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. 2008 Dec;82(24):12325-34.
doi: 10.1128/JVI.02082-08. Epub 2008 Oct 15.

Mouse hepatitis virus liver pathology is dependent on ADP-ribose-1''-phosphatase, a viral function conserved in the alpha-like supergroup

Klara Kristin Eriksson  1 , Luisa Cervantes-Barragán, Burkhard Ludewig, Volker Thiel
Affiliations

Mouse hepatitis virus liver pathology is dependent on ADP-ribose-1''-phosphatase, a viral function conserved in the alpha-like supergroup

Klara Kristin Eriksson et al. J Virol. 2008 Dec.

Abstract

Viral infection of the liver can lead to severe tissue damage when high levels of viral replication and spread in the organ are coupled with strong induction of inflammatory responses. Here we report an unexpected correlation between the expression of a functional X domain encoded by the hepatotropic mouse hepatitis virus strain A59 (MHV-A59), the high-level production of inflammatory cytokines, and the induction of acute viral hepatitis in mice. X-domain (also called macro domain) proteins possess poly-ADP-ribose binding and/or ADP-ribose-1''-phosphatase (ADRP) activity. They are conserved in coronaviruses and in members of the "alpha-like supergroup" of phylogenetically related positive-strand RNA viruses that includes viruses of medical importance, such as rubella virus and hepatitis E virus. By using reverse genetics, we constructed a recombinant murine coronavirus MHV-A59 mutant encoding a single-amino-acid substitution of a strictly conserved residue that is essential for coronaviral ADRP activity. We found that the mutant virus replicated to slightly reduced titers in livers but, strikingly, did not induce liver disease. In vitro, the mutant virus induced only low levels of the inflammatory cytokines tumor necrosis factor alpha and interleukin-6 (IL-6). In vivo, we found that IL-6 production, in particular, was reduced in the spleens and livers of mutant virus-infected mice. Collectively, our data demonstrate that the MHV X domain exacerbates MHV-induced liver pathology, most likely through the induction of excessive inflammatory cytokine expression.

