Hepatitis B virus RNA-binding proteins associated with cytokine-induced clearance of viral RNA from the liver of transgenic mice - PubMed (original) (raw)

Hepatitis B virus RNA-binding proteins associated with cytokine-induced clearance of viral RNA from the liver of transgenic mice

T Heise et al. J Virol. 1999 Jan.

Abstract

Hepatitis B virus (HBV) gene expression is downregulated in the liver of HBV transgenic mice by a posttranscriptional mechanism that is triggered by the local production of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) during intrahepatic inflammation (hepatitis). The molecular basis for this antiviral effect is unknown. In this study, we identified three HBV RNA-binding liver nuclear proteins (p45, p39, and p26) the relative abundance of which correlates with the abundance of HBV RNA in response to the induction of IFN-gamma and TNF-alpha. All three proteins bind to a 91-bp element located at the 5' end of a previously defined posttranscriptional regulatory element that is thought to mediate the nuclear export of HBV RNA. The presence of p45 correlates directly with the presence of HBV RNA, being detectable under baseline conditions when the viral RNA is abundant and undetectable when the viral RNA disappears in response to IFN-gamma and TNF-alpha. In contrast, p26 is inversely related to HBV RNA, being detectable only when the viral RNA disappears following cytokine activation. Finally, p39 is constitutively expressed, and its abundance and mobility appear to be slightly increased by cytokine activation. These results suggest a model in which hepatocellular HBV RNA content might be controlled by the stabilizing and/or destabilizing influences of these RNA-binding proteins whose activity is regulated by cytokine-induced signaling pathways.

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Figures

FIG. 1

FIG. 1

(A) Schematic map of the HBV genome showing the 3.5-kb pregenomic RNA and the 2.1-kb envelope RNA. (B) Location of the PRE (nt 1239 to 1805) (34) and position of in vitro RNA-B (nt 1243 to 1333) used in this study.

FIG. 2

FIG. 2

HBV RNA-binding protein p45 is detectable primarily in liver nuclear extracts from untreated mice, while p26 is detectable mainly in liver nuclear extracts from CTL-injected mice. Northern blot (top) and UV cross-linking (bottom) analyses of 20 μg of total liver RNA or 5 μg of liver nuclear extracts isolated and prepared from the same liver of HBV transgenic mice lineages 219 and 1.3.32 were performed as described in Materials and Methods. (A) Three sex- and serum HBsAg-matched mice (lineage 219) were intravenously injected with 107 CTLs and sacrificed at 5 days later. Results were compared with those for a mouse that was sacrificed 5 days after saline injection. (B) Three sex- and serum HBsAg-matched lineage 1.3.32 mice were intraperitoneally injected with 250 μg of hamster monoclonal antibodies against TNF-α and IFN-γ 24 h before and 2 days after intravenous injection of 107 HBsAg-specific CTLs. Two control mice were injected either with saline or with 250 μg irrelevant anti-hamster immunoglobulin G (Hα IgG). Mice were sacrificed on day 5 after CTL administration.

FIG. 3

FIG. 3

Characteristics of RNA-binding proteins p45, p39, and p26. Standard UV cross-linking experiments were performed under conditions described in Materials and Methods. Liver nuclear extracts (NE) from untreated (ut; 3 μg) or CTL-injected (2 μg) HBV transgenic mice were incubated with 40 fmol of 32P-labeled RNA-B. (A) Pretreatment of extracts with proteinase K (20 μg) was performed for 30 min at 37°C, or extracts were heated at 45°, 55°, and 75°C for 10 min before the binding reaction. UV cross-linking samples in lanes 14 and 15 were analyzed under nonreducing conditions. (B) Nuclear extracts from untreated (2 μg) or CTL-injected (2 μg) HBV transgenic mice were incubated with 40 fmol of 32P-labeled RNA-B without NaCl or at increasing concentrations of NaCl as indicated.

