no further increase in infection (Fig. 4G and
fig. S12), suggesting that the effects of BADGE
on cathelicidin production from adipocytes was
responsible for the increase in S. aureus infection in vivo.
These results show that a local increase in
subcutaneous adipocytes is an important host
defense response against skin infection. This
observation is consistent with prior observations that adipocytes secrete a variety of bioactive adipokines and cytokines that mediate
immune responses after injury ( 28) and now
shows that adipocytes produce an AMP that can
directly kill bacteria. Local expansion of dermal
fat produces cathelicidin in response to infection, but this response appears to decline as
adipocytes mature. The expansion of dermal fat
in response to infection may also indirectly benefit immune defense by influencing other processes such as neutrophil oxidative burst, thus
further amplifying the importance of the subcutaneous preadipocyte pool in preventing infections. Defective AMP production by mature
adipocytes may explain observations of elevated
susceptibility to infection during obesity and insulin resistance ( 29). Cathelicidin has also been
shown to be proinflammatory ( 30). Therefore,
the production of cathelicidin by adipocytes may
also participate in the chronic, low-level inflammation observed in obesity ( 28).
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This study was supported by NIH R01 AI083358, R01AI052453,
and AR052728 (R.L.G.). Human serum collections were funded by
The Atopic Dermatitis Research Network (HHSN272201000020C).
S.P.B. was supported by the NIH (DK096828). M.V.P. is supported
by the Edward Mallinckrodt Jr. Foundation Research Grant, the
Dermatology Foundation Research Grant, and NIH NIAMS
R01-AR067273; C.F.G.-J. is supported by the NIH MBRS-IMSD
training grant (GM055246) and NSF Graduate Research Fellowship
number DGE-1321846; and R.R. is supported by California
Institute for Regenerative Medicine training grant (TG2-01152).
The authors declare no competing financial interests. We thank
T. Nakatuji for advice relating to bacterial infections and C. Aguilera
for mouse technical expertise, UCSD Bio-Core facility for reagents,
UCSD mouse hematology core laboratory for serum studies, and
J. M. Olefsky for comments on the manuscript. Zfp423-GFP mice
are available from R. Gupta under a material transfer agreement
with Dana-Farber Cancer Institute. All the data reported in this
manuscript are presented in the main paper and in the
Materials and Methods
Figs. S1 to S12
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8 September 2014; accepted 21 November 2014
Structure and inhibition of EV-D68,
a virus that causes respiratory
illness in children
Yue Liu,1 Ju Sheng,1 Andrei Fokine,1 Geng Meng,1 Woong-Hee Shin,1 Feng Long,1
Richard J. Kuhn,1 Daisuke Kihara,1,2 Michael G. Rossmann1*
Enterovirus D68 (EV-D68) is a member of Picornaviridae and is a causative agent of
recent outbreaks of respiratory illness in children in the United States. We report here the
crystal structures of EV-D68 and its complex with pleconaril, a capsid-binding compound
that had been developed as an anti-rhinovirus drug. The hydrophobic drug-binding pocket
in viral protein 1 contained density that is consistent with a fatty acid of about 10 carbon
atoms. This density could be displaced by pleconaril. We also showed that pleconaril
inhibits EV-D68 at a half-maximal effective concentration of 430 nanomolar and might,
therefore, be a possible drug candidate to alleviate EV-D68 outbreaks.
Picornaviruses constitute a large family of small icosahedral viruses with a single positive- stranded RNA genome and an external diameter of about 300 Å. The Enterovirus genus includes medically important human
pathogens, such as human rhinoviruses (HRVs),
polioviruses (PVs) and coxsackieviruses (CVs)
(table S1) (1, 2). Many of these enteroviruses (EVs)
have been well characterized structurally and
functionally ( 3–10). However, the species EV-D
remains poorly characterized.
An upsurge of EV-D68 cases in the past few
years has shown clusters of infections worldwide
( 11). In August 2014, an outbreak of mild to severe respiratory illnesses occurred among thousands of young children in the United States, of
which 1116 cases have been confirmed to be caused
by EV-D68. This virus has also been associated
with occasional neurological infections ( 12). Although EV-D68 has emerged as a considerable
global public health threat, there is no available
vaccine or effective antiviral treatment.
The capsids of EVs consist of 60 copies of each
of four different viral proteins: VP1, VP2, VP3,
and VP4 (Fig. 1A). Of these, VP1 (about 300
amino acids), VP2 (about 260 amino acids), and
VP3 (about 240 amino acids) each have a “jelly
roll” fold arranged in the capsid with pseudo T =
3 icosahedral symmetry, where T represents the
triangulation number ( 13). Their organization in
the capsid is similar to that of the T = 3 RNA
plant viruses, except that the three subunits re-
lated by quasi-threefold symmetry have different
amino acid sequences in picornaviruses ( 6, 10).
Each roughly 70–amino–acids-long VP4 mole-
cule forms an extended peptide on the internal
surface of the capsid shell. The jelly roll fold
consists of two antiparallel b sheets, which face
each other to form a b barrel with a hydrophobic
interior (Fig. 1B).
EVs have a deep surface depression (“canyon”)
circulating around each of the 12 pentameric
vertices (Fig. 1A). The canyon was predicted to be
the site of receptor binding, because the amino
acids outside the canyon that form the external
surface of the virus were more exposed and were
shown to be accessible to neutralizing antibodies
(Fig. 1A) ( 10, 14). The virus thus could remain
faithful to a specific receptor molecule that binds
into the canyon while evading the host’s immune
system ( 10, 15). The prediction that the canyon
would be the site of binding to cellular receptor
molecules was subsequently confirmed for numerous different EVs ( 16, 17). All of the receptor
1Department of Biological Sciences, Hockmeyer Hall of
Structural Biology, 240 South Martin Jischke Drive, Purdue
University, West Lafayette, IN 47907, USA. 2Department of
Computer Science, 305 North University Street, Purdue
University, West Lafayette, IN 47907, USA.
*Corresponding author. E-mail: firstname.lastname@example.org