IFN response suppresses HDV persistence during hepatocytes proliferation in vitro

Hepatitis B virus (HBV) and D virus (HDV) co-infections cause the most severe form of viral hepatitis. The long-tern persistence of both viruses makes it challenging to develop curative therapies. HDV spread through cell division was reported recently (Giersch K, et al. Gut. doi:10.1136/gutjnl-2017 – 314713.), which is suggested to contribute to HDV persistence. However, the modulation of this process is unclear. Our previous investigations showed that HDV infection strongly induces IFN response via MDA5 in hepatocytes (Zhang Z, et al. J Hepatol. 2018, 69(1):25 – 35). To evaluate the role of the IFN response in the regulation of HDV spread during proliferation of hepatocytes, innate immune competent undifferentiated HepaRGNTCP cells were infected with HDV and splitted (1:6) every 5 days. HDV positive cells and viral markers (HDV RNA and antigens) were quantified throughout 6 passages. We found that all HDV-specific markers were profoundly lost during HepaRGNTCP cell division. Through BrdU labeling of DNA synthesis, we found HDV replication doesn't impair cell division. Then we blocked the IFN response by knocking down of MDA5 (the pattern recognition receptor for HDV). In contrast to the native cell line, MDA5 depleted HepaRGNTCP cells supported efficient HDV spread during cell division. MDA5 reconstitution completely reverted this phenotype. In addition, inhibitors blocking the IFN signaling pathway also significantly enhanced the HDV spread in HepaRGNTCP cells during cell division, indicating that HDV spread during cell division is restricted by innate immune responses. This finding was further confirmed in HuH7NTCP cells which are defective to produce IFNs. In contrast to HepaRGNTCP, HuH7NTCP cell division efficiently amplified HDV markers, and this amplification was significantly inhibited by exogenous IFN-alpha, -beta and -lambda1 treatment. Of note, although the endogenous IFN response suppresses HDV spread in HepaRGNTCP cells, exogenous IFN treatment can significantly promote the suppression in a dose depend manner when the innate immune system is not saturated in the case of moderate or low HDV infection rate. These results suggest that IFN response is an important restriction factor against HDV persistence during cell division. This finding also suggests that the combination therapies using IFN and drugs blocking extracellular HDV spread (e.g. entry inhibitor Myrcludex B (Bogomolov P. et al. J Hepatol. 2016. 65(3):490 – 8)) may synergistically eliminate HDV infection.