key: cord-1022251-6t8jrm66
authors: Dokland, Terje; Walsh, Martin; Mackenzie, Jason M.; Khromykh, Alexander A.; Ee, Kim-Huey; Wang, Sifang
title: West Nile Virus Core Protein Tetramer Structure and Ribbon Formation
date: 2004-07-31
journal: Structure
DOI: 10.1016/j.str.2004.04.024
sha: 072432df924e23c10834a328fabc03c3ca14f8fc
doc_id: 1022251
cord_uid: 6t8jrm66

Abstract We have determined the crystal structure of the core (C) protein from the Kunjin subtype of West Nile virus (WNV), closely related to the NY99 strain of WNV, currently a major health threat in the U.S. WNV is a member of the Flaviviridae family of enveloped RNA viruses that contains many important human pathogens. The C protein is associated with the RNA genome and forms the internal core which is surrounded by the envelope in the virion. The C protein structure contains four α helices and forms dimers that are organized into tetramers. The tetramers form extended filamentous ribbons resembling the stacked α helices seen in HEAT protein structures.

The start and end of the trypsin-cleaved fragment (bullets) as well as the site of maturation cleavage (arrowhead) are indicated. The position of the ␣ helices in the C structure are shown above the sequence (spirals) (Gouet et al., 1999) . of the C protein from the Kunjin subtype of WNV. This yielded stable crystals which diffracted synchrotron rais the first crystal structure of a flavivirus core protein.

diation anisotropically to about 2.8 Å resolution. The The C protein forms dimers, similar to those of dengue crystals belong to space group I4 1 with a ϭ 85.7 Å and virus C, which are organized into tetramers with highly c ϭ 214.4 Å , with eight protein monomers in the asympositively charged surfaces. The ␣ helices in the tetrametric unit and V M ϭ 2.8. The structure was solved to mers stack up to form long filamentous ribbons in the 3.2 Å resolution by MAD on a Se-Met derivative, giving crystal. a final R cryst ϭ 0.25 and R free ϭ 0.31 (Table 1) and has been deposited in the Protein Data Bank with accession code 1SFK (Figures 2A and 2B ).

The C protein structure consists of four ␣ helices interspersed by short loops (Figures 1, 2B, and 3A) . The The first 103 amino acids of the C protein of Kunjin virus protein forms a tight 2-fold symmetric dimer, in which (strain MRM61C) were cloned with an N-terminal 6ϫHis the helices ␣1, ␣2, and ␣4 form three distinct "layers" tag and purified by affinity and ion exchange chromatogand ␣3 forms a short, connecting helix flanking the dimer raphy. Crystals of the full-length C protein were unstable ( Figure 3A) . A DALI search for structural homologs (Holm and disordered and did not diffract X-rays to high resoluand Sander, 1995) revealed a topological resemblance tion. Assuming that the crystal disorder was caused by with the cyclin A-like domain, like that of archeal tranflexible regions in the protein, we treated the full-length scription factor B (TFB; PDB code 1AIS; Z ϭ 4.0) (Kosa protein with trypsin, resulting in a stable fragment starting at Val23 and ending at Arg98 (Figure 1 ). This fragment et al., 1997), and with the HEAT repeat domain of human each of the two 4-fold screw axes in the crystal. This this helix undergoes an order-disorder transition upon crystallization. Such order-disorder transitions are often leaves a large space between adjacent tubules, which is reflected in the relatively high solvent content (57%).

of functional importance and may suggest a role in the conformational switching that is required during core Interestingly, the resulting repeated stacking of ␣ helices in the tubules resembles the long, filamentous structures assembly (Dokland, 2000) . The density is very poor for the overlapping ␣ helices in subunit C, perhaps due to commonly formed by HEAT and ARM repeat proteins, multimeric structures typically involved in protein-pro-two alternative orientations coexisting in the crystal. The GH dimer forms similar interactions with EЈFЈ; in this tein interactions (Andrade et al., 2001) .

The AB dimer encloses a hydrophobic pocket formed case ␣1 is missing from H where it would overlap with FЈ. The different orientation of ␣1 in the dengue C protein by Leu29, Leu36, Phe45, Leu49, Phe52, and Phe53 from the ␣1 and ␣2 helices ( Figure 4A ). The symmetry related (Ma et al., 2004 ) also suggests that this helix is flexible, perhaps reflecting a functional role in assembly or RNA CЈDЈ dimer approaches the pocket, so that the ␣1 helices from A, CЈ, and DЈ form a trimeric bundle. This would binding ( Figure 2D ). The hydrophobic pocket contains additional density that was not interpretable at 3.2 Å cause helix ␣1 from B and CЈ to overlap; consequently, density for ␣1 in subunit B is missing, suggesting that resolution, but could conceivably represent part of the . Indeed, the the crystallographic 2-fold axis between 2-fold related Thr43 residues and an anion, possibly Cl Ϫ , held between C tetramer has a strongly positively charged surface ( Figure 4B ). In this model, the tetramers would form the 2-fold related Arg31residues, about 9.5 Å from the cation, mediating the interactions between tubules in adja-building blocks for capsid assembly. The order-disorder transition in helix ␣1 ( Figure 4A) 

The WNV C protein dimer presumably represents the of dengue or WNV could mean that there is no ordered shell, and that the RNA is instead packed together with building block for core assembly, which also involves interactions with the viral RNA. , 1997) . In the C structure, this region forms cleaved off by the trypsin treatment, and the C-terminal region corresponds to helix ␣4, which forms an 11 Å wide the hydrophobic pocket that is partially protected by helix ␣1. Movement of ␣1 might render the hydrophobic tunnel in the tetramer ( Figure 3B) . The tunnel contains a number of positively charged residues, consistent with region in a position able to interact with the membrane. Part of the hydrophobic region of TBE C (residues 28-a role in RNA binding ( Figure 4B ). The N-and C-terminal RNA binding regions are located at opposite ends of the 43), which corresponds to parts of helices ␣1 and ␣2 in WNV, could be removed while still retaining some viabil-dimer. While a conformational switch in the N-terminal region, perhaps reflected in the order-disorder transition ity (Kofler et al., 2002) . This suggests that the RNA binding properties of the C-terminal domain are more impor-in helix ␣1, could bring the two termini together on one side, the organization would appear to be very different tant than the specific fold of the protein, or that RNA binding is more unspecific in nature. Furthermore, viabil-from that of the togaviruses or the arteriviruses, which have a clear "inside" or RNA binding face (Choi et al., ity was partially restored by mutations that increased tector (Mar USA, Inc.) and processed using HKL2000 (Otwinowski the hydrophobic nature of the remaining sequence, indiand Minor, 1997) to a resolution of 2.8 Å for the native data and cating that membrane localization is done through non-3.6 Å for the Se-Met data ( Table 1) 

Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States

Molecular biology of flaviviruses

Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes

Solution structure of dengue virus capsid protein reveals another fold

Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively

A conserved HEAT domain within eIF4G directs assembly of the translation initation machinery

A conserved internal hydrophobic domain mediates the stable membrane integration of the dengue virus capsid protein

A ligand-binding pocket in the dengue virus envelope glycoprotein

Improved methods for the building of protein models in electron Synchrotron Radiation Facility beamline BM14 using a marCCD de-