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DOI: 10.1126/science.1251249, 849 (2014);343 Science
Pei-Yong ShiUnraveling a Flavivirus Enigma
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www.sciencemag.org SCIENCE VOL 343 21 FEBRUARY 2014 849
PERSPECTIVES
Direct communication between adjacent cells underlies tissue organization. Com-municating with a dispersed community of cells presents quite different challenges: Decisions must be made about which cells to communicate with, and how. Cytonemes, nanotubes, and other filopodia-like struc-tures can be used for long-distance commu-nication, but there is still limited information about their biological importance. The most informative experiments, like selective dis-ruption of connections and detailed analy-sis of the consequences, are also the hardest to carry out. Direct communication allows private cell-to-cell conversations: Freely
transmitting the signal makes it simpler to reach many cells. Employing a combina-tion of these two signaling mechanisms may optimize strategies for decision-making in development. Even the nervous system, with its elaborate and sophisticated use of long-distance cell-cell connections, also uses dis-persed signals to modulate general outputs such as mood and other emotions.
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10.1126/science.1250885
STRUCTURAL BIOLOGY
Pei-Yong Shi
There is growing concern about the spread of flaviviruses, such as den-gue virus and West Nile virus, to new
geographic areas as they can cause major epi-demics and represent global public health threats. Controlling these viruses requires a better molecular understanding of how they infect cells. Nonstructural protein 1 (NS1) is perhaps the most enigmatic fl avivirus pro-tein. During infection, NS1 exists in two dis-tinct forms, travels to various compartments, decorates itself with different molecular dis-guises, and plays numerous roles in its infec-tious cycle and disease pathogenesis ( 1). How this protein manages all of this has been a puzzle since its discovery in 1970 ( 2). Crys-tallizing NS1 has daunted many researchers because of the heterogeneity of its glyco-sylation and association with lipids, but as reported on page 881 of this issue, Akey et al. ( 3) have accomplished this task. The unusual structural details revealed about NS1 may guide the design of compounds that inhibit viral replication and provide clues as to how it contributes to different stages of the virus life cycle and disease.
Flavivirus NS1 is a glycoprotein with a molecular mass of 46 to 55 kD, depending on its glycosylation status. The crystal struc-tures of dengue virus NS1 (3 Å resolution) and West Nile virus NS1 (2.8 Å resolution) exhibit a similar hexameric arrangement of three dimers, confi rming the hexameric
structure (30 Å resolution) indicated by a cryoelectron-microscopy analysis ( 4). Each monomer displays an unusual fold consisting of three regions: a “β-roll” domain that dimer-izes with that of another monomer; a “wing” domain that resembles a helicase domain; and a “β-ladder” domain that aligns with that of another NS1 molecule to form an extended β-sheet ladder. The ladder forms the plane of the NS1 dimer, with a hydrophobic side (exemplifi ed by a “greasy fi nger” loop) that can associate with the membrane. The hydro-phobic side of each dimer faces the interior
of the hexamer. Remarkably, recombinant NS1, which does not possess any transmem-brane domain, can convert large liposomes into smaller lipid-protein nanoparticles. This demonstrates that NS1 can directly modulate the lipid membrane without additional cellu-lar proteins. Such lipid-modulation activity and its underlying structure could account for the myriad functions of NS1.
After fl avivirus entry into a cell by endo-cytosis, the virus particle is released into the cytoplasm. Viral genomic RNA is translated into proteins and replicated, and virus assem-C
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Novartis Institute for Tropical Diseases, 10 Biopolis Road, 05-01 Chromos, Singapore 138670. E-mail: [email protected]
Virus entry andreplication
Cytoplasm
ER lumenER
NS1 NS1
NS3
NS5
NS2B
NS4BNS4A
NS2A
� RNA Virus assemblyand release
+ RNA
Assist before leaving. Flavivurus replicates at the ER surface in the infected cell. Viral NS1 protein forms dimers in the ER lumen, yet assist the replication complex on the opposite side of the membrane. Seven non-structural proteins, together with host proteins (not shown), form the replication complex. Once immature viral particles bud into the secretory pathway, NS1 protein forms hexamers that are secreted as lipoproteins.
The structure of a fl avirirus nonstructural
protein provides mechanistic understanding
for many of its functions.Unraveling a Flavivirus Enigma
Published by AAAS
21 FEBRUARY 2014 VOL 343 SCIENCE www.sciencemag.org 850
PERSPECTIVES
The benzene ring is one of the most
prevalent structural motifs found in
organic compounds, and the devel-
opment of effi cient and selective methods
for the synthesis of benzene derivatives has
attracted the interest of organic chemists
for more than a century. When introducing
a new substituent onto substituted benzene
derivatives, one critical issue is regioselec-
tivity (i.e., which particular C-H bonds will
react). One strategy for addressing this issue
is to use bulky substituents on the ring—
which often are added deliberately—to limit
access of a reagent or a catalyst to adjacent
C-H bonds, thus directing the reaction to
other positions. On page 878 of this issue,
Cheng and Hartwig ( 1) report that the steric
bulk of the substituent can be used to achieve
an unusually high selectivity among the C-H
bonds that are located at more remote posi-
tions of the benzene ring.
