Interleukin-1 and tumor necrosis factor-α trigger restriction of hepatitis B virus infection via a cytidine deaminase activation-induced cytidine deaminase (AID).
This effect was mediated by activation of the NF-κB signaling pathway.Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity.The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus.
Affiliation: From the Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.
Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.
- Cytidine Deaminase/biosynthesis*/genetics/immunology
- Gene Expression Regulation, Enzymologic*
- Hepatitis B/genetics/immunology/metabolism*
- Hepatitis B virus/genetics/immunology/metabolism*
- Tumor Necrosis Factor-alpha/genetics/immunology/metabolism*
- DNA, Viral/biosynthesis/genetics/immunology
- Hep G2 Cells
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Figure 5: AID played a significant role in IL-1-mediated anti-HBV activity.A and B, left panels, HepaRG cells were transduced with a lentiviral vector carrying the expression plasmid for AID (RG-AID), AID(M139V) mutant (RG-AID(M139V)) (B), or the control vector (RG-EV). Protein expression for AID (upper panel) and actin (lower panel) in these cells, the parental HepaRG cells (HepaRG), and those transiently transfected with AID expression plasmid (AID overexpression) (A) was examined by immunoblot. Right panels, these cells were infected with HBV followed by detection of secreted HBs protein as Fig. 1A. AID-transduced cells were less susceptible to HBV infection. C, HepaRG cells were transduced with lentiviral vector carrying shRNAs for AID (RG-shAID#1 and RG-shAID#2) or for cyclophilin A (RG-shCyPA) as a control. AID mRNA (left panel) and protein (right panel) were quantified by real time RT-PCR and immunoblot analysis. D, cells produced in C were infected with HBV in the absence or presence of IL-1β or heparin, and HBs was detected in the medium as in Fig. 1A to examine the anti-HBV effect of IL-1β and heparin. The fold reduction of HBV infection by IL-1β treatment is shown as IL-1β anti-HBV above the graph. The white, gray, and black bars indicate HBs value of the cells without treatment and with heparin and IL-1β treatment, respectively. The anti-HBV activity of IL-1β but not heparin was reduced in the AID-knockdown cells. E, AID and its mutant suppressed HBV replication. HepG2 cells were cotransfected with GFP-tagged AID, AID(H56Y), A3G, and GFP itself along with an HBV-encoding plasmid. Following 3 days, cytoplasmic nucleocapsid HBV DNA was quantified (upper graph), and the overexpressed proteins as well as actin were detected (lower panels). F, lentiviral vectors carrying AID, AID(M139V) mutant, A3G, or an empty vector (empty vector) were transduced or left untransduced (no transduction) into HepG2.2.15 cells. Nucleocapsid associated HBV DNA in these cells or in HepG2 cells (HBV−) was detected by Southern blot (upper panel). AID (middle panel) and A3G protein (lower panel) were also detected by immunoblot. G, HBV core interacted with AID. HepAD38 cells transduced without (no transduction) or with AID-expressing vector or the empty vector (empty vector) were lysed and treated with anti-core antibody (1st panel) or control normal IgG (2nd panel) for immunoprecipitation (IP). Total fraction without immunoprecipitation (3rd to 5th panels) was also recovered to detect AID (1st to 3rd panels), HBV core (5th panel), and actin (5th panel) by immunoblot. WB, Western blot. H, HBV RNA in core particles was extracted as shown under “Experimental Procedures” in HepG2 cells overexpressing HBV DNA together with or without AID or A3G.
To examine the function of AID during HBV infection, we transduced AID ectopically into HepaRG cells using a lentiviral vector (Fig. 5A, left panel). The susceptibility of these AID-overexpressing cells to HBV was decreased by approximately one-third compared with the parental or empty vector-transduced HepaRG cells (Fig. 5A, right panel), suggesting that AID can restrict HBV infection. An AID mutant AID(M139V), with reported diminished activity to support class switching (48), also decreased the susceptibility to HBV infection, although the reduction in HBV susceptibility was moderate compared with the case of the wild type AID (Fig. 5B).