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. 1997 Dec 23;94(26):14707-12.
doi: 10.1073/pnas.94.26.14707.

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis

L W Elmore  1 , A R Hancock, S F Chang, X W Wang, S Chang, C P Callahan, D A Geller, H Will, C C Harris
Affiliations

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis

L W Elmore et al. Proc Natl Acad Sci U S A. .

Abstract

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.

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Figures

Figure 1
Figure 1
The C-terminal domain of HBx is critical for in vitro association with GST-p53. (A) In vitro translated full-length HBx protein (lanes 1–4) and HBx deletion mutants (lanes 5–8) were incubated with glutathione-Sepharose beads loaded with either GST-p53 (lanes 2, 4, 6, 8) or GST (lanes 1, 3, 5, 7). Lanes 1–4 and 5–8, along with their respective binding input, are representative data from two independent assays. (B) To reference input for binding, 20% of the volume of the various in vitro translated HBx proteins used for binding were immunoprecipitated by anti-HBx antibody. (C) Schematic representation of full-length and truncated HBx, as described in Materials and Methods, along with a summary of their binding to p53. Percent binding represents the mean ± SD from at least three independent binding assays with values made relative to SK1–154x.
Figure 2
Figure 2
Effects of HBx expression on p53-mediated apoptosis and on p53 localization in normal human hepatocytes. Primary hepatocytes were microinjected with a p53 expression vector or coinjected with p53 and HBx (adr and ayw subtypes for donor 1; adr subtype for donor 2) expression vectors. (A) After 24 h, cells were immunostained for p53 and scored for apoptosis as described in Materials and Methods. Bar values represent the percentage of apoptotic hepatocytes from one to three individual experiments. The total number of p53 immunopositive cells scored for donors 1 and 2 were 108 and 119, respectively. ND, not determined. ∗, Fisher’s exact test comparing the levels of p53-mediated apoptosis in the absence versus presence of HBx expression, P ≤ 0.036. In the case of p53 ± HBx (adr) with donor 2, a χ2 test was performed because greater than 100 cells were analyzed, P ≤ 0.046. (B) Twenty-four hours after microinjection with p53 expression vector alone (Upper Left) or coinjection with p53 and full-length HBx expression vectors (Upper Right and Lower), hepatocytes were simultaneously immunostained for p53 (Texas red) and HBx (FITC) as described in Materials and Methods. Yellow regions (Lower Right) reflect overlapping areas of p53 and HBx immunostaining in a single hepatocyte also shown (Upper Right and Lower Left).
Figure 3
Figure 3
HBx via its distal C-terminal region sequesters p53 to the cytoplasm. (A) Normal human fibroblasts were microinjected with a p53 expression vector and either full-length HBx (CMV-1–154X) or a deletion mutant missing the last 44 amino acids (CMV-1–110X) followed by incubation for 24 h. Immunostaining was performed as described in Materials and Methods. (B) Confocal microscopic analysis of normal human fibroblasts coinjected with p53 and full-length HBx expression vectors. Yellow regions represent areas of colocalization. (Upper) Representative example of the degree of cytoplasmic sequestration typically observed in fibroblasts overexpressing p53 and HBx. (Lower) Fibroblast with all detectable p53 colocalizing with HBx in the cytoplasm.
Figure 4
Figure 4
Ability of full-length HBx (A; adr versus ayw subtypes) and various HBx deletion mutants (B; ayw subtype) to transcriptionally transactivate SV40- and/or human NOS2 promoter-driven luciferase reporter constructs in HepG2 cells. Thirty-six to 48 hours after transfection, whole cell lysates were prepared, and resonance light units per μg protein were determined as described in Materials and Methods. (A) Representative data from a single experiment testing each sample in triplicate (Student’s t test; all data points, P ≤ 0.003). (B) Bar values represent the mean ± SD of resonance light units per μg protein relative to the CMV–neomycin control vector from three independent experiments (Student’s t test: all data points for SV40, P ≤ 0.016 and for NOS2, P ≤ 0.005).
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