Hepatitis B Virus Maturation Is Sensitive to Functional Inhibition of ESCRT-III, Vps4, and γ2-Adaptin

Abstract

Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor ␥2-adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPase-defective Vps4A or Vps4B mutants, HBV assembly and egress were potently blocked. Each of the MVB inhibitors arrested virus particle maturation by entrapping the viral core and large and small envelope proteins in detergent-insoluble membrane structures that closely resembled aberrant endosomal class E compartments. In contrast, HBV subvirus particle release was not affected by MVB inhibitors, hinting at different export routes used by viral and subviral particles. To further define the role ␥2-adaptin plays in HBV formation, we examined the effects of its overexpression in virus-replicating cells. Intriguingly, excess ␥2-adaptin blocked HBV production in a manner similar to the actions of CHMP and Vps4 mutants. Moreover, overexpressed ␥2-adaptin perturbed the endosomal morphology and diminished the budding of a retroviral Gag protein, implying that it may act as a principal inhibitor of the MVB sorting pathway. Together, these results demonstrate that HBV exploits the MVB machinery with the aid of ␥2-adaptin.In order to be released from cells, nonlytic enveloped viruses must undergo budding from either the plasma membrane or intracellular membranes, followed by pinching off or fission. The fission event is facilitated by virus-encoded late assembly domains that recruit and hijack the cellular budding machinery of multivesicular bodies (MVBs) (3, 31). In the cell, MVBs have the unique ability to generate intraluminal vesicles that bud away from the cytosol, a process topologically equivalent to that of enveloped virus budding. Sorting of cellular proteins toward internal MVB vesicles for either degradation, lysosomal functions, or exosomal release requires the coordinated actions of at least three hetero-oligomeric complexes, referred to as ESCRT (endosomal sorting complex required for transport) complexes I, II, and III (ESCRT-I, -II, and -III) (1,2,20,35,40). They are sequentially recruited to the late endosomal membrane and drive the formation of MVBs. After sorting, the process is terminated by the AAA-type ATPase Vps4, which disassembles and thereby recycles the ESCRT machinery. The functional loss of individual subunits of this machinery results in a malformed, dysfunctional MVB known as the "class E compartment" (4, 9, 11).In recent years, intense research has begun to uncover how enveloped RNA viruses utilize components of th...

Ultrastructural Evidences of Hepatitis B Infection in Human Liver Biopsies Disclose Complex Assembly and Morphogenesis Pathways for Hepatitis B Virus

“…Nevertheless, results from this work showed that Dane's-like particles and SVP localize in different cellular compartments. This is in agreement with recent evidences showing that the viral and subviral HBV assembly pathways seemingly differ in their requirement for cell functions and trafficking routes [25,26] . While HBV virions budding required the involvement of MVB functions, the mechanisms of SVP production showed to be clearly distinct and likely independent of MVB functions [25,26] .…”

“…Most of these viruses access the MVB machinery through its virusencoded late assembly domains and bud through the plasma membrane. In addition, recent communications have reported the involvement of MVB functions in HBV virion maturation and egress using virusreplicating liver cell lines [25,26] . HBV virions have been suggested to bud into internal MVB-related compartments and exit the cell by the exosome pathway.…”

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

“…A DN ATPase-defective mutant of Vps4, in which the active site glutamic acid has been mutated to glutamine, leads to defective MVB sorting and formation of aberrant endosomes in BHK cells (Bishop & Woodman, 2000) and also in Huh7 cells. Production of infectious particles of HIV-1 (Garrus et al, 2001), HSV-1 (Crump et al, 2007), HBV (Lambert et al, 2007) and ASV (Pincetic et al, 2008) is inhibited upon expression of the Vps4DN protein. The wild-type or DN forms of Vps4, tagged with GFP (Vps4WT-GFP or Vps4DN-GFP) were transfected into Huh7 cells, the distribution of Vps4DN-GFP was distinct from Vps4WT ( Fig.…”

“…In the absence of Vps4 activity, the ESCRT complex cannot function and aberrant endosomes are formed. Numerous enveloped RNA viruses such as human immunodeficiency virus type 1 (HIV-1) (Garrus et al, 2001), Ebola virus (Martin-Serrano et al, 2001) and avian sarcoma virus (ASV) (Pincetic et al, 2008) and, more recently, enveloped DNA viruses including hepatitis B virus (HBV) (Lambert et al, 2007) and herpes simplex virus 1 (HSV-1) (Crump et al, 2007) have been shown to utilize ESCRT complexes during virion morphogenesis. In most cases viruses interact with the ESCRT machinery via late (L) domains, short motifs normally found in capsid or matrix proteins, which act as docking sites for ESCRT components.…”

Serological and molecular epidemiological outcomes after two decades of universal infant hepatitis B virus (HBV) vaccination in Nunavut, Canada

“…Positive-sense strand synthesis by viral polymerase and pregenomic mRNA degradation by a ribonuclease follows. The encapsidated viral genome may now be enveloped by a lipid envelope containing embedded HBsAg and be secreted from the host hepatocyte [47][48][49].…”