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Figures

FIG. 1.
FIG. 1.
Generation of MHV-N1348. (a) Alignment of macro domain sequences. The presented alignment was produced by using AlignX of Vector-NTI-9 with manual adjustment and cross verification with previously published data (9, 10, 20, 22). Highlighted are amino acid stretches that line the substrate binding pocket (asterisks) (22) and MHV-A59 asparagine1348 (N1348A, arrowhead). MHV-A59, AY700211; SARS-CoV Frankfurt-1, AY291315; HCoV-229E, AF304460; infectious bronchitis virus Beaudette (IBV), NC_001451; SFV clone 4 (SFV-4), NC_003215; HEV genotype 1 (HEV-1), D10330; human poly-ADP-ribose-polymerase-9 (hPARP9), NM_031458; rubella virus (RubV), Q6X2V4. (b) Graphic representation of MHV-A59 genome organization. Replicase open reading frames and nsps 1 to 16 are depicted with papain-like proteinase (black triangles) and 3C-like proteinase (gray triangles) cleavage sites. Domains shown include ADRP; acidic domain (Ac); papain-like proteinase 1 (PL1); papain-like proteinase 2 (PL2); a domain of unknown function (Y) (10); 3C-like proteinase (3CL); RNA-dependent RNA polymerase (RdRp); NTPase (N); RNA helicase (Hel); 3′-to-5′ exoribonuclease (ExoN); endoribonuclease (En); and 2′-O-methyltransferase (MT). (c) Growth kinetics of MHV-A59 (MHV wt) and MHV-N1348A in L929 and 17Cl1 cells (MOI = 1). Data shown are mean values ± standard errors of the means (SEMs) from representative experiments performed in quadruplicate (L929) or duplicate (17Cl1).
FIG. 2.
FIG. 2.
MHV-N1348A does not cause acute viral hepatitis. (a) B6 mice (n = 3 to 10) were infected with 5, 500, or 50,000 PFU of wild-type MHV-A59 (MHV wt) or MHV-N1348A. Viral titers in spleens and livers and serum ALT values were determined at the indicated time points p.i. Statistical analyses of titers and ALT values were performed, using Student's t test and the Mann-Whitney nonparametric test, respectively (*, P < 0.05; **, P < 0.01). d.p.i., day p.i. (b) Hematoxylin and eosin staining of 4% formaldehyde-fixed liver sections at day 5 p.i of B6 mice infected with 500 PFU (magnification, ×200; inset, ×400) of wild-type MHV-A59 or MHV-N1348A.
FIG. 3.
FIG. 3.
MHV-N1348A replication in macrophages and DCs. (a) Peritoneal macrophages, a Kupffer cell line, and bone marrow-derived cDCs and pDCs were infected with wild-type MHV-A59 or MHV-N1348A (MOI = 1). Viral titers in supernatants were measured at the indicated time points. The data shown represent mean values ± SEMs from two independent experiments. (b) Flow cytometric analysis of frequency of GFP-expressing bone marrow-derived pDCs (B220+ CD11chigh mPDCA+) after infection with MHV-GFP or MHV-N1348A-GFP (MOI = 10). The values indicated in the upper right quadrants are the percentages of GFP-positive cells.
FIG. 4.
FIG. 4.
MHV-N1348A and type I IFNs. (a) Bone marrow-derived pDCs and cDCs, as well as peritoneal macrophages, were infected with wild-type MHV-A59 (MHV wt) or MHV-N1348A (MOI = 1). The levels of IFN-α in the supernatants were measured at the indicated time points. For macrophages and cDCs, the data represent the mean values ± SEMs from two independent experiments. For pDCs, the data represent the mean values ± SEMs from one experiment representative of three. (b) Bone marrow-derived cDCs and peritoneal macrophages were pretreated with the indicated amounts of IFN-α for 4 h and then infected with wild-type MHV-A59 or MHV-N1348A (MOI = 0.01 and 1). The titers in the supernatants were determined at 18 h p.i. The data represent the mean values ± SEMs from a quadruplicate experiment. (c) B6, B6-IFNAR−/−, 129Sv, and A129 mice (n = 6 to 9) were infected with 5 PFU of wild-type MHV-A59 or MHV-N1348A. The viral titers in the spleen and liver and serum ALT levels were measured at 48 h p.i. Statistical analyses of titers and ALT levels were performed, using Student's t test and the Mann-Whitney nonparametric test, respectively (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (d) Hematoxylin and eosin staining of 4% formaldehyde-fixed liver sections at 48 h p.i of A129 mice infected with 5 PFU of wild-type MHV-A59 or MHV-N1348A (×200; inset, ×400).
FIG. 5.
FIG. 5.
MHV-N1348A-induced inflammatory cytokine expression in vitro. (a and b) Peritoneal macrophages, a Kupffer cell line, and bone marrow-derived cDCs and pDCs were infected with wild-type MHV-A59 (MHV wt) or MHV-N1348A (MOI = 1). TNF-α and IL-6 production in supernatants was measured by ELISA at the indicated time points. The data show mean values ± SEMs for representative experiments performed in triplicate (macrophages, Kupffer cell line, and cDC) or duplicate (pDC). Statistical analyses were performed using Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG. 6.
FIG. 6.
MHV-N1348A-induced IFN-α and inflammatory cytokine expression in vivo. 129Sv mice (n = 5) were infected with 5 × 104 PFU of wild-type MHV-A59 (MHV wt) or MHV-N1348A. The levels of IFN-α (a), TNF-α (b), and IL-6 (c) in spleens, livers, and sera were determined 4 days p.i. by ELISA. Statistical analyses were performed by using the Mann-Whitney nonparametric test (*, P < 0.05).
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