Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein

Abstract

Human MxA is an alpha/beta interferon-inducible intracytoplasmic protein that mediates antiviral activity against several RNA viruses. We had previously shown that overexpression of the hepatitis B virus (HBV) capsid led to selective downregulation of MxA gene expression, suggesting a mechanism by which the virus escapes from the host defense system (O. Rosmorduc, H. Sirma, P. Soussan, E. Gordien, P. Lebon, M. Horisberger, C. Brechot and D. Kremsdorf, J. Gen. Virol. 80:1253-1262, 1999). In the present study, we investigated the antiviral activity of MxA protein against HBV. MxA-expressing HuH7 clones were established and transiently transfected with HBV, and viral replication was then studied. Viral protein secretion was profoundly reduced in MxA-expressing clones by 80% for HBV surface antigen (HBsAg) and 70% for HBV e antigen (HBeAg). The levels of intracytoplasmic HBsAg and HBeAg were reduced by about 80 and 50% in the two MxA-positive clones tested. A nearly complete disappearance of HBV DNA replicative intermediates was observed in MxA-expressing clones. Although the expression of total viral RNAs was not modified, two- to fourfold reductions in HBV cytoplasmic RNAs were found in MxA-expressing clones. This suggests the inhibition of HBV replication at a posttranscriptional level. Indeed, using the well-characterized posttranscriptional regulation element (PRE) reporter system, we were able to demonstrate a marked reduction (three- to eightfold) in the nucleocytoplasmic export of unspliced RNA in MxA-expressing clones. In addition, MxA protein did not interact with HBV nucleocapsid or interfere with HBV nucleocapsid formation. Our results show an antiviral effect of MxA protein on a DNA virus for the first time. MxA protein acts, at least in part, by inhibiting the nucleocytoplasmic export of viral mRNA via the PRE sequence.

FIG. 1

Analysis of MxA clones. (a) Expression of MxA protein in stably transfected HuH7 clones. Western blot analysis of cytoplasmic extracts from HuH7 clones expressing the neomycin resistance gene, untreated (Neo), or treated (Neo + IFN) with IFN-α for 20 h, and from two MxA-expressing clones (MxA6 and MxA10) using a monoclonal mouse anti-MxA antibody. Level of protein expression was monitored by using a mouse monoclonal anti-GAPDH antibody. (b) Typical curve of cell growth obtained for neo (Neo) and MxA-positive (MxA6 and MxA10) clones. Cells were seeded at a confluence of 20,000 cells per well of six-well plates, trypsinized, and counted daily. Experiments were done in duplicate at least twice, and bars show standard errors.

FIG. 2

Inhibition of HBV protein synthesis in MxA-expressing clones. MxA clones (MxA6 and MxA10) and untreated (Neo) or IFN-treated (Neo + IFN) neo clones were transfected with pTHBV1.1 plasmid as described in Materials and Methods. Two (d2) or 3 days (d3) after transfection, culture supernatants (a) and cell lysates (b) were collected and assessed for HBs, HBc, and HBe Ag expression. The values shown are percentages of the value obtained for the HBV-transfected neo clone. They were calculated as the mean of the optical density of each experiment normalized to the β-galactosidase activity, as described in Materials and Methods. Bars show the standard errors of at least three independent experiments.

FIG. 3

MxA inhibits HBV DNA replication. Southern blot analysis of core-extracted HBV DNA after transient transfection of untreated (Neo) or IFN-treated (Neo + IFN) neo clone and MxA clones (MxA6 and MxA10) with the pTHBV1.1 plasmid. The DNA was loaded onto a 0.8% agarose gel according to the transfection efficiency and then blotted to nylon membranes. Blots were hybridized with a ³²P-labeled HBV probe. The arrows indicate relaxed circular (RC), linear double-stranded (DS), and single-stranded (SS) HBV DNA forms. Histograms express the values of the scanned bands as described in Materials and Methods.

FIG. 4

MxA selectively reduces cytoplasmic HBV RNAs. Northern blot analysis of total (a) and cytoplasmic (b) RNAs after transient transfection of MxA clones (MxA6 and MxA10) and untreated (Neo) or IFN-treated (Neo + IFN) neo clones with the pTHBV1.1 plasmid. Bands corresponding to the 3.5-, 2.4-, and 2.1-kb viral RNAs are indicated (arrows). The blots were stripped and rehybridized with a ³²P-labeled GAPDH probe for normalization. Histograms express the values of the scanned specific HBV bands, normalized to the corresponding scanned GAPDH bands and β-galactosidase values.

FIG. 5

MxA inhibition of the nuclear export of RNA mediated by the HBV PRE sequence. (a) neo clone, untreated (Neo) or treated with IFN (Neo/IFN), and MxA-positive clones (MxA6 and MxA10) were transfected with pRSVPDM138-CAT [PRE(−)] and with pRSVPRE-CAT [PRE(+)]. (b) neo, MxA6, and MxA10 clones were transfected with the pRSV-CAT (CAT) plasmid. Histograms represent the values of CAT activity (counts per minute) normalized to the transfection efficiency (β-galactosidase [β-Gal] activity). Bars show the standard errors of at least two independent experiments, each performed in duplicate.

FIG. 6

MxA protein does not interact with HBV nucleocapsid. (a) MxA protein interactions with HBV core protein were analyzed by cosedimentation assay. Lysates from 3T3 cells stably expressing the MxA protein alone (upper panel) or mixed with purified HBV core protein (middle panel) or with THOV-infected 3T3 cell lysate (lower panel) were subjected to glycerol gradient ultracentrifugation as described in Materials and Methods. The seven fractions collected were analyzed by Western blot using polyclonal antibodies against MxA protein (MxA), HBV core protein (HBc), and THOV nucleoprotein (NP). (b) The influence of the MxA protein on HBV capsid self-assembly into particles was analyzed by sucrose gradient assay. HuH7 cells (control) and MxA10 clone (MxA10) were transiently transfected with a vector expressing the capsid protein. Cleared lysates were subjected to ultracentrifugation through a discontinuous (15 to 60%) sucrose gradient as described in Materials and Methods. Eleven fractions were collected and analyzed for HBcAg expression by western blot using a polyclonal rabbit anti-HBe/c antibody. The size of each protein is indicated.