RESEARCH ARTICLE

HBV secretion is regulated through the activation of endocytic andautophagic compartments mediated by Rab7 stimulation

Jun Inoue1,2, Eugene W. Krueger1, Jing Chen1, Hong Cao1, Masashi Ninomiya1,2 and Mark A. McNiven1,*

ABSTRACT

The cellular mechanisms by which hepatitis B virus (HBV) is

assembled and exported are largely undefined. Recently, it has

been suggested that these steps require the multivesicular body

(MVB) and the autophagic machinery. However, the mechanisms by

which HBV might regulate these compartments are unclear. In this

study, we have found that by activating Rab7a, HBV alters its own

secretion by inducing dramatic changes in the morphology of MVB

and autophagic compartments. These changes are characterized by

the formation of numerous tubules that are dependent upon the

increase in Rab7 activity observed in the HBV-expressing

HepG2.2.15 cells compared to HepG2 cells. Interestingly,

transfection-based expression of the five individual viral proteins

indicated that the precore protein, which is a precursor of HBeAg,

was largely responsible for the increased Rab7 activity. Finally, small

interfering RNA (siRNA)-mediated depletion of Rab7 significantly

increased the secretion of virions, suggesting that reduced delivery of

the virus to the lysosome facilitates viral secretion. These findings

provide novel evidence indicating that HBV can regulate its own

secretion through an activation of the endo-lysosomal and

autophagic pathway mediated by Rab7 activation.

KEY WORDS: HBV, Rab7, MVB, Autophagosome, Tubule

INTRODUCTIONHepatitis B virus (HBV) infection is a global health problem as

infected individuals are at risk of chronic hepatitis, liver cirrhosis

and hepatocellular carcinoma. It is estimated that 350–400

million people are chronically infected with HBV (McMahon,

2005), despite the availability of effective vaccines. Although

nucleoside and nucleotide analogues and peginterferon are used

to prevent the progression as standard therapies (Rijckborst et al.,

2011), these drugs cannot eradicate HBV completely and their

efficacies are limited. Thus, a better understanding of the HBV

life cycle and the mechanisms by which it usurps established host

cell pathways is essential to enable the identification of new

antiviral targets.

The infectious HBV virion (or Dane particle) is a spherical

particle that is 42 nm in diameter. The virion consists of an inner

icosahedral nucleocapsid, which contains circular partially

double-stranded genomic DNA, and an outer envelope

composed of cellular lipids and three HBV surface proteins

[small (SHBs), medium (MHBs) and large (LHBs)]. These

surface proteins form also an excess of empty subviral particles

(SVPs). Although the mature nucleocapsid is known to originate

in the cytosol and contains the HBV core protein (HBc) (Bruss,

2007), the mechanisms by which the nucleocapsid is assembled,

enveloped within the endoplasmic reticulum (ER) membrane

(Eble et al., 1987) and trafficked to the cell surface for release are

poorly understood. Recently, a compartment of the late endocytic

pathway, the multivesicular body (MVB), has been shown to

participate in the final stages of HBV maturation and release. The

HBV appears to reside transiently in the MVB and disruption of

this compartment results in a decrease of released HBV (Kian

Chua et al., 2006; Lambert et al., 2007; Stieler and Prange, 2014;

Watanabe et al., 2007). Although the structural interactions

between the HBV and this organelle remain unclear, it appears

that virus buds inwards into invaginations of the MVB limiting

membrane, forming intraluminal vesicles (ILVs), a process that is

topologically equivalent to enveloped viral budding from the cell

surface (Prange, 2012). Subsequently, the MVBs are believed to

dock at the hepatocyte surface and release their intraluminal

viral cargo in a process that might resemble exosomal release

(Hanson and Cashikar, 2012). More recently, the autophagic

machinery has been implicated in HBV replication both

morphologically and functionally, as targeted knockdowns of

the autophagic proteins Atg5, Atg7 or Beclin1 reduce HBV

release (Li et al., 2011; Sir et al., 2010; Tian et al., 2011).

A physical and dynamic relationship is known to exist between

MVBs and autophagosomes as they appear to interact (Berg et al.,

1998; Fader et al., 2008) prior to fusion with a terminal lysosome

and exchange a variety of different components (Fader and

Colombo, 2009). The mechanisms that support the interactions

between these organelles are a topic of intense study, and the

processes by which the HBV utilizes these organelles to its own

end are almost completely undefined.

The Rab proteins are small GTPases that act as molecular

switches, orchestrating the formation, transport, tethering and

fusion of vesicles in the secretory and endocytic pathways. As for

most switch enzymes, the GTP-bound state of a Rab regulates its

binding to a variety of distinct effector proteins that facilitate

vesicular traffic (Stenmark, 2009). In humans, there are more than

60 Rabs, many with unknown functions, although Rab7 (for

which there are two isoforms, Rab7a and Rab7b) has been

demonstrated to play a central role in regulating endo-lysosomal

membrane traffic (Bucci et al., 2000; Cantalupo et al., 2001).

Rab7 is required for the maturation of late endosomes/MVBs and

also autophagosomes, directing the trafficking of cargos along

microtubules and participating in the fusion step with lysosomes

(Hyttinen et al., 2013; Vanlandingham and Ceresa, 2009). We

1Department of Biochemistry and Molecular Biology, and Center for BasicResearch in Digestive Diseases, Mayo Clinic, Rochester, MN 55905, USA.2Division of Gastroenterology, Tohoku University Graduate School of Medicine,Sendai, Miyagi 980-8574, Japan.

*Author for correspondence (mcniven.mark@mayo.edu)

Received 14 June 2014; Accepted 6 March 2015

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1696–1706 doi:10.1242/jcs.158097

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hypothesized that Rab7 could play an important role in theregulation of HBV assembly through the late endocytic pathway

of hepatocytes. In this study, we show that the HBV itself iscapable of activating Rab7 leading to the formation ofpronounced tubular networks extending from MVBs andautophagosomes. These tubules appear to promote the fusion of

MVBs, autophagosomes and lysosomes and facilitate thedegradation of HBV. These findings indicate that the HBValters the endo-lysosomal and autophagic pathways through the

regulation of a specific Rab protein and provides novel insightsinto host–pathogen interactions.

RESULTSHBV resides within markedly tubulated Rab7-associatedMVB compartmentsBased on the findings of others, HBV appears to utilize the MVB(Kian Chua et al., 2006; Lambert et al., 2007; Watanabe et al.,2007) and autophagic compartments (Li et al., 2011; Sir et al.,

2010; Tian et al., 2011) during assembly, maturation and release.As Rab7 is known to regulate the dynamics and traffic betweenthese late endocytic compartments and the lysosome (Cantalupo

et al., 2001; Jager et al., 2004; Stenmark, 2009), we testedwhether alterations in Rab7 expression and/or function might

Fig. 1. HBV components colocalize with MVBs and autophagosomes of virus-expressing hepatocytes. (A,B) HepG2.2.15 cells were transfected toexpress different GFP-tagged late endosome, MVB, lysosome or autophagosome markers including Rab7 (GFP–Rab7wt), GFP–LC3 and GFP–LAMP1 (green).Cells were double labeled with antibodies to two HBV antigens including anti-PreS1 of LHBs (A) or an anti-HBc antibody (red) (B), then analyzed by confocalmicroscopy. The viral antigens can be found in small putative MVB compartments (Rab7-positive) but appear most prominent in large autophagosomeorganelles positive for LAMP1 and LC3. Scale bars: 10 mm. (C,D) Transmission electron microscopy (TEM) of HepG2.2.15 cells showing electron-dense virus-like particles (arrows) in MVBs (C) and in a large autophagosome (AP) (D).

