Research Article: Human apolipoprotein E promotes hepatitis B virus infection and production

Date Published: August 8, 2019

Publisher: Public Library of Science

Author(s): Luhua Qiao, Guangxiang George Luo, Jianming Hu.


Hepatitis B virus (HBV) is a common cause of liver diseases, including chronic hepatitis, steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). HBV chronically infects about 240 million people worldwide, posing a major global health problem. The current standard antiviral therapy effectively inhibits HBV replication but does not eliminate the virus unlike direct-acting antivirals (DAA) for curing hepatitis C. Our previous studies have demonstrated that human apolipoprotein E (apoE) plays important roles in hepatitis C virus infection and morphogenesis. In the present study, we have found that apoE is also associated with HBV and is required for efficient HBV infection. An apoE-specific monoclonal antibody was able to capture HBV similar to anti-HBs. More importantly, apoE monoclonal antibody could effectively block HBV infection, resulting in a greater than 90% reduction of HBV infectivity. Likewise, silencing of apoE expression or knockout of apoE gene by CRISPR/Cas9 resulted in a greater than 90% reduction of HBV infection and more than 80% decrease of HBV production, which could be fully restored by ectopic apoE expression. However, apoE silencing or knockout did not significantly affect HBV DNA replication or the production of nonenveloped (naked) nucleocapsids. These findings demonstrate that human apoE promotes HBV infection and production. We speculate that apoE may also play a role in persistent HBV infection by evading host immune response similar to its role in the HCV life cycle and pathogenesis. Inhibitors interfering with apoE biogenesis, secretion, and/or binding to receptors may serve as antivirals for elimination of chronic HBV infection.

Partial Text

Hepatitis B virus (HBV) infection continues to pose a major global health problem despite of the availability of effective HBV vaccine and antiviral drugs consisting of interferon (IFN) and nucleoside analogs (NAs). Currently, there are more than 240 million people chronically infected with HBV worldwide [1]. HBV vaccine has greatly reduced the number of new HBV infections and hepatocellular carcinoma (HCC) cases but does not offer therapeutic benefit to those chronically infected with HBV. Current antiviral regimens with NAs can effectively suppress HBV replication but are not curative unlike direct-acting antivirals (DAAs) for hepatitis C [2, 3]. Individuals with chronic HBV infection are at a substantial risk for progression to cirrhosis and HCC [4]. The World health organization has called for the elimination of viral hepatitis as a public health threat by 2030 [5].

In the present study, we have obtained substantial evidence demonstrating that human apoE is associated with infectious HBV and plays an important role in HBV infection. ApoE was found to be enriched in purified HBV, as demonstrated by its co-existence with HBcAg, LHBsAg, and HBV DNA in the same fractions (Fig 1A, 1B and 1D). There was a close correlation between apoE and HBV infection as shown by the peak level of HBV infectivity in fractions 6 and 7 (Fig 1C), which also contained the highest levels of apoE (Fig 1A). The association of apoE with HBV was further supported by a specific capture of HBV with an apoE monoclonal antibody similar to anti-HBs. Most of purified HBV could be specifically pulled down by anti-apoE and anti-HBs, as determined by the levels of HBV DNA and HBcAg in the immunoprecipitated virus (Fig 2A). Interestingly, the HBV-associated apoE was sensitive to trypsin digestion unlike HBcAg which only became sensitive to trypsin digestion upon disruption of the viral envelope by treatment with triton X-100 (Fig 2B). These findings suggest that apoE is likely exposed on the HBV envelope and may play a role in HBV infection. Indeed, HBV infectivity in PHHs and HepG2NTCP could be efficiently neutralized by an apoE monoclonal antibody, which was added during HBV infection or during and after HBV infection, resulting in a greater than 90% reduction of HBcAg and HBV cccDNA in the cell and HBeAg and HBV DNA in the cell culture supernatants (Fig 3). However, HBV infection was not significantly affected when the apoE-blocking antibody was added to cell culture media after HBV infection (Fig 3A). These findings demonstrate that apoE on the viral envelope does play a critical role in HBV infection. Apart from the importance of the virus-associated apoE in HBV infection, apoE expression from target cells is also required for efficient HBV infection. Both silencing of apoE expression and knockout of apoE gene greatly decreased the permissiveness of HepG2NTCP cells to HBV infection (Figs 4 and 5). However, silencing of apoB expression did not affect HBV infection (Fig 5), suggesting a specific requirement of apoE for HBV infection. We have not examined the effect of apoE silencing or knockout on HBV infection in PHH or other human hepatocyte cell lines besides HepG2NTCP cell lines available in the lab. More importantly, the defect of HBV infection in the apoE-/- HepG2NTCP cells could be completely restored by ectopic expression of apoE (Fig 9A and 9B). However, HBV protein expression and DNA replication were not significantly affected in apoE knockdown or knockout HepAD38 cells (Figs 7 and 8), demonstrating that apoE does not play a role in HBV protein expression or DNA replication. These findings are similar to those observed for HCV. ApoE gene knockout was also found to greatly impair the susceptibility of Huh7.5 cells to HCV infection but not replication [54, 55]. Thus, it is likely that apoE may mediate HBV infection via a similar mechanism to HCV infection, which is warranted for future investigation.