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: pei_yong.shi@novartis.com

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: tobisu@chem.eng.osaka-u.ac.jp; chatani@chem.eng.osaka-u.ac.jp

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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