Dengue, Japanese encephalitis, West Nile and yellow fever belong to the Flavivirus genus, which is a member of the Flaviviridae family. They are human pathogens that cause large epidemics and tens of thousands of deaths annually in many parts of the world. The structural organization of these viruses and their associated structural proteins has provided insight into the molecular transitions that occur during the viral life cycle, such as assembly, budding, maturation and fusion. This review focuses mainly on structural studies of dengue virus.

The first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map. The virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein E dimers. The three E monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. Difference maps indicate the location of the small membrane protein M relative to the overlaying scaffold of E dimers. The structure suggests that flaviviruses, and by analogy also alphaviruses, employ a fusion mechanism in which the distal beta barrels of domain II of the glycoprotein E are inserted into the cellular membrane.

SummaryThe first structure of a flavivirus has been determined by using a comThe first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map. The virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein E dimers. The three E monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. Difference maps indicate the location of the small membrane protein M relative to the overlaying scaffold of E dimers. The structure suggests that flaviviruses, and by analogy also alphaviruses, employ a fusion mechanism in which the distal β barrels of domain II of the glycoprotein E are inserted into the cellular membrane.

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Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by G q and G i ␣ subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. We report here that an N-terminal domain of RGS4 discriminated among receptor signaling complexes coupled via G q . Accordingly, deletion of the N-terminal domain of RGS4 eliminated receptor selectivity and reduced potency by 10 4 -fold. Receptor selectivity and potency of inhibition were partially restored when the RGS4 box was added together with an N-terminal peptide. In vitro reconstitution experiments also indicated that sequences flanking the RGS4 box were essential for high potency GAP activity. Thus, RGS4 regulates G q class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by G␣ q and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled G q signaling.Heterotrimeric G proteins of the G q class are mediators of Ca 2ϩ responses in animal cells. Signaling is initiated by agonist binding to heptahelical transmembrane receptors complexed with G q ␣␤␥ and phospholipase C-␤ (PLC␤) 1 (1), which generates IP 3 to trigger Ca 2ϩ release from internal stores (2).Many cells express several G q -coupled receptors that regulate the location, intensity, and propagation of intracellular Ca 2ϩ waves. For example, pancreatic acini respond to acetylcholine, bombesin, and cholecystokinin by activating the same set of G q class proteins and mobilizing the same Ca 2ϩ pool, but each receptor evokes distinct patterns of Ca 2ϩ waves (3). Ca 2ϩ release may be regulated by intracellular proteins that interact with guanine nucleotide binding proteins, such as regulators of G protein signaling (RGS) proteins.2 RGS proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by G q and G i ␣ subunits, thus attenuating signaling (5-8). Mammals express over 20 different RGS proteins, of which RGS4 has received the most extensive biochemical characterization (5, 7-12). RGS4 is composed of a central domain of 120 amino acids that is homologous to other RGS proteins, termed the RGS box, flanked by less well conserved N-and C-terminal sequences (13). In rat pancreatic acinar cells, RGS4 preferentially inhibited G q/11 -mediated signaling evoked by carbachol relative to bombesin and cholecystokinin regardless of the identity of the G q class ␣ subunit. 2Regulatory specificity was apparently conferred by direct or indirect interaction between RGS4 and the receptor.In the present study, we used deletion mutations to identify two domains in RGS4 that regulate agonist-dependent Ca 2ϩ signaling. The RGS box accelerates GTP hydrolysis by G␣ q whereas the N terminus conveys high affinity and receptorselective inhibition. These combined activities are required for receptor selectivity and high potency i...

The structure of the lipid-enveloped Sindbis virus has been determined by fitting atomic resolution crystallographic structures of component proteins into an 11-Å resolution cryoelectron microscopy map. The virus has T‫4؍‬ quasisymmetry elements that are accurately maintained between the external glycoproteins, the transmembrane helical region, and the internal nucleocapsid core. The crystal structure of the E1 glycoprotein was fitted into the cryoelectron microscopy density, in part by using the known carbohydrate positions as restraints. A difference map showed that the E2 glycoprotein was shaped similarly to E1, suggesting a possible common evolutionary origin for these two glycoproteins. The structure shows that the E2 glycoprotein would have to move away from the center of the trimeric spike in order to expose enough viral membrane surface to permit fusion with the cellular membrane during the initial stages of host infection. The well-resolved E1-E2 transmembrane regions form ␣-helical coiled coils that were consistent with T‫4؍‬ symmetry. The known structure of the capsid protein was fitted into the density corresponding to the nucleocapsid, revising the structure published earlier.

Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 Å by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The α-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane α-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus.Knowledge of protein structures within cellular membranes is limited to only a few examples of membrane proteins whose structures have been determined in situ [1][2][3] . Although X-ray crystallography has succeeded in the analysis of the PRD1 membrane-containing bacteriophage 4 , in general it is difficult to crystallize integral membrane proteins or enveloped viruses. Improved technology for reconstructing cryo-EM images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. We report here the structure and disposition of the membrane proteins in mature dengue virus.Dengue viruses belong to the Flavivirus genus of the family Flaviviridae. The flaviviruses are insect-transmitted, icosahedral, enveloped RNA viruses that infect vertebrates and Note added in proof: The structure of the dengue virus E protein was published 39 subsequent to the submission of our manuscript. The r.m.s. deviation between Cα atoms with respect to the homology model used in the above manuscript is 3.5 Å. Competing Interests Statement:The authors declare that they have no competing financial interests. 5,6 . Other viruses belonging to the same genus are West Nile, yellow fever, Japanese encephalitis and tick-borne encephalitis virus (TBEV). During the last half of the twentieth century, instances of dengue hemorrhagic fever, which usually results from the sequential infection by more than one of the four dengue virus serotypes, have spread from southeast Asia to most tropical and semitropical regions on Earth. As there is no effective dengue virus vaccine or antiviral agent, the spread of dengue virus infection has become a major health concern and a subject of special interest to the World Health Organization. Similarly, the spread of the closely related West Nile virus into North America has become a prominent public health issue in the United States 7 . NIH Public AccessThe positive-sense, 10.2-kb RNA genome of dengue virus has...

The 9 A resolution cryo-electron microscopy map of Sindbis virus presented here provides structural information on the polypeptide topology of the E2 protein, on the interactions between the E1 and E2 glycoproteins in the formation of a heterodimer, on the difference in conformation of the two types of trimeric spikes, on the interaction between the transmembrane helices of the E1 and E2 proteins, and on the conformational changes that occur when fusing with a host cell. The positions of various markers on the E2 protein established the approximate topology of the E2 structure. The largest conformational differences between the icosahedral surface spikes at icosahedral 3-fold and quasi-3-fold positions are associated with the monomers closest to the 5-fold axes. The long E2 monomers, containing the cell receptor recognition motif at their extremities, are shown to rotate by about 180 degrees and to move away from the center of the spikes during fusion.