The classical method for benzene func-
tionalization is electrophilic aromatic sub-
stitution, in which the electronic nature of a
substituent controls the regioselectivity for
further substitution reactions. An electron-
donating group such as methoxy (–OCH3)
results in the ortho and para positions being
substituted, whereas an electron-withdraw-
ing group such as nitro (–NO2) delivers a
Remote Control by Steric Effects
CHEMISTRY
Mamoru Tobisu 1 and Naoto Chatani 2
A rhodium-catalyzed reaction places a silicon substituent on the site farthest away from the
largest group present on an aromatic ring.
1Center for Atomic and Molecular Technologies, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan. 2Department of Applied Chemistry, Faculty of Engineering, Osaka University, Suita, Osaka 565-0871, Japan. E-mail: [email protected]; [email protected]
bly occurs on the surface of the endoplas-
mic reticulum (ER). Viral particles bud into
the ER and mature as they are transported
through the secretory pathway for release
from the cell.
NS1 protein is translated from viral RNA
and translocated into the ER lumen, where it
is glycosylated. NS1 dimers then form and
associate with the luminal side of the ER
membrane at a virus-induced vesicle packet
(see the figure). Although dimeric NS1 is
required for viral RNA synthesis, the repli-
cation complex resides on the cytoplasmic
side of the ER membrane. Two factors could
facilitate the recruitment of NS1 to the rep-
lication complex: the membrane-association
of NS1, and the specifi c interactions between
NS1 and viral transmembrane proteins NS4A
and NS4B ( 5, 6).
What happens after the NS1 dimer has
facilitated viral replication? It is eventually
released by the infected cell. A model pro-
poses that the assembly of hexameric NS1
( 4) is key to this process. Newly synthe-
sized monomeric NS1 is water-soluble. As
its concentration and glycosylation increase
in the ER lumen, NS1 dimerizes, creating
the hydrophobicity needed for its interaction
with the membrane ( 7). Three NS1 dimers
juxtapose on the lipid bilayer and pinch off
the membrane, resulting in a water-soluble
hexamer. Host lipids become trapped within
the central channel of the hexamer, forming a
lipoprotein particle. The particle is then trans-
ported and released from the cell through the
secretory pathway.
In dengue virus–infected patients, the
concentration of extracellular NS1 can
reach 15 µg/ml in sera ( 1). NS1-based tests
have been developed for rapid, point-of-care
diagnosis. The concentration of serum NS1
correlates with the amount of the viral RNA
present in the patient, and high amounts of
circulating dengue virus NS1 early in illness
correlate with severe disease outcome ( 8).
Mounting evidence indicates that secreted
NS1 modulates disease pathogenesis. Pre-
incubation of hepatocytes with soluble NS1
enhances homologous dengue virus infec-
tion ( 9). Secreted NS1 interacts with host
proteins, many of which are involved in the
immune complement pathway ( 10, 11); this
may allow fl aviruses to evade the immune
system. Secreted NS1 also is highly immu-
nogenic. Some antibodies against NS1 are
cross-reactive with cellular components;
these auto-antibodies may contribute to
platelet and endothelial cell damage, lead-
ing to vascular leakage, the hallmark of
severe dengue hemorrhagic fever and den-
gue shock syndrome.
The critical roles of NS1 in fl avivirus rep-
lication and pathogenesis implicate NS1 as
an attractive antiviral target. A few tangible
approaches can be envisioned. Cells express-
ing NS1 could be screened for inhibitors of
NS1 dimerization and hexamerization, and
libraries could be screened for compounds
that block the ability of NS1 to convert lipo-
somes into lipoprotein particles. The crys-
tal structure will greatly facilitate structure-
based rational design of antiviral compounds.
In fact, inhibitors of cellular glucosidases
that are required for NS1 glycosylation sup-
press fl avivirus replication in cell culture and
in a mouse model ( 12). Future studies should
define how NS1 physically interacts with
the replication complex and its specifi c role
in RNA replication. The molecular details
remain to be determined as to when, where,
and how the conversion of NS1 monomer
to dimer and then to hexamer is controlled.
One question concerns the NS1 “wing”
domain, whose folding is similar to that seen
in two proteins [retinoic acid–inducible gene
I (RIG-I) and melanoma differentiation–
associated gene 5 (MDA5)] that function
as viral sensors in the innate immune sys-
tem. Does this somehow allow fl aviviruses
to evade the host immune response? Another
intriguing question is why, within the family
Flaviviridae, only members of the genus Fla-
vivirus encode the NS1 protein; members of
the other two genera, Hepacivirus and Pes-
tivirus, do not contain a gene equivalent to
NS1. The reason may be that most fl avivi-
ruses transfer between insects and mammals.
If so, it raises the question of how fl avivirus
NS1 play distinct roles when replicating in
different host cells. Perhaps more interesting
is how the essential role of NS1 in fl avivirus
replication is compensated in hepacivirus and
pestivirus. The answers to these questions
will unravel more mysteries of this fascinat-
ing protein.
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10.1126/science.1251249
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