2009 Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease Ubiquitin-Like Domain and Catalytic Domain Regul

17 Pages·2009·1.03 MB·English

JOURNAL OF VIROLOGY, July 2009, p. 6689–6705
Vol. 83, No. 13
0022-538X/09/$08.00�0
doi:10.1128/JVI.02220-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease
Ubiquitin-Like Domain and Catalytic Domain Regulate Antagonism of
IRF3 and NF-�B Signaling�†

Matthew Frieman,1 Kiira Ratia,2 Robert E. Johnston,3 Andrew D. Mesecar,2 and Ralph S. Baric1*

Department of Epidemiology, Department of Microbiology and Immunology, University of North Carolina, Chapel Hill,
North Carolina 275991; Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,
University of Illinois, Chicago, Illinois 606072; and Carolina Vaccine Institute and Department of Microbiology and
Immunology, University of North Carolina, Chapel Hill, North Carolina 275993

Received 21 October 2008/Accepted 8 April 2009

The outcome of a viral infection is regulated in part by the complex coordination of viral and host
interactions that compete for the control and optimization of virus replication. Severe acute respiratory
syndrome coronavirus (SARS-CoV) intimately engages and regulates the host innate immune responses during
infection. Using a novel interferon (IFN) antagonism screen, we show that the SARS-CoV proteome contains
several replicase, structural, and accessory proteins that antagonize the IFN pathway. In this study, we focus
on the SARS-CoV papain-like protease (PLP), which engages and antagonizes the IFN induction and NF-�B
signaling pathways. PLP blocks these pathways by affecting activation of the important signaling proteins in
each pathway, IRF3 and NF-�B. We also show that the ubiquitin-like domain of PLP is necessary for pathway
antagonism but not sufficient by itself to block these pathways regardless of the enzymatic activity of the
protease. The potential mechanism of PLP antagonism and its role in pathogenesis are discussed.

The outcome of a viral infection is mediated, in part,
through the complex interplay of viral and cellular components
that coordinate the innate immune response. During virus
entry and replication, the innate immune response machinery
senses the invading virus and activates a cascade of signaling
pathways that ultimately concludes with the translation of sev-
eral hundred antiviral proteins to convert the intracellular en-
vironment into a suboptimal context for replication (32). In
response to this powerful selective environment, viruses have
evolved strategies to disable the host’s innate immune arsenal
and optimize the intracellular environment for efficient virus
replication and release. Screens for interferon (IFN) antago-
nist activity of various viruses have shown that most viruses,
including all highly pathogenic human viruses tested, attempt
to modulate the innate immune response early in infection
(25).
The innate immune response is a series of signaling cascades
that are activated once a foreign pathogen is detected in the
host (34, 45). The response is initiated by cytoplasmic protein
sensors, such as RIG-I (retinoic acid-inducible gene I) and
MDA5, which bind to by-products of viral entry and replication
in the cell (2, 28, 51, 59). During viral infection, RIG-I and
MDA5 bind to viral single-stranded RNA and/or double-
stranded RNA (dsRNA) and signal through a protein called
MAVS (mitochondrial antiviral signaling protein) (51, 59).
MAVS activation leads to the activation of IKKi and TBK1,

the kinases which phosphorylate IRF3 to induce its dimeriza-
tion and import into the nucleus (34). Once phosphorylated by
the kinases, IRF3 dimerizes and is imported into the nucleus.
Nuclear IRF3, in complex with several other proteins, induces
type I IFN to alert neighboring cells of the infection. Once
IRF3 induces transcription of IFN-�, this powerful cytokine is
secreted from the cells. The NF-�B pathway is also activated
via these mediators as well. Homologous to the IRF3 signaling
pathway, kinases play a key role in activating the NF-�B path-
way as well. The IKK�, IKK�, and IKK� kinases phosphory-
late the NF-�B inhibitor I���. After I��� is phosphorylated,
it is targeted for degradation, which allows NF-�B to be im-
ported into the nucleus. There it induces IFN-� and complexes
with IRF3 to induce antiviral genes. The STAT1 signaling
pathways are activated via the binding of type I (IFN-�), II
(IFN-�), and III (IFN-�) IFN binding to their receptors on the
surface of cells. This binding initiates either the formation of
the STAT1/STAT2/IRF9 complex (called ISGF3) after type I
or type III binding or STAT1 homodimerization after type II
IFN binding to receptors. STAT1 complex formation leads to
its import into the nucleus and activation of antiviral genes.
The regulation of these signaling cascades is crucially impor-
tant for the survival of the host. Disregulation of these events
can lead to increased infection and disease.
Many viruses encode proteins that block, modulate, or slow
several important signaling pathways that are involved in in-
duction or amplification of the innate immune response (3, 7,
10, 19, 35, 37, 38, 49, 55, 56). The Ebola virus encodes the
VP35 and VP24 proteins that block the dsRNA sensing and
STAT1 signaling pathways, respectively (7, 43). Coxsackievirus
B3 encodes the 3a protein, which blocks almost total protein
secretion during infection (9). The severe acute respiratory
syndrome coronavirus (SARS-CoV) encodes several innate
immune antagonists, including the open reading frame 6 (ORF6)

* Corresponding author. Mailing address: Department of Epidemi-
ology, Department of Microbiology and Immunology, University of
North Carolina, Chapel Hill, NC 27599. Phone: [phone]. Fax:
[phone]. E-mail: