HBx-induced phenotype: disturbing the cell cycle progression

The ability of HBx to interfere with cell viability, a property documented to reflect its potential to induce apoptosis, requires association of HBx with DDB1 in the nuclear compartment [85]. Previous work (Annexin V staining) and this work (caspase-mediated proteolytic and DNA cleavage) did not allow to detect any induction of apoptotic cell death upon expression of HBx. When ectopically expressed in human cells, HBx induces severe morphological changes and cell death. The observed morphological abnormalities include the appearance of increasing numbers of nuclear aberrations, basically multinucleated cells and micronuclei (Figure S2A). This phenotype prompted us to investigate the DNA content of HBx-expressing cells at different time points after transfection. Drug-resistant cells were stained by propidium iodide and analyzed for their DNA content by flow cytometry. Three days after HBx transfection, the staining revealed an increased number of cells with 4N, whereas HBx(R96E)-expressing cells showed a typical profile of asynchronous cells with the

Figure S2: HBx-expressing cells show altered morp majority of cells in Go/G1 (2N) (data not shown).

hology and aberrant DNA content

rug-resistant cells were selected with hygromycin. Nuclei were visualized by staining with the DNA

cells were stained with propidium iodide and their DNA content assessed by FACS analysis.

A

Mock

Vect GFP-HBx GFP-HBx

(R96E)

B

1 2 3 4

A

Mock

Vect GFP-HBx GFP-HBx

(R96E)

B

1 2 3 4

A) Altered morphology upon HBx expression (panels 1-4). HeLa cells were transfected with HBx and d

marker Hoechst 3342 (Blue, panel 2 and 4). Panel 1 shows a light microscopy picture of HBx-expressing cells whereas panel 3 and 4 show tubulin immunofluorescence (green) staining.

B) Expression of HBx leads to cells with more than 4N DNA content. Flow cytometric analysis of HeLa cells transfected with GFP-HBx variants. Ten days after transfection, drug-resistant

A large number of cells with a more than 4N DNA content could be observed in a DDB1-dependant manner after 10 days of HBx expression (Figure S2B). The cells

er of cells in anaphase and

es ere stained with TPX2 (red) and DNA with TOTO-3 (white). Cells with lagging chromosomes and

aberrant centrosome numbers depends on HBx teracting with DDB1, since the HBx(R96E) mutant unable to bind DDB1 did not how this phenotype. In principle, lagging chromosomes and chromosomal bridges with a DNA content superior to 4N could already be detected as soon as day 3 post HBx transfection. These multinucleated cells may arise either through aborted cell division or as a result of cell fusion. To test the latter hypothesis, cells expressing GFP-HBx were mixed with RFP expressing cells. Almost no double-colored cells were observed, even when multinucleated (data not shown), arguing in favour of a defect in cell cycle regulation rather than a fusion event.

Microscopy studies showed that as early as 48h after transfection in HeLa cells, HBx expression produces a significant increase in the numb

telophase showing lagging chromosomes and/or chromosomal bridges (Figure S3).

defects.

(By courtesy of Silvia Martin-Lluesma) Two days after GFP-HBx transfection in HeLa cells, spindl Lagging

Figure S3: Binding of HBx to DDB1 affects normal cell growth by inducing mitotic

w

multipolar spindles were scored in GFP-positive cells. These features are also observed in the hepatoma cell line HepG2 (data not shown).

Abnormal spindles architecture due to in

s

could be generated either by stalled replication forks or sustained cohesion. Both cases would ultimately lead either to proper resolution of the knots, to chromosomal

breaks or to complete mitotic failure, producing a mixed cell population including normal cells, aneuploid cells, micronuclei and multinucleated cells with multiple centrosomes. After a number of cell divisions, this population will show increased number of aberrations, and eventually cell death. Available data from the lab (Silvia Martin-Lluesma, personal communication) suggest that HBx expression leads to a longer S phase probably by acting at the level of replication initiation.

GFP-HBx

Figure S4: HBx-mediated toxicity is p53-independent.

epG2 cells were transduced with a siRNA lentiviral vector direct against p53 (LV-THsi-p53) or a transduced with a second lentiviral cells were stained with crystal violet

hese mitotic defects induced by HBx as well as HBx toxicity were observed in HeLa ells that express low levels of wild-type p53 protein, which is presumed to be altered

H

control vector (LV-TH). 12 days after transduction, the cells were vector expressing either GFP-HBx or GFP-HBx(R96E). Surviving 16 days after HBx expression.

T c

in function as a consequence of its association with HPV E6 oncoproteins [178]. The p53 protein was shown to be important for the control of different cell cycle checkpoints. We therefore asked whether p53 was involved in HBx-mediated cytotoxicity. First, presence of multiple spindles and lagging chromosomes could also be detected in a p53-positive hepatoblastoma cell line, HepG2 (data not shown). To further assess the role of p53 in HBx activity, HepG2 cells were first transduced with a lentiviral vector directing synthesis of a siRNA against p53 (kindly provided by M.Wiznerowicz) or with empty vector [179]. In a second stage, cells expressing close to undetectable levels of p53 (data not shown) were transduced with different HBx-expressing viral vectors. The HBx-mediated cytotoxic phenotype was shown to be

p53-independent since both; p53 positive and p53 knockdown, cells showed reduced viability (Figure S4). This effect was again dependent on HBx binding to DDB1 since HBx(R96E) did not alter cell growth. Additionally, the Hep3B cells in which a functional p53 is absent showed the same HBx-mediated cytotoxicity (data not shown) further confirming that this phenotype is p53-independent as reported for other HBx-mediated activities.

4.2 Hepatitis B virus X protein and simian virus 5 V protein exhibit