2013 Cell-Type-Specific Activation of the Oligoadenylate Synthetase-RNase L Pathway by a Murine Coronavirus

12 Pages·2013·2.01 MB·English

Published Ahead of Print 22 May 2013.
2013, 87(15):8408. DOI: 10.1128/JVI.00769-13.
J. Virol.
Grinspan, Robert H. Silverman and Susan R. Weiss
Kristine M. Rose, Judith M. Phillips, Yize Li, Judith
Ling Zhao, L. Dillon Birdwell, Ashley Wu, Ruth Elliott,
Pathway by a Murine Coronavirus
RNase L
−
Oligoadenylate Synthetase
Cell-Type-Specific Activation of the

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Cell-Type-Specific Activation of the Oligoadenylate Synthetase–RNase
L Pathway by a Murine Coronavirus

Ling Zhao,a* L. Dillon Birdwell,a Ashley Wu,a Ruth Elliott,a Kristine M. Rose,a Judith M. Phillips,a Yize Li,a Judith Grinspan,b,c

Robert H. Silverman,d Susan R. Weissa

Departments of Microbiology,a Neurology,b Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA; Department of Neurology, c

Children’s Hospital of Philadelphia, Pennsylvania, USA; Department of Cancer Biology,d Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA

Previous studies have demonstrated that the murine coronavirus mouse hepatitis virus (MHV) nonstructural protein 2 (ns2) is a
2=,5=-phosphodiesterase that inhibits activation of the interferon-induced oligoadenylate synthetase (OAS)-RNase L pathway.
Enzymatically active ns2 is required for efficient MHV replication in macrophages, as well as for the induction of hepatitis in
C57BL/6 mice. In contrast, following intranasal or intracranial inoculation, efficient replication of MHV in the brain is not de-
pendent on an enzymatically active ns2. The replication of wild-type MHV strain A59 (A59) and a mutant with an inactive phos-
phodiesterase (ns2-H126R) was assessed in primary hepatocytes and primary central nervous system (CNS) cell types—neurons,
astrocytes, and oligodendrocytes. A59 and ns2-H126R replicated with similar kinetics in all cell types tested, except macrophages
and microglia. RNase L activity, as assessed by rRNA cleavage, was induced by ns2-H126R, but not by A59, and only in macro-
phages and microglia. Activation of RNase L correlated with the induction of type I interferon and the consequent high levels of
OAS mRNA induced in these cell types. Pretreatment of nonmyeloid cells with interferon restricted A59 and ns2-H126R to the
same extent and failed to activate RNase L following infection, despite induction of OAS expression. However, rRNA degrada-
tion was induced by treatment of astrocytes or oligodendrocytes with poly(I·C). Thus, RNase L activation during MHV infection
is cell type specific and correlates with relatively high levels of expression of OAS genes, which are necessary but not sufficient for
induction of an effective RNase L antiviral response.

T

he murine coronavirus mouse hepatitis virus (MHV) is an
enveloped, positive-strand RNA virus of the coronavirus fam-
ily within the nidovirus order. MHV is a collection of strains with
tropisms for different organs, including the liver and central ner-
vous system (CNS), and thus provides models for the study of
acute encephalitis and hepatitis, as well as chronic demyelinating
disease. The MHV-A59 strain (A59) used in this study induces
mild encephalitis and moderate hepatitis. Studies of the patho-
genesis of MHV strains and recombinant chimeric MHVs have
shown that postentry virus-host interactions have significant im-
pact on organ tropism and virulence in MHV-infected mice (1, 2).
The type I interferon (IFN) response is an early innate response
that is crucial to survival of mice following infection with many
viruses, including MHV (3–5). During infection, viral double-
stranded RNA (dsRNA) is recognized by pattern recognition re-
ceptors, such as MDA5 in the case of MHV in most cell types
(3–5); this leads to the synthesis of type I IFN (Fig. 1). Alpha/beta
IFN (IFN-�/�) induces expression of interferon-stimulated genes
(ISGs) encoding pattern recognition receptors, transcription fac-
tors, and antiviral effectors, including multiple oligoadenylate
synthetase (OAS) proteins. Viral dsRNA directly binds to and ac-
tivates OAS to synthesize 2=,5=-linked oligoadenylates (2-5A),
which induce RNase L dimerization and activity (6–9). RNase L is
a particularly potent antiviral effector in that it both directly
cleaves viral and host RNA, thereby reducing the amount of viral
RNA available for replication and packaging of the genome into
progeny virus, and inhibits host and viral protein synthesis. In
addition, RNase L-mediated cleavage generates additional small
RNAs that can be recognized by cytoplasmic pattern recognition
receptors, further amplifying IFN synthesis (7). Importantly, and
unlike most other IFN-induced activities, activation of the OAS-
RNase L pathway requires both viral infection (production of

dsRNA) and type I IFN exposure (upregulation of OAS) in the
same cell in order to produce sufficient 2-5A to activate RNase L.
Many viruses, including MHV, have evolved mechanisms to
avoid and/or antagonize host type I IFN responses (10), including
the OAS-RNase L pathway (7). We have shown previously that the
A59 accessory protein nonstructural protein 2 (ns2) is a type I IFN
antagonist. ns2 has 2=,5=-phosphodiesterase activity that cleaves
2-5A, thereby antagonizing the activation of the OAS-RNase L
pathway (11). An ns2 mutant of A59 (ns2-H126R), expressing an
inactive phosphodiesterase, was unable to replicate efficiently in
macrophages derived from C57BL/6 (B6) mice, indicating that an
active ns2 is required for robust replication of A59 in that cell type.
However, ns2-H126R was able to replicate efficiently in macro-
phages derived from mice deficient in either type I interferon re-
ceptor expression (IFNAR�/�) or RNase L expression (RNase
L�/�) (2, 11). These and other published data (2, 11) led us to
conclude that ns2 antagonizes IFN signaling by downregulating
RNase L activation. Furthermore, in vivo, ns2 acts as an organ-
specific virulence factor that is required for efficient hepatic virus
replication and pathology in B6 mice (2, 12). Macrophage deple-

Received 19 March 2013 Accepted 15 May 2013

Published ahead of print 22 May 2013

Address correspondence to Susan R. Weiss,