The hepatitis B virus X protein modulates hepatocyte proliferation pathways to stimulate viral replication

Abstract

Worldwide, there are over 350 million people who are chronically infected with the human hepatitis B virus (HBV); chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). The results of various studies suggest that the HBV X protein (HBx) has a role in the development of HBV-associated HCC. HBx can regulate numerous cellular signal transduction pathways, including those that modulate cell proliferation. Many previous studies that analyzed the impact of HBx on cell proliferation pathways were conducted using established or immortalized cell lines, and when HBx was expressed in the absence of HBV replication, and the precise effect of HBx on these pathways has often differed depending on experimental conditions. We have studied the effect of HBx on cell proliferation in cultured primary rat hepatocytes, a biologically relevant system. We demonstrate that HBx, both by itself and in the context of HBV replication, affected the levels and activities of various cell cycle-regulatory proteins to induce normally quiescent hepatocytes to enter the G(1) phase of the cell cycle but not to proceed to S phase. We linked HBx regulation of cell proliferation to cytosolic calcium signaling and HBx stimulation of HBV replication. Cumulatively, our studies suggest that HBx induces normally quiescent hepatocytes to enter the G(1) phase of the cell cycle and that this calcium-dependent HBx activity is required for HBV replication. These studies identify an essential function of HBx during HBV replication and a mechanism that may connect HBV infections to the development of HCC.

FIG. 1.

Confirmation of differentiated primary rat hepatocytes in culture. RNA was extracted from freshly isolated hepatocytes or hepatocytes cultured for 72 h as described in Materials and Methods. RNA was then reverse transcribed and PCR amplified using primers specific for transferrin (TFN), albumin (ALB), hepatocyte nuclear factor 4 (HNF4), connexin 26 (CNX 26), or connexin 43 (CNX 43). The expected band sizes are 914 bp for ALB, 530 bp for TFN, 770 bp for HNF4, 663 bp for CNX 26, and 642 bp for CNX 43. Note that primers for TFN and ALB were combined in the same reaction due to the substantial difference in PCR product size. (−) signifies samples where isolated RNA was directly PCR amplified to confirm the absence of DNA contamination.

FIG. 2.

HBx induces quiescent hepatocytes to enter, and stall in, the G₁ phase of the cell cycle. Hepatocytes were transfected with FL1-154 HBx or pcDNA3.1(−) (vector control) and collected at 24 h posttransfection. Lysates were resolved via SDS-PAGE and subjected to Western analysis for α-tubulin, HBx, p15, p16, p21, p27, cyclin D1, cyclin E, cyclin A, and PCNA (A to D). Results shown are representative samples from at least 3 experiments performed in duplicate. *, statistically significant fold difference ± standard error between FL1-154 HBx and pcDNA3.1(−) as determined using Student's t test (P ≤ 0.05).

FIG. 3.

HBx differentially regulates CDK4 and CDK2 in primary rat hepatocytes. Hepatocytes were transfected with FL1-154 HBx or pcDNA3.1(−) and collected at 24 h posttransfection. (A) Hepatocytes were analyzed for CDK4 activity by using a standard CDK4 kinase assay as described in Materials and Methods. IP, immunoprecipitation; WB, Western blot. (B) Hepatocytes plated at 50% confluence and treated with HGF or 1× PBS (CON) were analyzed for CDK2 activity by using a standard kinase assay as described in Materials and Methods. Results shown are representative samples from at least 3 experiments performed in duplicate. *, statistically significant fold difference ± standard error between FL1-154 HBx and pcDNA3.1(−) as determined using Student's t test (P ≤ 0.05).

FIG. 4.