FIG. 4

FIG. 4

(A) p45, p39, and p26 bind specifically to HBV RNA-B. A 10-, 30-, or 60-fold molar excess of unlabeled in vitro RNA-B (upper panel, lanes 2 to 4 and 6 to 8) or 500-, 1,000-, or 1,500-fold molar excess of unlabeled mouse β-actin in vitro transcript (114 nt) (lower panel, lanes 2 to 4 and 6 to 8) was added to 1 μg of liver nuclear extract from untreated or CTL-injected mice in binding buffer just before addition of 40 fmol of [32P]UTP-labeled in vitro RNA-B. After irradiation, samples were RNase treated and analyzed by SDS-PAGE as described in Materials and Methods. (B) HBV RNA-binding proteins are present in mouse and human hepatocytes. Liver nuclear extracts (Liv.; 2 μg) from untreated HBV transgenic mice and CTL-injected HBV transgenic mice, nuclear extracts from partially (60%) purified hepatocytes prepared from untreated HBV transgenic mice (Hep; 1 μg), and nuclear extracts prepared from human liver (1 μg) and the human hepatocyte cell line HepG2 (1 μg) were used in the standard UV cross-linking analysis as described in Materials and Methods.

FIG. 5

FIG. 5

Kinetics of p45, p39, and p26 binding activities during MCMV infection. HBV transgenic mice were infected with MCMV, and livers were harvested from groups of two mice sacrificed on days 1 (d1), 3, 5, 7, 14, and 28 after infection, as indicated. Total hepatic RNA and liver nuclear extracts were prepared and then analyzed by Northern blotting, UV cross-linking (UV-Cross.), and RNase protection assay (RPA) as described in Materials and Methods. Northern blots were probed for the expression of HBV RNA, GAPDH mRNA, and 2′,5′-OAS mRNA and compared with total liver RNA prepared from two saline-injected animals. Nuclear extract (5 μg) from each mouse was incubated with 40 fmol in vitro RNA-B, processed, and analyzed by SDS-PAGE. Total RNA (10 μg) from the same livers was analyzed by RNase protection assay for the expression of TNF-α and IFN-γ. The mRNA encoding the ribosomal protein L32 was used to normalize the amount of RNA loaded in each lane.

FIG. 6

FIG. 6

RNA binding activities of p45, p39, and p26 after CTL and IL-12 injection and after MCMV, LCMV, and adenovirus infection. Groups of sex- and serum HBeAg-matched transgenic mice (two mice per group) were injected with saline or CTL and sacrificed on day 5 after injection (lineage 1.3.32; NaCl or CTL d5), infected with LCMV and sacrificed on day 5 after infection (lineage 1.3.32; LCMV d5), infected with MCMV and sacrificed on day 5 after infection (lineage 1.3.32; (MCMV d5), infected with adenovirus and sacrificed on day 7 after infection (lineage 1.3.32; Adeno d7), or injected with IL-12 daily for 3 consecutive days and sacrificed on 1 day after the third injection (IL-12 d3). Total hepatic RNA and liver nuclear extracts were prepared and analyzed as described in the legend to Fig. 5.

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References

    1. Alonso S, Minty A, Bourlet Y, Buckingham M. Comparison of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates. J Mol Evol. 1986;23:11–22. - PubMed
    1. Ando K, Guidotti L G, Cerny A, Ishikawa T, Chisari F V. CTL access to tissue antigen is restricted in vivo. J Immunol. 1994;153:482–488. - PubMed
    1. Besancon F, Przewlocki G, Baro I, Hongre A S, Escande D, Edelman A. Interferon-gamma downregulates CFTR gene expression in epithelial cells. Am J Physiol. 1994;267:C1398–C1404. - PubMed
    1. Binder R, Horowitz J A, Basilion J P, Koeller D M, Klausner R D, Harford J B. Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3′ UTR and does not involve poly(A) tail shortening. EMBO J. 1994;13:1969–1980. - PMC - PubMed
    1. Blyn L B, Swiderek K M, Richards O, Stahl D C, Semler B L, Ehrenfeld E. Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5′ noncoding region: identification by automated liquid chromatography-tandem mass spectrometry. Proc Natl Acad Sci USA. 1996;93:11115–11120. - PMC - PubMed

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