Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies

Abstract

In addition to infectious viral particles, hepatitis B virus-replicating cells secrete large amounts of subviral particles assembled by the surface proteins, but lacking any capsid and genome. Subviral particles form spheres (22-nm particles) and filaments. Filaments contain a much larger amount of the large surface protein (LHBs) compared to spheres. Spheres are released via the constitutive secretory pathway, while viral particles are ESCRT-dependently released via multivesicular bodies (MVBs). The interaction of virions with the ESCRT machinery is mediated by ␣-taxilin that connects the viral surface protein LHBs with the ESCRT component tsg101. Since filaments in contrast to spheres contain a significant amount of LHBs, it is unclear whether filaments are released like spheres or like virions. To study the release of subviral particles in the absence of virion formation, a core-deficient HBV mutant was generated. Confocal microscopy, immune electron microscopy of ultrathin sections and isolation of MVBs revealed that filaments enter MVBs. Inhibition of MVB biogenesis by the small-molecule inhibitor U18666A or inhibition of ESCRT functionality by coexpression of transdominant negative mutants (Vps4A, Vps4B, and CHMP3) abolishes the release of filaments while the secretion of spheres is not affected. These data indicate that in contrast to spheres which are secreted via the secretory pathway, filaments are released via ESCRT/MVB pathway like infectious viral particles. IMPORTANCEThis study revises the current model describing the release of subviral particles by showing that in contrast to spheres, which are secreted via the secretory pathway, filaments are released via the ESCRT/MVB pathway like infectious viral particles. These data significantly contribute to a better understanding of the viral morphogenesis and might be helpful for the design of novel antiviral strategies.T he human hepatitis B virus (HBV) is a spherical particle, 42 nm in diameter, consisting of an outer envelope and an inner icosahedral nucleocapsid. The nucleocapsid is formed by the core protein and harbors the viral genomic DNA. The HBV genome encodes at least four different open reading frames, coding for the viral polymerase, the core and the e antigen (HBcAg and HBeAg), the regulatory X protein (HBx), and the three different surface proteins (HBsAg): the large HBV surface protein (LHBs), the middle surface protein (MHBs) and the small surface protein (SHBs) (1). LHBs encompasses the PreS1 domain, the PreS2 domain, and the S domain, MHBs consists of the PreS2 and the S domain, and SHBs contains the S domain. These surface proteins are not only constitutive components of the envelope of viral particles but also assemble to capsid-free subviral particles lacking any viral genome having the shape of spheres and filaments (2) that are secreted in 1,000-to 100,000-fold excess relative to infectious viral particles. SHBs, the predominant part of these subviral particles, can assemble to 22-nm spherical particles. The incorporation ...

The Intracellular Cholesterol Transport Inhibitor U18666A Inhibits the Exosome-Dependent Release of Mature Hepatitis C Virus

“…Due to the relevance of the MVB system for the release of infectious viral particles and of filaments, an inhibitory effect on the release of these particles was observed. In contrast to this, the secretion of spheres that occurs dependent on the ER/Golgi via the constitutive secretory pathway was not affected, indicating that the observed effects of U18666A on the release of HCV are not due to an effect on the Golgi system (45). This was further confirmed by analyzing the subcellular distribution of ER and Golgi markers that is not affected by the presence of U18666A (see Fig.…”

Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application

“…There are three HBV envelope proteins, called large (L), middle (M), and small (S) (Figure 1), among which S is the predominant protein present in both virions and HBsAg particles and L is enriched relatively in virions and filaments and barely detectable in spheres [26]. In accordance with their relative abundance of L, virions and filaments appear to share a similar pathway of secretion involving the host cell endosomal sorting complexes required for transport (ESCRT) components and the multivesicular bodies (MVBs), whereas spheres are thought to be secreted via the constitutive secretory pathway of the host cell [27,28]. …”

“…To accomplish this selective virion formation, only the “correct” NCs, i.e., mature ones containing RC DNA, but not immature ones containing SS DNA or pgRNA, are selected for envelopment during virion formation (Figure 1) [3,4,5,6,8,29]. Mature NCs are thought to acquire the host-derived lipid bilayer studded with the viral envelope proteins via budding into the lumen of an intracellular membrane thought to represent MVBs [27,28,30] for extracellular secretion. As mentioned earlier, complete virions are known to be enriched (relative to HBsAg spheres) for the L protein, which along the S protein (but not the M protein), are required for envelopment of mature NCs and secretion of complete virions [31].…”

Formation of semi‐enveloped particles as a unique feature of a hepatitis B virus PreS1 deletion mutant

“…HBsAg‐containing particles after polyethylene glycol (PEG) precipitation were dialysed against STE buffer (0.1 mol/L NaCl, 0.01 mol/L Tris‐HCl, 0.001 mol/L EDTA, pH 8.0) and then layered on preformed gradients composed of 2 mL 10, 15, 20, 25 and 30% sucrose (w/w) in STE. Ultracentrifugation was performed in a Beckman SW41 Ti rotor at 40 000 rpm for 4 hours at 4°C . Fractions of 0.5 mL were collected from above, and their refractive indexes were measured to guarantee the fractionation.…”