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. 2016 Oct 14;90(21):9826-9832.
doi: 10.1128/JVI.01423-16. Print 2016 Nov 1.

Antagonism of RNase L Is Required for Murine Coronavirus Replication in Kupffer Cells and Liver Sinusoidal Endothelial Cells but Not in Hepatocytes

Yize Li  1 , Susan R Weiss  2
Affiliations

Antagonism of RNase L Is Required for Murine Coronavirus Replication in Kupffer Cells and Liver Sinusoidal Endothelial Cells but Not in Hepatocytes

Yize Li et al. J Virol. .

Abstract

Mouse hepatitis virus strain A59 infection of mice is a useful tool for studying virus-host interaction during hepatitis development. The NS2H126R mutant is attenuated in liver replication due to loss of phosphodiesterase activity, which the wild-type (WT) virus uses to block the 2',5'-oligoadenylate synthetase (OAS)-RNase L (RNase L) antiviral pathway. The activation of RNase L by NS2H126R is cell type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We tested the hypothesis that the resident liver macrophages, Kupffer cells (KC), represent the cell type most likely to restrict NS2H126R and prevent hepatitis. As found previously, A59 and NS2H126R replicate similarly in hepatocytes and neither activates RNase L, as assessed by an rRNA degradation assay. In contrast, in KC, A59 exhibited a 100-fold-higher titer than NS2H126R and NS2H126R induced rRNA degradation. Interestingly, in liver sinusoidal endothelial cells (LSEC), the cells that form a barrier between blood and liver parenchymal cells, NS2H126R activates RNase L, which limits viral replication. Similar growth kinetics were observed for the two viruses in KC and LSEC from RNase L-/- mice, demonstrating that both use RNase L to limit NS2H126R replication. Depletion of KC by gadolinium(III) chloride or of LSEC by cyclophosphamide partially restores liver replication of NS2H126R, leading to hepatitis. Thus, during mouse hepatitis virus (MHV) infection, hepatitis, which damages the parenchyma, is prevented by RNase L activity in both KC and LSEC but not in hepatocytes. This may be explained by the undetectable levels of RNase L as well as by the OASs expressed in hepatocytes.

Importance: Mouse hepatitis virus infection of mice provides a useful tool for studying virus-host interactions during hepatitis development. The NS2H126R mutant is attenuated in liver replication due to loss of phosphodiesterase activity, by which the wild-type virus blocks the potent OAS-RNase L antiviral pathway. RNase L activation by NS2H126R is cell type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We showed that the hepatocytes that comprise the liver parenchyma do not activate RNase L when infected with NS2H126R or restrict replication. However, both Kupffer cells (KC) (i.e., the liver-resident macrophages) and the liver sinusoidal endothelial cells (LSEC) which line the sinusoids activate RNase L in response to NS2H126R These data suggest that KC and LSEC prevent viral spread into the parenchyma, preventing hepatitis. Furthermore, hepatocytes express undetectable levels of OASs and RNase L, which likely explains the lack of RNase L activation during NS2H126R infection.

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Figures

FIG 1
FIG 1
Isolation and characterization of hepatocytes (Hep), KC, and LSEC. Hep, KC, and LSEC were isolated as described in Materials and Methods. (A) Hep were stained with mouse anti-human HNF-4α followed by secondary Alexa Fluor 488-conjugated goat anti-mouse antibody (green). (B) DAPI staining (blue). (C) The two stains merged. (D and E) KC (D) and LSEC (E) were stained with Alexa Fluor 488-conjugated anti-CD68 antibodies (green) and with DAPI (blue) to identify the nuclei. (F) LSEC were incubated with Alexa Fluor 488-conjugated LDL (green) in serum-free media at 5 μg/ml for 4 h, and Hoechst (blue) was added into the media to stain the nucleus 30 min before imaging.
FIG 3
FIG 3
RNase L is activated in BMM, KKC, and LSEC but not in hepatocytes (Hep) during infection with NS2H126R. BMM, hepatocytes, KC, and LSEC were infected with MHV A59 and ns2H126R (MOI = 1). At 12 h (BMM and KC), 13 h (LSEC), or 15 h (Hep) postinfection, RNA was harvested and analyzed by a Bioanalyzer for rRNA integrity. The positions of 28S and 18S rRNA are indicated. Data shown are from one representative of two independent experiments.
FIG 2
FIG 2
The replication of NS2H126R is limited in KC and LSEC isolated from B6 mice but not from RNase L−/− mice. Hepatocytes (Hep) from B6 mice (A), KC from B6 mice (B), KC from RNase L−/− mice (C), LSEC from B6 mice (D), and LSEC from RNase L−/− mice (E) were infected with A59 and NS2H126R (MOI = 1, n = 3). At the indicated time points, infectious virus was quantified by plaque assay on L2 cells from the combined cell lysates and supernatants. Data shown were analyzed by the two-tailed Student t test and expressed as means ± standard deviations (SD) (***, P<0.001) and are from one representative of two independent experiments.
FIG 4
FIG 4
Depletion of KC or LSEC partially restores NS2H126R replication and liver pathogenicity. B6 mice were intravenously injected with 20 mg/kg of gadolinium(III) chloride (KC depletion) or intraperitoneally injected with 300 mg/kg of cyclophosphamide (LSEC depletion) or with saline solution as a control (n = 5 for each group). Mice were infected with 200 PFU of virus intrahepatically at 24 h postinjection and sacrificed 5 days postinfection. (A) Virus was quantified from liver lysates by plaque assay on L2 cells. The data were analyzed by the Mann-Whitney test and expressed as means ± SD (**, P < 0.01; ***, P < 0.001 [titers in depleted mice compared to the saline control]). (B) Liver sections from NS2H126R-infected mice were stained with hematoxylin and eosin for histological analysis. Arrows indicate foci of pathology. Data shown are from one representative of two independent experiments.
FIG 5
FIG 5
Expression levels of OAS1a, OAS2, OAS3, and RNase L were below the level of detection in hepatocytes. Hepatocytes (Hep), KC, and LSEC were mock treated or treated with IFN-α (1,000 U/ml) overnight. Cells were lysed, and proteins were analyzed by immunoblotting with antibodies against OAS1a, OAS2, OAS3, RNase L, and GAPDH. The positions and molecular masses of the indicated proteins are indicated with arrows. Data shown are from one representative of two independent experiments. The additional bands detected with OAS2 antibody in all cell types and with OAS3 antibody in Hep and LSEC are not induced by IFN as expected for OAS proteins and are nonspecific (10).
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