Hepatitis B Virus Precore Protein p22 Inhibits Alpha Interferon Signaling by Blocking STAT Nuclear Translocation

Abstract

Antagonism of host immune defenses against hepatitis B virus (HBV) infection by the viral proteins is speculated to cause HBV persistence and the development of chronic hepatitis. The circulating hepatitis B e antigen (HBeAg, p17) is known to manipulate host immune responses to assist in the establishment of persistent viral infection, and HBeAg-positive (HBeAg+) patients respond less effectively to IFN-α therapy than do HBeAg-negative (HBeAg−) patients in clinical practice. However, the function(s) of the intracellular form of HBeAg, previously reported as the precore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we report that the cytosolic p22 protein, but not the secreted HBeAg, significantly reduces interferon-stimulated response element (ISRE) activity and the expression of interferon-stimulated genes (ISGs) upon alpha interferon (IFN-α) stimulation in cell cultures. In line with this, HBeAg+ patients exhibit weaker induction of ISGs in their livers than do HBeAg− patients upon IFN-α therapy. Mechanistically, while p22 does not alter the total STAT1 or pSTAT1 levels in cells treated with IFN-α, it blocks the nuclear translocation of pSTAT1 by interacting with the nuclear transport factor karyopherin α1 through its C-terminal arginine-rich domain. In summary, our study suggests that HBV precore protein, specifically the p22 form, impedes JAK-STAT signaling to help the virus evade the host innate immune response and, thus, causes resistance to IFN therapy. IMPORTANCE Chronic hepatitis B virus (HBV) infection continues to be a major global health concern, and patients who fail to mount an efficient immune response to clear the virus will develop a life-long chronic infection that can progress to chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma. There is no definite cure for chronic hepatitis B, and alpha interferon (IFN-α) is the only available immunomodulatory drug, to which only a minority of chronic patients are responsive, with hepatitis B e antigen (HBeAg)-negative patients responding better than HBeAg-positive patients. We herein report that the intracellular HBeAg, also known as precore or p22, inhibits the antiviral signaling of IFN-α, which sheds light on the enigmatic function of precore protein in shaping HBV chronicity and provides a perspective toward areas that need to be further studied to make the current therapy better until a cure is achieved.

Biogenesis and molecular characteristics of serum hepatitis B virus RNA

“…HBV wild-type infectious particles were collected from the supernatant of induced HepAD38 cells, HBV RNA particles were collected from the 3TC- or L-FMAU-treated HepAD38 cells upon induction, HBV naked capsids were collected from the supernatant of untreated and 3TC-treated HepDES19 cells upon induction. The infection of HepG2-NTCP12 cells, intracellular HBc immunofluorescence microscopy and HBeAg ELISA were conducted as previously published [ 51 ].…”

The Hepatitis B Virus Pre-Core Protein p22 Activates Wnt Signaling

“…P22 is further truncated, losing the arginine-rich C-terminal domain, to yield HBe (p17), which is secreted [ 19 ]. Expression of p22 was confirmed by immunoblot on whole cell lysates prepared from transfected Huh7 cells using an anti-HBc antibody and, as shown by others [ 19 ], neither p17 nor p25 were detected by immunoblot ( Figure 1 b and Figure S2 ). HBV p17 and p25 were detected by confocal immunofluorescence in transfected Huh7 cells ( Figure S3 ).…”

“…During natural HBV infection, p22 is processed to p17 or HBV e antigen (HBeAg) and secreted into the extracellular space [ 19 ]. We confirmed that the transfected p22 is processed to p17 by detecting and quantifying HBeAg in the supernatant of transfected Huh7 cells ( Figure S6 ).…”

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus

“…It is possible that in the setting of high levels of plasmid, even a less-optimal binding alternative may still favour production despite potential structural controls. Therefore, this model may not be representative of natural processes despite successes with this approach for other pathway studies (73)(74)(75). (2) It is also possible that the quadruplex structure is not needed for binding and that the primary nucleotide sequence is the sole influencer of the subtle finding in this model.…”