Ultrastructural analysis of hepatitis B virus in HepG2-transfected cells with special emphasis on subviral filament morphogenesis

Abstract

The intracellular accumulation of empty hepatitis B virus (HBV) particles of filamentous shape leads to a direct cytopathic effect in so-called ground-glass hepatocytes. The aim of this study was to investigate how these filaments can be structurally formed at the cellular level. By electron microscopy, we reexamined the HBV-producer HepG2T-14 cells, which have been described as producing a substantial amount of empty HBV filaments compared with the other forms of HBV particles. Examination of ultrathin sections of HepG2T14 cells revealed the presence of HBV virions and filaments at the periphery of extremely large intracellular cisternae, probably related to a pre-Golgi compartment. Very long filaments appeared to be formed by a tubular budding of a long portion of the cisterna membrane. This phenomenon may be identical to that observed in the hepatocytes of HBV chronic carriers, in which the inability of the infected cell to export long HBV filamentous particles through the cellular secretion pathway seems to be at the origin of a direct cytopathic effect. (HEPATOLOGY 1998;28: 1128-1133.)Hepatitis B virus (HBV) is an enveloped hepatotropic DNA virus that infects a large number of people worldwide. 1 Its genome is encapsulated with a virus-encoded polymerase in a nucleocapsid surrounded by a host-derived lipid envelope bearing three viral surface proteins. 2 The large (L) protein, containing the preS1, preS2, and S domains, is translated from the first start codon of the surface open reading frame. The middle (M) protein, containing the preS2 and S domain, and the small (S) protein are translated from in-frame start codons further downstream. 2 During synthesis, all three proteins are cotranslationally inserted into the endoplasmic reticulum (ER) as transmembrane proteins that can form disulfide bridges with each other. 3 The S protein traverses the ER membrane at least twice to form a cytosolic loop and a luminal domain. 4 The M protein has a similar topology with the additional N-terminal preS2 domain located in the ER lumen. 5 The L protein can adopt two different transmembrane topologies, allowing the exposure of the N-terminal preS domain at either the luminal or the cytosolic side of the ER membrane. 6 This latter form is essential during HBV morphogenesis to establish a protein-protein interaction between the cytoplasmic core particle and a specific sequence within the preS domain of the L protein. 7,8 One peculiarity of HBV morphogenesis compared with that of other viruses is that the surface proteins not only envelop the nucleocapsid to form a mature virion, they are capable of forming subviral empty spherical or filamentous particles in the absence of nucleocapsid. 2 Although the envelopes of all types of HBV particles contain the M and S proteins, filamentous and virion envelopes are richer in the L protein. 2 It is thought that during morphogenesis, the M and S proteins, in the absence of any other viral proteins, can bud into a post-ER/pre-Golgi compartment to form 20-nm spherical particles that ca...

Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles

“…The same may apply for HBV, because the different virus particle types were found within the cisternae of ER. [44][45][46] The excessive formation of a novel cellular compartment during the course of a DHBV infection is strong evidence for a virus-induced process. In line with this notion is that the ectopic expression of the HBV envelope protein L alone induces extensive reorganization of the hepatocellular endomembranes in transgenic mice, retention and accumulation of subviral particles, and cytotoxic demise of hepatocytes, leading to formation of hepatocellular carcinoma.…”

Hepatitis C virus ultrastructure and morphogenesis

“…tion of hepatoma cell lines with cloned hepatitis B virus (HBV) genomes has led to the establishment of an in vitro system for studying HBV morphogenesis (Roingeard et al, 1990;Roingeard and Sureau 1998), this approach has turned out to be very difficult for HCV (Bartenschlager and Lohman, 2000). Some virion-like structures were reported in HeLa (Mizuno et al, 1995) and HepG2 cells (Dash et al, 1997) transfected with a full-length HCV genome.…”

Vacuolization in hepatitis B virus-infected hepatocytes

“…Interestingly, they refer to the HepG2 T-14 cells, which have been previously described to contain very large vacuoles harboring a substantial amount of HBV envelope filaments. 8,9 Figure 1 shows a typical HepG2 T-14 cell, exhibiting a large vacuole in which HBV virions and associated subviral envelope filaments bud from the membrane towards the vacuolar lumen. As previously discussed, 9 it seems that these long HBV subviral envelope filaments are formed in the HepG2 T-14 cells by a tubular budding of the vacuole membrane where HBV envelope proteins are accumulated.…”

“…8,9 Figure 1 shows a typical HepG2 T-14 cell, exhibiting a large vacuole in which HBV virions and associated subviral envelope filaments bud from the membrane towards the vacuolar lumen. As previously discussed, 9 it seems that these long HBV subviral envelope filaments are formed in the HepG2 T-14 cells by a tubular budding of the vacuole membrane where HBV envelope proteins are accumulated. The discrepancy with the HuH-7 cells described by Foo et al might be explained by the difference in the cell system.…”