclei have been counterstained with DAPI (blue).

Figure 6.

Biogenesis of extracellular quasi-enveloped (eHAV) and nonenveloped virions. Following assembly and packaging of the viral RNA, capsids are recruited to the surface of endosomes through VP2 and VP1pX interactions with components of the endosomal sorting complexes required for transport (ESCRT) system (apoptosis-linked gene 2-interacting protein X [ALIX] and others). Inward budding of the capsids into the endosome results in intraluminal vesicles (ILVs) and formation of multivesicular bodies (MVBs). The MVBs undergo transport to the plasma membrane, where membrane fusion leads to the release of membrane-wrapped eHAV virions to the extracellular space. eHAV virions that are shed from the basolateral hepatocyte membrane into hepatic sinusoids circulate in the blood, whereas eHAV secreted across the apical membrane undergoes conversion to naked, nonenveloped virions mediated by bile salts in the proximal biliary system.