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interfere with the viral life cycle. First, HepG2.2.15 cells, whichstably express HBV, but are not re-infectable with released virus,

were transfected with GFP-tagged wild-type Rab7 (GFP–Rab7wt)and the relationship between the virus and the Rab7-positivecompartment was assessed by confocal microscopy. Rab7 localizedalong numerous large punctate late endosomes and MVBs

that also contained large quantities of HBV proteins (LHBs andHBc; Fig. 1A,B). As expected, mCherry-tagged Rab-interactinglysosomal protein (RILP), which is a classic effector protein of

active Rab7 and has been used to visualize active Rab7 in the cell(Cantalupo et al., 2001), also colocalized with these HBV proteins(supplementary material Fig. S1A). We further confirmed that

HBV proteins reside within MVBs and other late endosomal

structures by colocalization studies of these viral antigens incompartments positive for the GFP-tagged autophagosome marker

LC3 (also known as MAP1LC3) and GFP-tagged LAMP1 as alysosomal marker (Fig. 1A,B). In addition, cells were stained withantibodies against the MVB marker Hrs, the tetraspanin MVBmarker CD63 or the autophagosomal protein LC3. Cells were then

co-stained with for the viral LHBs (supplementary material Fig.S1B–D). In addition, transmission electron microscopy (TEM)images of HepG2.2.15 showed numerous electron-dense virus-like

particles of 40 nm in diameter residing within both MVBs andautophagosome compartments (Fig. 1C,D). However, these virus-like structures were not observed in the untransfected HepG2 cells

(data not shown). These particles do not look like the typical Dane

Fig. 2. HBV-producing cells possess highly tubulated MVB-autophagosome membrane networks containing viral antigens. (A,B) Parental HepG2 cellsand HepG2.2.15 cells (A9,A0,B9,B0) were transfected with GFP–Rab7wt (A) or GFP–LC3 (B) and analyzed by confocal microscopy. The MVB andautophagosome compartments are spherical and punctate in the parental cells (A,B), but exceptionally large tubules can be seen extending from Rab7- andLC3-positive compartments (arrows) in the virus-producing cells. Rab7-positive tubules (A-) and LC3-positive tubules (B-) in HepG2 and HepG2.2.15 cells werequantified in three independent experiments. The mean6s.e.m. (n53) percentage of cells with Rab7 tubules in Rab7-transfected cells, and the percentage ofcells with LC3-positive tubules in cells with LC3 puncta are shown. (C) HepG2 cells were transfected with 1.3-fold HBV full genome and GFP–LC3 and analyzedby confocal microscopy. Arrows indicate LC3-positive tubules. Scale bars: 10 mm. (D,E) TEM images of MVBs in virus-expressing HepG2.2.15 that extendmultiple tubules of uniform diameter (arrows). (F) Quantification (n511 cells per condition) of MVB tubules in HepG2 and HepG2.2.15 cells in TEM imagesreveals a tenfold increase of tubules in virus-producing cells compared to parental control cells. **P,0.01; ***P,0.001.

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particles, which are observed with negative staining. It wasconsidered that this is due to the difference in staining technique.

Surprisingly, although some HBV-expressing cells displayednormal spherically shaped MVBs and autophagosomes containingviral proteins (Fig. 1A,B), many possessed large and very longRab7- or LC3-positive tubules extending from these compartments,

which were not found in control cells (Fig. 2A,B; supplementarymaterial Fig. S1E,F). The tubules were also observed in HepG2cells transfected with 1.3-fold HBV full genome (Fig. 2C). In many

instances, these tubules interconnected to form reticular networksthat could also be resolved by TEM (Fig. 2D–F; supplementarymaterial Fig. S1G–J). The viral protein (LHBs) was observed

in these tubules (supplementary material Fig. S2A). Importantly,HBV infection also induced the formation of Rab7 tubules inprimary human hepatocytes (supplementary material Fig. S2B–E),

indicating that this dramatic response was not an aberration thatoccurs only in a neoplastic cell line.

To test whether other Rab-centric endocytic pathways arealtered by HBV infection, HepG2.2.15 and HepG2 cells were

transfected to express GFP-tagged Rab5 or Rab11. We speculatedthat changes in these compartments within the infected cells thatwould represent activation of these Rab proteins similar to that

observed for Rab7. No changes in these compartments wereobserved in infected versus control cells. Because it is thoughtthat MVB functions are associated with the HBV life cycle, we

investigated whether there were changes in the distribution ofendosomal sorting complexes required for transport (ESCRT)proteins. As well as staining for Hrs, GFP–Tsg101 (ESCRT1),

GFP–Vps25 (ESCRT2) or RFP–CHMP6 (ESCRT3) wasexpressed in these cells but no differences in their distributionwas observed (data not shown).

HBV infection increases Rab7 activity and subsequenttubulation of the MVB and autophagosome compartmentsTo test whether the dramatic morphological changes observed inthe late endocytic compartments of HBV-producing cells (Fig. 2)required the participation of Rab7, both control HepG2 and virus-expressing HepG2.2.15 cells were treated with Rab7-specific

small interfering RNA (siRNA) to reduce the levels of this smallGTPase. The efficiency of Rab7 depletion was good (seeFig. 5A). Indeed, we observed in TEM images very little MVB

tubulation following Rab7 depletion (Fig. 3A,B). As assessed byconfocal microscopy, Rab7-depleted cells had significantly lessLC3-positive tubules extending from LC3-positive organelles

than control cells, suggesting that these membrane dynamicsrequired functional Rab7 (Fig. 3C,D).

To test whether the observed Rab7-dependent tubule formation

was a result of a specific interaction with its effector protein RILP,which is known to bind active Rab7 and mediate an interactionwith microtubule-based motors (Jordens et al., 2001; van der Kantet al., 2013), we reduced RILP levels in HepG2.2.15 cells by

siRNA-mediated knockdown (supplementary material Fig.S2F,G). This manipulation resulted in a significant reduction inRab7-dependent tubule formation and induced a vesiculation of the

Rab7 compartment compared to infected control cells(supplementary material Fig. S2H–J). Additionally, expression ofexogenous mCherry-tagged RILP also reduced tubule formation

substantially (supplementary material Fig. S2K,L). This responsewas seen both upon expression of the wild-type protein, an effectwe observe often with an overexpression of tagged proteins, and

was accentuated by expression of a tagged Rab7-binding domain(RBD, amino acids 241–320). Interestingly, expression of an RILPC-terminal deletion protein (amino acids 1–220, supplementary

Fig. 3. The formation of membranous tubules extending from MVBs and autophagosomes in HBV-expressing cells is dependent upon Rab7 andfacilitates fusion with the lysosome. (A) TEM images of HepG2.2.15 cells treated with non-targeting (NT) control siRNA or Rab7 siRNA. Representativeimages of MVBs from NT control and Rab7-depleted cells are shown. Note the MVB with extending tubules (arrows) in NT siRNA-treated cells, whereasvirus-producing cells with reduced levels of Rab7 have spherical MVBs with no tubulation. (B) Quantification (n512 cells per condition) of MVBs with tubules percell in control and Rab7-deficient cells. (C) Rab7 is also required for the dramatic tubulation of the LC3-positive autophagic compartments. HepG2.2.15 cellswere transfected with NT siRNA or Rab7 siRNA, and subsequently transfected to express GFP–LC3. Cells were fixed after 48 h and analyzed with by confocalmicroscopy. Cells without Rab7 do not show autophagosome tubulation. (D) Percentage of cells with LC3 tubules emanating from LC3 puncta, and numbers oftubules per cell with LC3 puncta are quantified in HepG2.2.15 cells treated with NT siRNA or Rab7 siRNA in three independent experiments. ***P,0.001.

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material Fig. S2K) restored Rab7-based MVB tubulation inHepG2.2.15 cells substantially to levels seen in cells that had not

been transfected with exogenous RILP (supplementary materialFig. S2L), suggesting that expression of a 79 amino acid fragment(amino acids 241–320) makes the protein inert and unrecognizableby the cellular machinery. Taken together, these findings are

consistent with the premise that a Rab7–RILP complex mediatesHBV-activated MVB tubulation.

As the tubulation of late endocytic compartments is between

three and ten times more prevalent in the virus-expressing versuscontrol cells (Fig. 2) and is dependent upon Rab7 expression(Fig. 3), we tested whether the HBV is responsible for its

activation. To this end, Rab7 activity in HepG2.2.15 cells wascompared with that in HepG2 cells using a GST–RILP pulldownassay. Surprisingly, HepG2.2.15 cells had five to eight times

higher Rab7 activity than HepG2 control cells (Fig. 4A,B)

although there was no difference in total Rab7 levels.Furthermore, HBV from HepG2.2.15 supernatant was added to

primary human hepatocytes and also increased Rab7 activity aswell as did the secreted viral protein HBsAg, which was detectedby an enzyme-linked immunosorbent assay (ELISA; Fig. 4C,D).To define the HBV component responsible for this dramatic

activation, HuH7 cells, which have a higher transfection efficiencythan HepG2 cells, were transfected individually with FLAG-taggedvariants of the five distinct proteins encoded by the HBV genome

[polymerase, LHBs, HBc, precore, and HBVx protein (HBx)].Interestingly, the precore protein alone was sufficient to increaseRab7 activity (by fourfold; Fig. 4E,F), although the expression

level was significantly lower than that of HBc and HBx and similarto LHBs (supplementary material Fig. S2M). The precore protein isa precursor of HBeAg, and the processed form of HBeAg, in which

both N-terminus and C-terminus are cleaved (supplementary

Fig. 4. Rab7 is activated by HBe, a specific gene product of theHBV. (A,B) Cell lysates of HepG2.2.15 cells or parental HepG2 cellswere subjected to a GST–RILP pulldown assay to assess Rab7 activity.RILP pulldowns from three independent experiments were subjected towestern blot analysis and quantification reveals a marked five toeightfold increase in Rab7 activity in the virus-producing versus parentalcells. (C) As a second model of HBV infection, primary humanhepatocytes (PHHs) were incubated with supernatant from HepG2.2.15cells for 4 h, washed and 7 days later cells were harvested to determineRab7 activity using a GST–RILP pulldown assay. The HBsAg levels inthe supernatant were determined with ELISA. (D) A quantification ofactive Rab7 as a proportion of total Rab7 for the experiment described inC, shows a greater than twofold increase in Rab7 activity above infectedcontrol cells. (E) The precore protein, a precursor of HBeAg, activatesRab7. Lysates of HuH7 cells, previously transfected to express fivedistinct FLAG-tagged HBV proteins (polymerase, LHBs, HBc, precoreand HBx), were subjected to GST–RILP pulldown assays to test for thecomponent that induces the highest Rab7 activity. (F) Quantification ofactive Rab7 levels in three independent experiments as described in Erevealed that cells expressing the viral precore protein activated four tofive times more Rab7 than the other expressed viral components.(G,H) To test whether the precore protein might activate Rab7 in otherepithelial cells, Hep3B or HeLa cells were also transfected with FLAG-tagged precore protein, lysed and subjected to a GST–RILP pulldownassays (G). A quantification of active Rab7 as a proportion of total Rab7in three independent experiments indicates that the precore proteinalone markedly activates Rab7 in other epithelial cells, including non-hepatocytes such as HeLa (H). Pol, polymerase; PreC,precore. *P,0.05.

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material Fig. S2N). To further confirm the Rab7-activating

capacity of this viral antigen, the FLAG-tagged precore proteinwas expressed in two additional epithelial cell models (Hep3B andHeLa). This protein significantly increased Rab7 activity in both

cell lines (Fig. 4G,H), and also the processed form of HBeAgincreased Rab7 activity in HuH7 cells (supplementary material Fig.S2N–P). Thus, our data suggests that precore- and HBe-induced

Rab7 activation promotes tubulation of late endosomes, MVBs andautophagosomes, which might help regulate virus maturation andsubsequent release. The specific knockdown of the precore proteinis technically difficult as an siRNA targeting the precore mRNA

can also affect the expression of core protein and polymerase.

Rab7 depletion increases HBV secretionAs HBV infection results in a profound remodeling of the Rab7-associated MVB and autophagosome compartments in cells itseemed likely that these changes could affect HBV assembly,

intracellular trafficking, and secretion from cells. To test this,Rab7 was depleted in HepG2.2.15 cells by siRNA and the levels

of internal viral proteins were assayed by western blot analysisand the level of secreted virus was assayed by real-time PCR.Surprisingly, we observed that Rab7 depletion induced athreefold increase in the intracellular accumulation of LHBs

(Fig. 5A,B) and also significantly increased the release of HBVDNA into the supernatant (Fig. 5C). To confirm that the increasein HBV DNA released from Rab7-depleted cells represents intact

enveloped virus, HepG2.2.15 cells were transfected with FLAG–HBs and the supernatant was subjected to an immunoprecipitationwith anti-FLAG antibody and then subjected to PCR for the HBV

genome. The rationale for this approach was that the tagged HBscould associate with and subsequently precipitate the viral genomefor detection only if packaged into a virion. As shown in Fig. 5D,

the levels of HBV DNA post-immunoprecipitation were alsoincreased in the supernatant from Rab7-depleted cells, anindication that Rab7 depletion increases the secretion ofenveloped HBV particles. In control experiments, the expression

of FLAG-tagged mock protein or an addition of control antibodydid not increase the HBV DNA levels after immunoprecipitation.There was no significant difference in the amount of HBsAg in the

supernatant from cells with or without Rab7 depletion (data notshown), suggesting that the secretion pathways of HBV particlesand HBsAg subviral particles (SVPs) are different, as reported

previously (Garcia et al., 2009), and Rab7 does not affect thesecretion pathway of SVPs. The expression of a GTPase-defectiveRab7 mutant (Rab7T22N) also increased the amount of

intracellular LHBs and released viral genome, as with the Rab7knockdown (Fig. 5E,F). Additionally, the expression of mCherry–RILP reduced the secretion of HBV DNA in the supernatant but theeffect was not seen after Rab7 knockdown (supplementary material

Fig. S3A,B).To further define the role of Rab7 function in the HBV life

cycle, ‘rescue’ experiments were performed in which GFP–

Rab7wt or GFP–Rab7T22N were re-expressed in the Rab7-depleted cells. As expected, we observed that GFP–Rab7wt re-expression, but not that of GFP–Rab7T22N, restored the LHBs

level (supplementary material Fig. S3C,D) as well as thecolocalization of LHBs and LAMP1 (supplementary materialFig. S3E,F). In addition, the reduced number of LC3-positivetubules seen after Rab7 depletion were restored by the expression

of FLAG–Rab7wt (supplementary material Fig. S3G–I). Thesedata provide additional support for the concept that the effects ofthe Rab7 depletion on HBV production and localization were not

due to off-target effects. More importantly, the viruses secretedfrom the Rab7-depleted cells appear to represent infectiousparticles because HuS-E/2 cells, which are susceptible to HBV

infection (Huang et al., 2012), show equal infectivity in responseto virus collected from either knockdown or control cells(supplementary material Fig. S4A–C). A successful infection

was confirmed by the gradual increase of HBV DNA in thesupernatant from HuS-E/2 cells (supplementary material Fig.S4A).

From the findings described above, we hypothesized that the

effect of decreased Rab7 levels leading to an increase in bothinternal and secreted virus could be a result of altered traffickingto the degradative lysosome. To test whether this transport occurs

in live cells, we analyzed HepG2.2.15 cells expressing GFP–LC3and mCherry–LAMP1 by confocal microscopy. As depicted inFig. 6A, time-lapse images revealed that LAMP1 puncta were

tethered to and then fused with LC3-positive tubules or,

Fig. 5. Inhibiting Rab7 function induces a marked increase in HBVsecretion. (A) HepG2.2.15 cells were transfected with non-targeting (NT)control siRNA or Rab7 siRNA, and 3 days after the transfection, the levels ofLHBs in the cells were determined by western blot analysis. Note that Rab7depletion substantially increases the amount of intracellular LHBs.(B) Quantification of LHBs in five independent experiments as described in Aconfirm a greater than threefold accumulation of LHBs protein in Rab7-deficient cells. (C) As a measure of viral secretion, total HBV DNA recoveredfrom the supernatant of cells with or without Rab7 depletion was determinedby real-time PCR in five independent experiments and showed asubstantial increase (.threefold) in the amount of viral genome released.(D) Rab7 depletion increases the release of intact enveloped virion. Rab7was knocked down in HepG2.2.15 cells, followed by transfection of FLAG–HBs 24 h later. Two days following the transfection procedure, envelopedHBV in the supernatant was precipitated with the anti-FLAG antibody and thelevels of HBV DNA from the precipitates were determined by real-time PCR.Three independent experiments revealed a significant increase in therelease of enveloped HBV from the cells with reduced Rab7 levels.(E,F) Inactive Rab7 increases HBV secretion. HepG2.2.15 cells weretransfected twice with GFP–Rab7wt or the GTPase-defective GFP–Rab7T22N mutant, and the levels of LHBs were determined by western blotanalysis (E) and total HBV DNA in the supernatant was determined with real-time PCR (F). Quantification of three independent experiments are shownand indicate an increase in HBV secretion in the mutant compared to thewild-type-expressing cells, similar to that observed in the Rab7-depletedcells. *P,0.05; **P,0.01; ***P,0.001.

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alternatively, were pulled towards an autophagosome. Therefore, itwas considered that the Rab7-dependent tubules induced by HBV

might facilitate the lysosomal degradation of virus in MVBs orautophagosomes. Accordingly, we tested for changes in thedistribution of viral proteins and lysosomes in virus-producing

cells in the context of Rab7 depletion. Knockdown of Rab7 in thesecells induced a significant (70%) decrease in the localization ofLHBs with LAMP1 compartments (Fig. 6B,C), suggesting that

Rab7 facilitates the transport to and subsequent degradation ofHBV in lysosomes. In addition and consistent with previousobservations (Vanlandingham and Ceresa, 2009), significantlymore MVBs were found in Rab7-depleted cells when assessed by

TEM (Fig. 6D–F), suggesting an induced defect in MVB–lysosome fusion. In support of this premise, we observedenlarged MVBs in Rab7-depleted cells that were not seen in

control cells (supplementary material Fig. S4D,E).To investigate the effect of HBV on endocytic uptake and

trafficking of non-viral endogenous receptors, we compared the

kinetics of the epidermal growth factor receptor (EGFR)

following stimulation with EGF ligand in HepG2 versus theinfected HepG2.2.15 cells as described previously (Schroeder

et al., 2010). Although HepG2.2.15 cells exhibited markedlyhigher levels of EGFR to begin with, the kinetics of degradationup to 6 h appeared very similar. Furthermore, we observed little

difference in the uptake and trafficking of rhodamine-taggedtransferrin (data not shown).

Inhibiting lysosome function increases HBV secretionBecause Rab7 appears to be mediating the transfer of nascentHBV from MVBs to the lysosome for potential degradation, wetested whether treating cells with CQ, which disrupts lysosome

function by preventing acidification, might also increase viralsecretion and therefore mimic the effects of the Rab7 knockdown.HepG2.2.15 cells were treated with 0–50 mM CQ for 24 h and the

levels of LHBs in the cells and HBV DNA in the supernatantwere assayed. As predicted, both were increased significantlyafter CQ treatment in a dose-dependent manner (Fig. 7A–C). In

addition, another lysosome inhibitor NH4Cl produced similar

Fig. 6. Rab7-dependent tubules facilitate thefusion of MVBs or autophagosomes withlysosomes. (A) LC3-positive tubules in HBV-producing cells interact with LAMP1-positivelysosomes. HepG2.2.15 cells were transfectedwith GFP–LC3 and mCherry–LAMP1, and time-lapse confocal images were obtained over 20–30 min. Two representative image series show aphysical interaction between the autophagosomesand lysosomes (arrows). (B,C) Altering Rab7function reduces the transport of viral proteinsfrom the MVB or autophagosome to the lysosome.HepG2.2.15 cells were treated with non-targeting(NT) siRNA or Rab7 siRNA followed bytransfection of mCherry–LAMP1, and then stainedwith an anti-PreS1 antibody labeling LHBs. Cellswith impaired Rab7 function showed a markedreduction of transported HBV proteins to theLAMP1-positive lysosomal compartment. Scalebars: 10 mm. The graph represents threeindependent experiments where the numbers ofLHBs- and LAMP1-positive vesicles as aproportion of the total LHBs-positive vesicles werecalculated. (D,E) TEM images of HepG2.2.15cells treated with NT siRNA (D) or Rab7 siRNA(E) showing a substantial increase in the numberof accumulated MVBs. (F) Quantification (n512cells per condition) of MVBs per cell with NTsiRNA or Rab7 siRNA. ***P,0.001.

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results in two independent experiments (supplementary material

Fig. S4F,G). This indicates that a substantial percentage of HBVis degraded in lysosomes under normal conditions. When the virus-containing cell supernatant was collected from the drug-treated

cells and added to HuS-E/2 cells (the CQ concentration was diluted300 times beyond its working concentration), we did not see asignificant difference in the infectivity compared to control cells

(data not shown), similar to our observations using virus-containing supernatant from Rab7-depleted cells (supplementarymaterial Fig. S4A–C). Most importantly, the CQ-treated cells no

longer exhibited a Rab7-depletion effect on the intracellular levelsof LHBs (Fig. 7D,E), supporting the concept that Rab7 mediatesthe transport of HBV to the lysosome to reduce viral productionand secretion.

DISCUSSIONIn this study, we provide novel findings implicating the small

GTPase Rab7 as a central regulator of HBV transport and secretion.We find that assembled virus accumulates in Rab7-positive MVBand autophagosome compartments (Fig. 1), which then become

markedly altered and tubulated upon infection of hepatocytes(Fig. 2). Equally surprising was the finding that HBV significantlyenhances Rab7 activity through the expression of the precore or

HBe protein encoded by its own invasive genome (Fig. 4). In thiscontext, Rab7-dependent activation of the late endosomal pathway(Fig. 3) appears to facilitate the interaction with, and potentiallyexchange of contents between (Fig. 6), the MVB, autophagosome

and lysosome (Figs 6,7), leading to an increase in HBV degradationand a subsequent attenuation of viral shedding (Fig. 5). Fig. 8illustrates our main concept, demonstrating that the HBV infection

promotes the formation of an interconnecting reticular networkbetween MVBs, autophagosomes and lysosomes driven by theactivation of Rab7. Although tubules extending from MVBs and

autophagosomes have been reported previously (Cooney et al.,

2002; Cortese et al., 2013; Gao et al., 2010), those induced by HBV

in the current study are much more prominent.Rab7 is a key trafficking regulator involved in multiple post-

endocytic processes including transport between late endosomes/

MVBs, autophagosomes, lysosomes and other lysosome-relatedorganelles (Wang et al., 2011). Activated Rab7 on the endosomalmembrane binds to RILP, a well-characterized effector of Rab7,

together forming a complex with p150Glued (also known as DCTN1)and dynein–dynactin to drive the transport of endosomes to theminus-end of microtubules (Jordens et al., 2001). Additionally,

Rab7 is known to associate with the FYVE coiled coil domainprotein (FYCO1) and the molecular motor kinesin to participate inthe microtubule plus-end-directed transport of endosomes (Pankivet al., 2010). The activation of this complex might promote the

formation of tubular extensions bidirectionally along microtubules,as discussed previously (Harrison et al., 2003), and, as observed byour electron micrographs (Fig. 3A; supplementary material Fig.

S1J). Although many tubules were found to extend from MVBs andautophagosomes in the HBV-producing cells (Fig. 2), we observedno alterations in the lysosomal structure (data not shown),

suggesting that Rab7 specifically acted in the MVB andautophagosome compartments.

Currently, the mechanism by which the precore or HBe protein

activates Rab7 is unclear as we have been unable to detect anyinteractions between the viral protein and Rab7 biochemically(data not shown). It is possible, if not likely, that the HBe proteincould interact with either guanine-nucleotide-exchange factors

(GEFs) or GTPase-activating proteins (GAPs) specific for Rab7(Wang et al., 2011). Interaction with these regulatory proteinscould activate Rab7 by enhancing the function of a GEF, or in

contrast, inhibiting the function of a GAP. Studies to test forinteractions between HBe and these accessory factors are ongoing.

A central finding of this study indicates that the HBV itself

activates Rab7 to promote exchange between MVBs,

Fig. 7. Inhibition of lysosomal function prevents the Rab7-dependent reduction of HBV. (A,B) HBV levels in cells are reduced by lysosomal degradation.HepG2.2.15 cells were treated with the lysosome inhibitor chloroquine (CQ) at various concentrations for 24 h (A) and the levels of LHBs were determinedand quantified by western blot analysis (B) from three independent experiments. A substantial, seven to eightfold retention of viral protein was observed in cellsupon disruption of lysosomal function. (C) Total HBV DNA in the culture supernatant of HepG2.2.15 cells treated with CQ was determined with real-time PCR(n53) and reveals a three to fourfold increase in viral secretion. (D,E) Rab7 supports lysosome-mediated degradation of HBV. HepG2.2.15 cells weretransfected with Rab7 or NT siRNA prior to treatment with 25 mM CQ. 24 h following treatment, LHBs levels were determined and quantified by western blotanalysis (D) in three independent experiments (E). The siRNA-mediated reduction of Rab7 resulted in a twofold increase in total cellular LHBs protein, an effectthat was negated by CQ treatment. *P,0.05; **P,0.01.

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autophagosomes and the lysosome (Fig. 6) resulting in reduced

viral production. The marked intracellular accumulation of viralproteins accompanied by the increase in viral secretion followingRab7 knockdown (Fig. 5) or inhibition of lysosome function(Fig. 7) supports this concept. Some possibilities exist to explain

how the virus would benefit from activating the pathway thatwould direct it into the lysosome for eventual destruction. First,processing of surface proteins by lysosomal enzymes might aid in

viral ‘maturation’ and increase infectivity, or, alternatively,decrease viral recognition by neutralizing antibodies in theblood. To test these concepts, we compared the infectivity of

virus released from cells with or without Rab7 depletion, as wellas with or without CQ treatment; however, no differences in

infectivity were observed (supplementary material Fig. S4A–C).Second, and perhaps more relevant, is the fact that HBV isconsidered a ‘stealth virus’ that is able to escape from the frontline of host defenses (Wieland and Chisari, 2005). HBe, which is

not required for viral replication, is considered to have a role inthe establishment of chronic infection (Milich and Liang, 2003).It has been shown that HBe modulates innate immune signal

transduction pathways through interaction with and targeting ofthe signaling pathways mediated by Toll-like receptors (Langet al., 2011). As a result of attenuated immune responses, the viral

load increases greatly in the early phase of chronic infection(Liang, 2009). We hypothesize that HBV might put a brake on itsown secretion through HBe-mediated Rab7 activation to reduce

the immune response. In support of this hypothesis, it has beenshown that HBe-negative strains have high replication capacity in

vitro (Inoue et al., 2011; Ozasa et al., 2006) and that they cancause vigorous immune responses resulting in fulminant hepatitis

(Milich and Liang, 2003).An alternative explanation as to why we observed this Rab7

activation is that the activation of a Rab7-mediated viral

degradation pathway rather than representing a host defensemechanism – that is, hepatocytes respond to the expression of theHBe antigen by grossly activating the tubulation and fusion of

MVBs and autophagosomes with the lysosome. Such membraneremodeling events could be part of an autophagy-mediatedclearance of invading pathogens (xenophagy), a well-established

cellular defense mechanism (Levine, 2005).Finally, it is important to note that the specific role of Rab7

described here might represent just one of several functions in theHBV life cycle. A recent paper has shown that the early entry

stages of HBV infection in HepaRG cells depend on both Rab5and Rab7 (Macovei et al., 2013). The HepG2.2.15 cell modelused in our current study stably expresses HBV and is not

susceptible to further infection because it expresses very lowlevels of the putative HBV receptor, the sodium taurocholatecotransporting polypeptide (NTCP) (Yan et al., 2012). Therefore,

HepG2.2.15 cells provide a useful model to study the productionand release of the virus rather than infection. Thus, Rab7activation by the HBe protein might also increase the efficiencyof the early stages of infection.

It is clear from this and other studies implicating the endosomalpathways in HBV infection that a more complete understandingof how this virus usurps the vesicle trafficking machinery from

the hepatocyte to suit its own ends will be a complex butrewarding challenge. Additional regulatory Rab GTPases, vesiclecoat and adaptor proteins, as well as fission enzymes, are likely to

participate in the HBV life cycle and thus will provide useful drugtargets for future therapy.

MATERIALS AND METHODSPlasmids and siRNATo obtain FLAG-tagged HBV individual protein constructs, individual

DNA sequences specific for each protein were amplified from a total

DNA extracted from the culture supernatant of HepG2.2.15 cells.

Nucleotides [nt, the numbers are in accordance with a genotype D HBV

sequence of 3182 nt from HepG2.2.15 (accession number U95551)]

2307–3182 and 1–1623, 2847–3182 and 1–835, 155–835, 1899–2453,

1814–2453, and 1374–1840 were amplified for FLAG–polymerase,

FLAG–LHBs, FLAG–HBs, FLAG–HBc, FLAG–precore and FLAG–

HBx, respectively. These PCR products were cloned into pcDNA3

(Invitrogen, Carlsbad, CA) modified to have a FLAG sequence upstream

Fig. 8. Model depicting the role of Rab7 in the regulation of HBVsecretion. (A) In uninfected cells with basal Rab7 activity, MVB andautophagosome (AP) tubules are less prominent resulting in normalexocytosis. (B) The viral HBe protein within infected cells activates Rab7,inducing the formation of an interconnected tubular-reticular network ofMVBs, autophagosomes and lysosomes along microtubules. This networkpromotes the transfer of virus between the various compartments, resultingin the degradation of nascent virus and reduced secretion. (C) In Rab7-depleted cells, the interaction between these compartments is substantiallyreduced, leading to an accumulation of MVBs and a subsequent increasethe levels of intracellular and released virus, a result of reduced viraltransport to the lysosome.

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of the multiple-cloning site. 1.3-fold wild-type HBV genome (nt 1051–

3215 and 1–1953, which is 1.3-fold longer than a circular HBV genome)

of genotype B, which was obtained from an acute hepatitis patient, was

described previously (Inoue et al., 2011). GFP–Rab7wt was as described

previously (Schroeder et al., 2012) and GFP–Rab7T22N was kindly

provided by Dr Bruce Horazdovsky (Department of Biochemistry and

Molecular Biology, Mayo Clinic, Rochester, MN). FLAG–Rab7wt was

made from a PCR product that was amplified from GFP–Rab7wt. GST–

RILP was kindly provided by Dr Cecilia Bucci (Universita del Salento,

Italy) and mCherry–RILP was provided by Dr Barbara Schroeder

(Department of Biochemistry and Molecular Biology, Mayo Clinic,

Rochester, MN) and obtained by cloning the RILP sequence into the

HindIII/XbaI sites of pmCherry-C3 (Takara Bio, Shiga, Japan). GFP–

LAMP1 and mCherry–LAMP1 were made as described previously

(Schulze et al., 2013). siRNA targeting human Rab7, whose sequence

was described previously (Jager et al., 2004), siRNA targeting human

RILP (59-GAUCAAGGCCAAGAUGUUAUU-39) and a non-targeting

(NT) control siRNA were purchased from Dharmacon (Thermo Fisher

Scientific, Lafayette, CO).

Antibodies and other reagentsAnti-PreS1 and anti-GST antibodies were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA). Anti-HBc was purchased from Abcam

(Cambridge, UK). Antibodies against Rab7, FLAG (monoclonal) and actin,

as well as chloroquine (CQ), were purchased from Sigma-Aldrich (St.

Louis, MO). Anti-FLAG (polyclonal) and anti-Myc (monoclonal and

polyclonal) antibodies were purchased from Cell Signaling (Danvers, MA).

Anti-Hrs antibody was purchased from Bethyl Laboratories (Montgomery,

TX). Anti-LC3 was purchased from Novus Biologicals (Littleton, CO).

Cell culture and transfectionThe HBV-expressing stable cell line HepG2.2.15, which was kindly

provided by Dr Andrea Cuconati (Institute of Hepatitis and Virus

Research, Doylestown, PA), and the parental human hepatoma cell line

HepG2 were incubated in RPMI 1640 with L-glutamine (Corning

Cellgro, Manassas, VA) supplemented with 10% fetal bovine serum

(FBS), 50 U/ml penicillin and 50 mg/ml streptomycin. HepG2.2.15 and

HepG2 cells were seeded onto coverslips or plates coated with poly-L-

lysine (Sigma-Aldrich) as described previously (Abdulkarim et al., 2003).

Cells were transiently transfected with Lipofectamine Plus (Invitrogen)

or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s

instructions. HepG2.2.15 cells were transfected twice in a 24 h interval to

increase the transfection efficiency. Transfection of HepG2.2.15 cells

with siRNA was performed using RNAiMAX (Invitrogen) following the

standard protocol. The re-expression was performed as above 24 h after

the siRNA transfection. HeLa and the human hepatoma cell lines HuH7

and Hep3B were incubated in MEM (Corning cellgro), supplemented

with 10% FBS, 1.5 g/l sodium bicarbonate, 16nonessential amino acids,

1 mM sodium pyrophosphate, 50 U/ml penicillin and 50 mg/ml

streptomycin. An immortalized human primary hepatocyte cell line

HuS-E/2, which was kindly provided by Dr Makoto Hijikata (Kyoto

University, Japan), was grown as described previously (Aly et al., 2007).

HBV infection experiments with HuS-E/2 cells were performed as

described previously (Huang et al., 2012). Primary human hepatocytes

(PHHs) were from Yecuris (Tualatin, OR) and maintained in DMEM

with high glucose and L-glutamine, supplemented with 10% FBS and

50 U/ml penicillin and 50 mg/ml streptomycin.

Western blot analysisCells were lysed in RIPA buffer [150 mM NaCl, 1% NP-40, 0.5%

deoxycholic acid, 0.1% SDS, 50 mM Tris-HCl pH 8.0 and complete

protease inhibitors (Roche Diagnostics, Indianapolis, IN)]. 30 mg of

soluble proteins were resolved by SDS-PAGE, and transferred onto a

PVDF membrane. After blocking with 10% milk in PBS, the membrane

was incubated with primary antibodies at room temperature for 2 h, and

incubated with secondary antibodies for 1 h. The signals were detected

with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher

Scientific).

GST–RILP pulldownGST–RILP was transformed into Escherichia coli BL21 cells and 4 ml of

an overnight culture was cultured further in 200 ml LB to an optical

density (OD) at 600 nm of 0.6–0.8. After the addition of isopropyl b-D-1-

thiogalactopyranoside (IPTG, final concentration of 1 mM), it was

incubated at room temperature for 3–4 h. The culture was spun down,

and the collected bacteria were sonicated in PBS on ice. They were

rotated at 4 C for 30 min with 1% Triton X-100 and GST–RILP protein

was purified using glutathione–Sepharose-4B beads (Amersham-

Pharmacia Biotech, GE Healthcare Bio-Sciences, Piscataway, NJ)

according to the manufacturer’s instructions. The beads and 0.25 mg of

cell lysate to be analyzed were rocked in 600 ml of Bing buffer (25 mM

Tris-HCI pH 7.4, 100 mM NaCI, 1 mM DTT, 1% NP-40, 2mM Na3VO4,

15 mM NaF, 0.1 mM EDTA and complete protease inhibitors) at 4 C for

1–2 h. The levels of active Rab7 binding to GST–RILP were quantified

by western blot analysis.

Real-time PCR and ELISATotal DNA was extracted with a QIAamp DNA Blood Mini Kit

(QIAGEN, Hilden, Germany) from 100 ml of the culture supernatant,

and 2 ml of the extracted DNA solution was subjected to real-time

PCR using a LightCycler 480 system (Roche Diagnostics) with SYBR

Green I Master (Roche Diagnostics), primers HBXF11 (59-ATG-

GCTGCTAGGCTGTGCTG-39) and HBXR4 (59-GTCCGCGTA-

AAGAGAGGTGC-39). The HBsAg levels in the culture supernatant

were quantified with Hepatitis B Surface Antigen ELISA Kit

(MyBioSource, San Diego, CA).

Immunoprecipitation of viral particlesHepG2.2.15 cells were transfected with FLAG–HBs, and 2 days

following transfection, enveloped HBV particles in the supernatant

were precipitated using the anti-FLAG antibody. The levels of HBV

DNA from the precipitates were determined by real-time quantitative

PCR.

Immunofluorescence and transmission electron microscopyFluorescence micrographs were acquired using a Zeiss Axio Observer.D1

microscope (Carl Zeiss, Oberkochen, Germany) with a 636objective, an

Orca II camera (Hamamatsu Photonics, Hamamatsu, Japan), and iVision

software (BioVision Technologies, Exton, PA), or an LSM780 confocal

microscope (Carl Zeiss) with a 636 objective and ZEN software (Carl

Zeiss). Time-lapse images of live cells were taken using the confocal

microscope every 30 s on a heated chamber set at 37 C and under 5%

CO2. Transmission electron microscopy (TEM) micrographs were

acquired as previously described (Henley et al., 1998) with a JEOL

1200 electron microscope (JEOL Ltd, Tokyo, Japan). Images were

adjusted with Adobe Photoshop software (Adobe, San Jose, CA).

Statistical analysisResults of quantitative variables are expressed as mean6s.e.m. Statistical

comparisons were made by using a Student’s t-test unless otherwise

indicated and P,0.05 was considered significant.

AcknowledgementsThe authors would like to thank members of the McNiven laboratory for theircontributions, especially Barbara Schroeder and Ryan Schulze for criticalreadings of the manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsJ.I. designed and performed experiments, analyzed data and wrote the paper;E.W.K. performed microscopy experiments; J.C. assisted with protein assays;H.C. performed experiments of RILP knockdown and expression, EGFRtrafficking and transferrin trafficking; M.N. assisted with HBV infection andquantification; M.A.M. designed experiments and wrote the paper.

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1696–1706 doi:10.1242/jcs.158097

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FundingThis study was supported by the Japan Society for the Promotion of Science (toJ.I.); the Mochida Memorial Foundation for Medical and Pharmaceutical Research(to J.I.); and by the National Institute of Diabetes and Digestive and KidneyDiseases [grant number DK44650 to M.A.M.]. Deposited in PMC for release after12 months.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.158097/-/DC1

ReferencesAbdulkarim, A. S., Cao, H., Huang, B. and McNiven, M. A. (2003). The large

GTPase dynamin is required for hepatitis B virus protein secretion fromhepatocytes. J. Hepatol. 38, 76-83.

Aly, H. H., Watashi, K., Hijikata, M., Kaneko, H., Takada, Y., Egawa, H.,Uemoto, S. and Shimotohno, K. (2007). Serum-derived hepatitis C virusinfectivity in interferon regulatory factor-7-suppressed human primaryhepatocytes. J. Hepatol. 46, 26-36.

Berg, T. O., Fengsrud, M., Strømhaug, P. E., Berg, T. and Seglen, P. O. (1998).Isolation and characterization of rat liver amphisomes. Evidence for fusion ofautophagosomes with both early and late endosomes. J. Biol. Chem. 273,21883-21892.

Bruss, V. (2007). Hepatitis B virus morphogenesis. World J. Gastroenterol. 13,65-73.

Bucci, C., Thomsen, P., Nicoziani, P., McCarthy, J. and van Deurs, B. (2000).Rab7: a key to lysosome biogenesis. Mol. Biol. Cell 11, 467-480.

Cantalupo, G., Alifano, P., Roberti, V., Bruni, C. B. and Bucci, C. (2001). Rab-interacting lysosomal protein (RILP): the Rab7 effector required for transport tolysosomes. EMBO J. 20, 683-693.

Cooney, J. R., Hurlburt, J. L., Selig, D. K., Harris, K. M. and Fiala, J. C. (2002).Endosomal compartments serve multiple hippocampal dendritic spines from awidespread rather than a local store of recycling membrane. J. Neurosci. 22,2215-2224.

Cortese, K., Howes, M. T., Lundmark, R., Tagliatti, E., Bagnato, P., Petrelli, A.,Bono, M., McMahon, H. T., Parton, R. G. and Tacchetti, C. (2013). TheHSP90 inhibitor geldanamycin perturbs endosomal structure and drivesrecycling ErbB2 and transferrin to modified MVBs/lysosomal compartments.Mol. Biol. Cell 24, 129-144.

Eble, B. E., MacRae, D. R., Lingappa, V. R. and Ganem, D. (1987). Multipletopogenic sequences determine the transmembrane orientation of the hepatitisB surface antigen. Mol. Cell. Biol. 7, 3591-3601.

Fader, C. M. and Colombo, M. I. (2009). Autophagy and multivesicular bodies:two closely related partners. Cell Death Differ. 16, 70-78.

Fader, C. M., Sanchez, D., Furlan, M. and Colombo, M. I. (2008). Induction ofautophagy promotes fusion of multivesicular bodies with autophagic vacuoles ink562 cells. Traffic 9, 230-250.

Gao, W., Kang, J. H., Liao, Y., Ding, W. X., Gambotto, A. A., Watkins, S. C.,Liu, Y. J., Stolz, D. B. and Yin, X. M. (2010). Biochemical isolation andcharacterization of the tubulovesicular LC3-positive autophagosomalcompartment. J. Biol. Chem. 285, 1371-1383.

Garcia, T., Li, J., Sureau, C., Ito, K., Qin, Y., Wands, J. and Tong, S. (2009).Drastic reduction in the production of subviral particles does not impair hepatitisB virus virion secretion. J. Virol. 83, 11152-11165.

Hanson, P. I. and Cashikar, A. (2012). Multivesicular body morphogenesis.Annu. Rev. Cell Dev. Biol. 28, 337-362.

Harrison, R. E., Bucci, C., Vieira, O. V., Schroer, T. A. and Grinstein, S. (2003).Phagosomes fuse with late endosomes and/or lysosomes by extension ofmembrane protrusions along microtubules: role of Rab7 and RILP. Mol. Cell.Biol. 23, 6494-6506.

Henley, J. R., Krueger, E. W., Oswald, B. J. and McNiven, M. A. (1998).Dynamin-mediated internalization of caveolae. J. Cell Biol. 141, 85-99.

Huang, H. C., Chen, C. C., Chang, W. C., Tao, M. H. and Huang, C. (2012).Entry of hepatitis B virus into immortalized human primary hepatocytes byclathrin-dependent endocytosis. J. Virol. 86, 9443-9453.

