Inhibition of hepatitis B virus replication by interferon requires proteasome activity

Abstract

Hepatitis B virus (HBV) replication is inhibited in a noncytopathic manner by alpha/beta interferon (IFN-alpha/beta) and IFN-gamma. We demonstrate here that inhibitors of cellular proteasome activity can block this antiviral effect. These results suggest that a critical component of the IFN-induced antiviral response may be the proteasome-dependent degradation of viral or cellular proteins that are required for HBV replication.

FIG. 1.

Lactacystin blocks IFN-β-mediated inhibition of HBV replication. Differentiated HBV-Met (clone 1-1.4) cells were pretreated for 1 h with 20 μM lactacystin followed by addition of 500 U of IFN-β/ml. Cells were harvested at the indicated time points post-addition of IFN-β, and total DNA and RNA were prepared for Southern blot (SB) and Northern blot (NB) analysis of HBV DNA replicative intermediates and 2′5′-OAS/GAPDH expression. Tg, integrated transgene; RC and SS, relaxed-circle and single-stranded HBV DNA replicative forms.

FIG. 2.

Inhibition of antiviral effect by additional proteasome inhibitors. Differentiated HBV-Met cells were pretreated for 1 h with 50 μM MG-132 or 7.5 μM epoxomicin (A) or 50 μM NLVS (B) followed by addition of 500 U of IFN-β/ml. Cells were harvested at the indicated time points post-addition of IFN-β, and total DNA and RNA were prepared for Southern blot (SB) and Northern blot (NB) analysis of HBV DNA replicative intermediates and 2′5′-OAS/GAPDH expression. Tg, integrated transgene; RC and SS, relaxed-circle and single-stranded HBV DNA replicative forms.

FIG. 3.

Lactacystin inhibits IFN-γ-induced antiviral effect. Differentiated HBV-Met cells were pretreated for 1 h with 20 μM lactacystin followed by addition of 1,000 U of IFN-γ/ml. Cells were harvested at the indicated time points post-addition of IFN-γ, and total DNA and RNA were prepared for Southern blot (SB) and Northern blot (NB) analysis of HBV DNA replicative intermediates and 2′5′-OAS/GAPDH expression. Tg, integrated transgene; RC and SS, relaxed-circle and single-stranded HBV DNA replicative forms.

FIG. 4.

Reversible and irreversible inhibition of antiviral effect. Differentiated HBV-Met cells were pretreated for 1 h with 20 μM lactacystin or 50 μM MG-132 followed by addition of 500 U of IFN-β/ml. In the “[12] + 24”-h lanes, the cells were cultured with IFN-β and the proteasome inhibitor for 12 h, followed by a 24-h incubation in the presence of IFN-β alone. Cells were harvested at the indicated time points post-addition of IFN-β, and total DNA and RNA were prepared for Southern blot (SB) and Northern blot (NB) analysis of HBV DNA replicative intermediates and 2′5′-OAS/GAPDH expression. Tg, integrated transgene; RC and SS, relaxed-circle and single-stranded HBV DNA replicative forms.

FIG. 5.

Proteasome inhibition blocks IFN-β-induced disappearance of HBV capsids. Differentiated HBV-Met cells were pretreated for 1 h with 5 μM epoxomicin followed by addition of 500 U of IFN-β/ml and were harvested at the indicated time points post-addition of IFN-β for DNA and cytoplasmic protein preparations. The amount of DNA replicative intermediates was determined by Southern blot (SB) analysis, while the levels of viral capsids and encapsidated HBV DNA were assessed by agarose gel electrophoresis followed by Southern blot for HBV DNA or Western blot (WB) with an anti-HBV core polyclonal antibody. The “No DMSO” lane corresponds to HBV-Met cells that were cultured in the absence of 2% DMSO and therefore do not contain HBV DNA or HBcAg. Tg, integrated transgene; RC and SS, relaxed-circle and single-stranded HBV DNA replicative forms; HBcAg, HBV core antigen.