HBx, in the context of HBV replication, modulates the levels and activities of various cell cycle-regulatory proteins. (A to C) Hepatocytes were infected with AdHBV(HBx−) or AdHBV and collected at 24 h postinfection. Lysates were resolved via SDS-PAGE and subjected to Western analysis for α-tubulin, p15, p16, p21, p27, cyclin D1, cyclin E, cyclin A, PCNA, and HBcAg. (D) Hepatocytes were transfected with pGEMHBV and pGEMHBV(HBx−) expression plasmids and collected at 24 h posttransfection. Lysates were resolved via SDS-PAGE and subjected to Western blot analysis for α-tubulin, p16, p21, cyclin A, and HBcAg. (E) Hepatocytes were infected with AdHBV(HBx−) or AdHBV, collected at 24 h postinfection, and analyzed for CDK4 activity by using a standard CDK4 kinase assay as described in Materials and Methods. (F) Hepatocytes plated at 50% confluence were infected with AdHBV(HBx−) or AdHBV, treated with either HGF or 1× PBS (CON), and collected at 24 h postinfection. Hepatocytes were analyzed for CDK2 activity by using a standard kinase assay as described in Materials and Methods. Results shown are representative samples from at least 3 experiments performed in duplicate. *, statistically significant fold difference ± standard error between AdHBV(HBx−) and AdHBV or pGEMHBV and pGEMHBV(HBx−) as determined using Student's t test (P ≤ 0.05).

FIG. 5.

HBx-induced modulation of the levels and activities of cell cycle-regulatory proteins requires cytosolic calcium signaling. Hepatocytes were transfected with FL1-154 HBx or pcDNA3.1(−) and treated with either 50 μM BAPTA-AM or a vehicle control. (A and B) Lysates were collected at 24 h posttransfection; resolved via SDS-PAGE; and subjected to Western analysis for α-tubulin, p15, p16, HBx, p21, p27, and cyclin E. (C) Hepatocytes were collected at 24 h posttransfection and analyzed for CDK4 activity by using a standard CDK4 kinase assay as described in Materials and Methods. Results shown are representative samples from at least 3 experiments performed in duplicate. *, statistically significant fold difference ± standard error between FL1-154 HBx and pcDNA3.1(−) as determined using Student's t test (P ≤ 0.05).

FIG. 6.

HBx, when expressed in the context of HBV replication, requires cytosolic calcium signaling to modulate the levels and activities of cell cycle-regulatory proteins in primary rat hepatocytes. Hepatocytes were infected with AdHBV(HBx−) or AdHBV and treated with either 50 μM BAPTA-AM or a vehicle control. (A and B) Lysates were collected at 24 h postinfection; resolved via SDS-PAGE; and subjected to Western analysis for α-tubulin, p15, p16, HBcAg, p21, p27, and cyclin E. (C) Hepatocytes were collected at 24 h postinfection and analyzed for CDK4 activity by using a standard CDK4 kinase assay as described in Materials and Methods. Results shown are representative samples from at least 3 experiments performed in duplicate. *, statistically significant fold difference ± standard error between AdHBV(HBx−) and AdHBV as determined using Student's t test (P ≤ 0.05).

FIG. 7.

HBV requires cell cycle entry for efficient HBV replication. (A) Primary rat hepatocytes were transfected with vector control or p16 overexpression plasmids and collected at 72 h posttransfection. Lysates were resolved via SDS-PAGE and analyzed for levels of α-tubulin and p16 via Western analysis. (B) Hepatocytes were cotransfected with FL1-154 HBx and vector control or FL1-154 HBx and p16 overexpression plasmids and collected at 24 h posttransfection. CDK4 activity was analyzed using a standard CDK4 kinase assay as described in Materials and Methods. (C) Primary rat hepatocytes were transfected with either vector control or p16 overexpression plasmids and coinfected with AdHBV. Lysates were collected at 72 h posttransfection or postinfection, subjected to SDS-PAGE, and analyzed for α-tubulin and HBcAg via Western analysis. (D) Primary rat hepatocytes transfected with either vector control or p16 overexpression plasmids and coinfected with AdHBV were collected at 72 h posttransfection or postinfection. HBV replication was analyzed with a standard HBV replication assay (see Materials and Methods). RC, relaxed circular; DL, double-stranded linear; SS, single stranded. Results shown are representative samples from at least 3 experiments performed in duplicate. *, statistically significant fold difference as determined using Student's t test (P ≤ 0.05).