Hyttinen, J. M., Niittykoski, M., Salminen, A. and Kaarniranta, K. (2013).Maturation of autophagosomes and endosomes: a key role for Rab7. Biochim.Biophys. Acta 1833, 503-510.

Inoue, J., Ueno, Y., Wakui, Y., Fukushima, K., Kondo, Y., Kakazu, E., Ninomiya,M., Niitsuma, H. and Shimosegawa, T. (2011). Enhanced replication of hepatitisB virus with frameshift in the precore region found in fulminant hepatitis patients.J. Infect. Dis. 204, 1017-1025.

Jager, S., Bucci, C., Tanida, I., Ueno, T., Kominami, E., Saftig, P. andEskelinen, E. L. (2004). Role for Rab7 in maturation of late autophagicvacuoles. J. Cell Sci. 117, 4837-4848.

Jordens, I., Fernandez-Borja, M., Marsman, M., Dusseljee, S., Janssen, L.,Calafat, J., Janssen, H., Wubbolts, R. and Neefjes, J. (2001). The Rab7effector protein RILP controls lysosomal transport by inducing the recruitment ofdynein-dynactin motors. Curr. Biol. 11, 1680-1685.

Kian Chua, P., Lin, M. H. and Shih, C. (2006). Potent inhibition of humanHepatitis B virus replication by a host factor Vps4. Virology 354, 1-6.

Lambert, C., Doring, T. and Prange, R. (2007). Hepatitis B virus maturation issensitive to functional inhibition of ESCRT-III, Vps4, and gamma 2-adaptin.J. Virol. 81, 9050-9060.

Lang, T., Lo, C., Skinner, N., Locarnini, S., Visvanathan, K. and Mansell, A.(2011). The hepatitis B e antigen (HBeAg) targets and suppresses activation ofthe toll-like receptor signaling pathway. J. Hepatol. 55, 762-769.

Levine, B. (2005). Eating oneself and uninvited guests: autophagy-relatedpathways in cellular defense. Cell 120, 159-162.

Li, J., Liu, Y., Wang, Z., Liu, K., Wang, Y., Liu, J., Ding, H. and Yuan, Z. (2011).Subversion of cellular autophagy machinery by hepatitis B virus for viralenvelopment. J. Virol. 85, 6319-6333.

Liang, T. J. (2009). Hepatitis B: the virus and disease. Hepatology 49 Suppl.,S13-S21.

Macovei, A., Petrareanu, C., Lazar, C., Florian, P. and Branza-Nichita, N.(2013). Regulation of hepatitis B virus infection by Rab5, Rab7, and theendolysosomal compartment. J. Virol. 87, 6415-6427.

McMahon, B. J. (2005). Epidemiology and natural history of hepatitis B. Semin.Liver Dis. 25 Suppl. 1, 3-8.

Milich, D. and Liang, T. J. (2003). Exploring the biological basis of hepatitis B eantigen in hepatitis B virus infection. Hepatology 38, 1075-1086.

Ozasa, A., Tanaka, Y., Orito, E., Sugiyama, M., Kang, J. H., Hige, S.,Kuramitsu, T., Suzuki, K., Tanaka, E., Okada, S. et al. (2006). Influence ofgenotypes and precore mutations on fulminant or chronic outcome of acutehepatitis B virus infection. Hepatology 44, 326-334.

Pankiv, S., Alemu, E. A., Brech, A., Bruun, J. A., Lamark, T., Overvatn, A.,Bjørkøy, G. and Johansen, T. (2010). FYCO1 is a Rab7 effector that binds toLC3 and PI3P to mediate microtubule plus end-directed vesicle transport. J. CellBiol. 188, 253-269.

Prange, R. (2012). Host factors involved in hepatitis B virus maturation, assembly,and egress. Med. Microbiol. Immunol. (Berl.) 201, 449-461.

Rijckborst, V., Sonneveld, M. J. and Janssen, H. L. (2011). Review article:chronic hepatitis B – anti-viral or immunomodulatory therapy? Aliment.Pharmacol. Ther. 33, 501-513.

Schroeder, B., Weller, S. G., Chen, J., Billadeau, D. and McNiven, M. A. (2010).A Dyn2-CIN85 complex mediates degradative traffic of the EGFR by regulationof late endosomal budding. EMBO J. 29, 3039-3053.

Schroeder, B., Srivatsan, S., Shaw, A., Billadeau, D. and McNiven, M. A.(2012). CIN85 phosphorylation is essential for EGFR ubiquitination and sortinginto multivesicular bodies. Mol. Biol. Cell 23, 3602-3611.

Schulze, R. J., Weller, S. G., Schroeder, B., Krueger, E. W., Chi, S., Casey,C. A. and McNiven, M. A. (2013). Lipid droplet breakdown requires dynamin 2for vesiculation of autolysosomal tubules in hepatocytes. J. Cell Biol. 203, 315-326.

Sir, D., Tian, Y., Chen, W. L., Ann, D. K., Yen, T. S. and Ou, J. H. (2010). Theearly autophagic pathway is activated by hepatitis B virus and required for viralDNA replication. Proc. Natl. Acad. Sci. USA 107, 4383-4388.

Stenmark, H. (2009). Rab GTPases as coordinators of vesicle traffic. Nat. Rev.Mol. Cell Biol. 10, 513-525.

Stieler, J. T. and Prange, R. (2014). Involvement of ESCRT-II in hepatitis B virusmorphogenesis. PLoS ONE 9, e91279.

Tian, Y., Sir, D., Kuo, C. F., Ann, D. K. and Ou, J. H. (2011). Autophagy requiredfor hepatitis B virus replication in transgenic mice. J. Virol. 85, 13453-13456.

van der Kant, R., Fish, A., Janssen, L., Janssen, H., Krom, S., Ho, N.,Brummelkamp, T., Carette, J., Rocha, N. and Neefjes, J. (2013). Lateendosomal transport and tethering are coupled processes controlled by RILPand the cholesterol sensor ORP1L. J. Cell Sci. 126, 3462-3474.

Vanlandingham, P. A. and Ceresa, B. P. (2009). Rab7 regulates late endocytictrafficking downstream of multivesicular body biogenesis and cargosequestration. J. Biol. Chem. 284, 12110-12124.

Wang, T., Ming, Z., Xiaochun, W. and Hong, W. (2011). Rab7: role of its proteininteraction cascades in endo-lysosomal traffic. Cell. Signal. 23, 516-521.

Watanabe, T., Sorensen, E. M., Naito, A., Schott, M., Kim, S. and Ahlquist, P.(2007). Involvement of host cellular multivesicular body functions in hepatitis Bvirus budding. Proc. Natl. Acad. Sci. USA 104, 10205-10210.

Wieland, S. F. and Chisari, F. V. (2005). Stealth and cunning: hepatitis B andhepatitis C viruses. J. Virol. 79, 9369-9380.

Yan, H., Zhong, G., Xu, G., He, W., Jing, Z., Gao, Z., Huang, Y., Qi, Y., Peng, B.,Wang, H. et al. (2012). Sodium taurocholate cotransporting polypeptide is afunctional receptor for human hepatitis B and D virus. eLife 1, e00049.

RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1696–1706 doi:10.1242/jcs.158097

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