Inhibition of hepatitis B virus replication during adenovirus and cytomegalovirus infections in transgenic mice

Abstract

We have previously demonstrated that hepatitis B virus (HBV) replication and gene expression are abolished in the livers of HBV transgenic mice by cytotoxic T lymphocytes (CTLs) and during lymphocytic choriomeningitis virus (LCMV) infection, stimuli that trigger the production of alpha/beta interferon, gamma interferon, and tumor necrosis factor alpha in the liver. We now report that hepatic HBV replication and gene expression are inhibited by the local induction of these cytokines during adenovirus- and murine cytomegalovirus (MCMV)-induced hepatitis. Further, we show that MCMV also blocks HBV replication and gene expression in the proximal convoluted tubules of the kidney by causing interstitial nephritis and inducing the same cytokines in the renal parenchyma. These results suggest that inflammatory cytokines probably contribute to viral clearance during acute viral hepatitis in humans, and they imply that induction of these cytokines in the liver and other infected tissues of chronically infected patients might have therapeutic value.

FIG. 1

Adenovirus infection inhibits hepatic HBV replication and induces the expression of cytokine genes and T-cell markers in the liver. Age-, sex-, and serum HBsAg-matched HBV transgenic mice were injected intravenously (via the lateral tail vein) with 5 × 10⁸, 1.5 × 10⁹, or 5.0 × 10⁹ PFU of Ad.CBlacZ, and livers were harvested from each of three mice sacrificed on days 1, 3, 7, 14, 24, and 42 after infection, as indicated. Northern blot analysis (A) was performed with 20 μg of total liver RNA from one representative mouse per group. The membrane was cohybridized with ³²P-labeled HBV- and GAPDH-specific DNA probes. The steady-state HBV and GAPDH mRNA content was compared with total hepatic RNA from two saline-injected control mice (time zero). Bands corresponding to the 3.5- and 2.1-kb HBV mRNAs are indicated. Southern blot analysis (B) was performed with 30 μg of total liver DNA isolated from the same mice. All DNA samples were treated with RNase A before their concentrations were determined. Bands corresponding to the integrated transgene (Int. Trans.) and the relaxed circular (RC), double-stranded (DS) linear, and single-stranded (SS) linear HBV DNA replicative forms are indicated. The integrated transgene can be used to normalize the amount of DNA bound to the membrane. The membrane was hybridized with a ³²P-labeled HBV-specific DNA probe. Total liver RNA (20 μg) from the same mice was analyzed by Northern blot analysis for the expression of 2′5′-OAS (C), a marker of IFN-α/β induction. The housekeeping enzyme GAPDH was used to normalize the amount of RNA loaded in each lane, and its expression was uniform in all mice (not shown). Total hepatic RNA (10 μg) from the same Ad.CBlacZ-infected transgenic mice was also analyzed by RNase protection (D) for the expression of IFN-γ and TNF-α cytokine transcripts and for the expression of CD3γ, CD4, and CD8α, as indicated. The mRNA encoding the ribosomal protein L32 was used to normalize the amount of RNA loaded in each lane. The sALT activity, measured at the time of autopsy, is indicated for each mouse and is expressed in units/liter. Fresh frozen liver sections from each animal were stained for β-galactosidase activity, and the percentage of lacZ-positive hepatocytes in each section is indicated.

FIG. 2

Expression of HBcAg in the livers and kidneys of HBV transgenic mice after adenovirus and MCMV infections, and lacZ expression in the same tissues of adenovirus-infected mice. Immunohistochemical analysis of HBcAg expression in liver (A to C) and kidney (E to G) sections from a saline-injected control animal (A and E) and from animals sacrificed 3 days after infection with 2.0 × 10⁴ PFU of MCMV (B and F) or 7 days after infection with 1.5 × 10⁹ PFU of Ad.CBlacZ (C and G) was performed. X-Gal histochemistry of a frozen liver section (D) from an animal sacrificed 1 day after infection with 5.0 × 10⁹ PFU of Ad.CBlacZ and of a frozen kidney section (H) from a mouse sacrificed 7 days after infection with the same dose is also shown. CV, central vein; PV, portal vein.

FIG. 3

MCMV infection inhibits hepatic HBV replication and gene expression and induces the expression of cytokine genes and T-cell markers in the liver. HBV transgenic mice were injected intraperitoneally with 2.0 × 10⁴ PFU of salivary gland-passaged MCMV, and livers were harvested from each of three mice sacrificed on days 1, 3, 5, 7, and 14 after infection, as indicated. Total hepatic RNA and DNA were extracted, analyzed by Northern and Southern blotting, respectively, for the expression of HBV mRNAs, 2′5′-OAS mRNA, and HBV DNA replicative forms, and compared with total liver RNA and DNA from two mice injected with a salivary gland homogenate from uninfected BALB/c mice (time zero). Northern blot (A and C) and Southern blot (B) analyses were performed exactly as described in the legend to Fig. 1 for two representative mice per group. Total RNA (10 μg) from the same livers was analyzed by RNase protection (D) for the expression of IFN-γ and TNF-α cytokine transcripts and for the expression of CD3γ, CD4, and CD8α, as indicated. The mRNA encoding the ribosomal protein L32 was used to normalize the amount of RNA loaded in each lane. The mean sALT activity, measured at the time of autopsy, is indicated for each group and is expressed in units/liter. A 10% (wt/vol) liver homogenate was prepared, and MCMV titers were quantitated by plaque assay on NIH 3T3 cell monolayers. The mean MCMV titers in the liver are shown for each group and are expressed in PFU/milliliter of tissue homogenate. Abbreviations are as defined in the legend to Fig. 1.

FIG. 4

Infection with MCMV, but not adenovirus, inhibits renal HBV replication. Kidneys were harvested from each of three to four HBV transgenic mice sacrificed 7 days after infection with 5.0 × 10⁹ PFU of Ad.CBlacZ or 3 days after infection with 2.0 × 10⁴ PFU of MCMV. Total renal DNA and RNA were extracted from one kidney of each animal, analyzed by Southern and Northern blotting, respectively, for the expression of HBV DNA replicative forms and 2′5′-OAS mRNA, and compared with total renal DNA and RNA from mock-infected control mice (time zero). Southern (A) and Northern (B) blot analyses were performed exactly as described in the legend to Fig. 1 for two representative mice per group. Abbreviations are as defined in the legend to Fig. 1.

FIG. 5

Early suppression of HBV replication by adenovirus and MCMV infections is mediated by IFN-α/β and TNF-α. Groups of three HBV transgenic mice were intraperitoneally injected with a combination of antibodies to IFN-α/β and TNF-α or with control antibodies 6 h before infection with 5.0 × 10⁴ PFU of MCMV or 1.5 × 10⁹ PFU of Ad.CBlacZ and were sacrificed 24 h after infection. Total hepatic DNA and RNA were extracted, analyzed by Southern and Northern blotting, respectively, for the expression of HBV DNA replicative forms and 2′5′-OAS mRNA, and compared with total hepatic DNA and RNA from two saline-injected control mice (lanes NaCl). Southern (A) and Northern (B) blot analyses were performed exactly as described in the legend to Fig. 1 for two representative mice per group. Abbreviations are as defined in the legend to Fig. 1.