Report

The Flavivirus Precursor Membrane-Envelope Protein Complex: Structure and Maturation

See allHide authors and affiliations

Science  28 Mar 2008:
Vol. 319, Issue 5871, pp. 1830-1834
DOI: 10.1126/science.1153263

Abstract

Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo–electron microscopy density of immature virus at neutral pH. The pr peptide β-barrel structure covers the fusion loop in E, preventing fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.

Many viruses, including flaviviruses (1), undergo a maturation step immediately before their release from the host; the evident purpose of this step is to maintain stability for the hazardous transfer to a new host while preparing virions for rapid fusion with, and entry into, a cell. Flaviviruses within the Flaviviridae family are major human pathogens that include dengue virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus. They have a positive-sense, 11-kb RNA genome that is packaged together with multiple copies of the capsid protein within a lipid envelope (2). The genome is translated as a polyprotein that has the capsid protein, the precursor membrane glycoprotein (prM), and the envelope glycoprotein (E) in its N-terminal region (Fig. 1A). The polyprotein is cleaved into component proteins by viral and cellular proteases (2). Partially assembled flavivirus nucleocapsids bud from the endoplasmic reticulum, thereby becoming enveloped with a lipid membrane that carries with it the E and prM glycoproteins (2). These immature particles are transported through the cellular secretory pathway, where the cellular furin protease cleaves prM, eventually resulting in the release of the pr peptide and formation of mature virions (3, 4).

Fig. 1.

The dengue virus polyprotein and the recombinant protein containing the prM and E proteins. (A) Threading of the dengue virus polyprotein N-terminal region through the endoplasmic reticulum membrane, showing the positions of the capsid, prM, and E structural proteins. Differentcolored arrows indicate various protease cleavage sites. (B) The order of viral proteins in the wild-type polyprotein and in the recombinant protein construct. The proteins are identified by the same colors as in (A). The mutations to inhibit furin cleavage in the recombinant protein are shown below (21). The linker between the prM and E proteins is labeled TEV.

The dengue virus prM glycoprotein consists of 166 amino acids. Cleavage by furin releases the N-terminal 91 “pr” residues during maturation, leaving only the ectodomain (residues 92 to 130) and C-terminal transmembrane region (residues 131 to 166) of “M” in the virion. The pr peptide protects immature virions against premature fusion with the host membrane (5, 6). The dengue virus E glycoprotein participates in the fusion of the virion with the endosomal membrane at low pH. It consists of an ectodomain (soluble E protein, sE), a stem region, and a transmembrane domain. The x-ray crystallographic structure of sE has been determined for a number of flaviviruses (712), all of which have three domains (DI, DII, and DIII) that consist mainly of β sheets with the fusion loop at the distal end of DII. The E protein is able to switch among different oligomeric states: as a trimer of prM-E heterodimers in immature particles, as a dimer in mature virus, and as a trimer when fusing with a host cell (8, 10).

The cryo–electron microscopy (cryoEM) structures of immature flaviviruses have been determined at neutral pH (6, 13). The “spiky” icosa-hedral immature virions have a diameter of about 600 Å and contain 60 trimeric prM-E spikes. In contrast, the final “smooth”-surfaced icosahedral mature particles have a diameter of about 500 Å andcontain 90 Edimers arrangedina herring-bone pattern and 180 copies of the M protein (14, 15). The transformation from immature to mature particles requires some large rearrangement of the E and M proteins (Fig. 2) (12, 13).

Fig. 2.

Rearrangement of the prM and E proteins during virus maturation. (A to D) Sequence of events as referenced in the text. The E proteins are shown as a Cα backbone; space-filling atoms show the pr peptide surfaces. The three independent heterodimers per icosahedral asymmetric unit are colored red, green, and blue. Although the diagram assumes knowledge of the relationship among the positions of specific heterodimers in the immature and mature viruses (red goes to red, green to green, and blue to blue), this is not known.

A recombinant fusion protein of prM and E from dengue virus 2 was constructed in which the transmembrane region of prM was replaced with an 8–amino acid linker (Fig. 1B) (16). The furin cleavage site of prM was mutated to prevent cleavage of the recombinant protein by intracellular proteases. The crystal structure of the recombinant protein was determined at pH 5.5 to 2.20 Å resolution, and also at pH 7.0 to 2.60 Å resolution (table S1). There were no significant structural differences between the two determinations, which had a root mean square difference of 1.0 Å between all pairs of equivalent atoms. Because of the slightly higher resolution of the low-pH structure, it was chosen for all subsequent calculations and discussions. The polypeptide chain of much of the prM protein (residues 1 to 81, corresponding to most of the pr peptide) and most of the sE protein could be traced in the electron density of the prM-E crystal structure. The pr peptide was positioned over the fusion loop at the distal end of DII (Fig. 3A), as anticipated given that it functions to prevent membrane fusion (5).

Fig. 3.

Structure of the prM-E protein. (A)Stereoview of the prM-E heterodimer in ribbon representation. The pr peptide is cyan, DI is red, DII is yellow, DIII is blue, and the fusion loop is green. Secondary structural elements of the E protein are labeled (7, 21). Secondary structural elements for the pr peptide are defined here as apr, bpr, ···, gpr. (B) Secondary structure of the pr peptide. Disulfide linkages are indicated with dashed lines. (C) Open-book view of the pr-E interactions showing charge complementarity. Positively and negatively charged surfaces in the contact areas are colored blue and red, respectively. Charged residues in the contact area are labeled. The fusion loop (residues 100 to 108) is outlined in green. Contact areas are defined by atoms less than 4.5 Å apart between the pr peptide and the E protein (21).

The pr peptide consists of seven β strands that are mostly antiparallel (Fig. 3). Three disulfide bonds (C34-C68, C45-C80, and C53-C66) stabilize the pr peptide structure, and the electron density map shows that Asn69 is glycosylated. A DALI search (17) did not find any structures with significant similarity to that of the pr peptide. The structure of the E protein in the prM-E heterodimer is similar to the crystal structure of the E protein in the dimeric, prefusion form (12). The hinge angle between DI and DII is only 5° different from the structure of E in immature virus (12), as compared to 23° with the mature virus, which suggests that the oligomeric state of the E protein determines the hinge angle. This similarity supports the biological relevance of the recombinant fusion protein structure. The contact area between pr and E is 865 Å2, representing 16% of the surface area of pr and 4% of E. There are three prominent complementary electrostatic patches (Fig. 3C) involving (Arg6), (Glu46, Asp47), and (Asp63, Asp65) on pr, and involving (Glu84), (Lys64), and (His244, Lys247) on E (table S2), respectively. Of these, the pr residues Asp63 and Asp65 and the E residue His244 are strictly conserved among all known flavivirus sequences.

A pseudo-atomic structure of the immature dengue virus at neutral pH (fig. S1A) was generated by fitting the prM-E crystal structure into the 12.5 Å resolution cryoEM density map (12). The structure of the pr peptide fits the density well, including the prominent carbohydrate moieties at residue Asn69 (fig. S1B and table S3). The surface area buried between pairs of heterodimers is 1052 Å2, 1445 Å2, and 0 Å2 in the “blue-red, ”“red-green, ” and “green-blue” interfaces, respectively, showing nonequivalent contacts between each of the three pairs of heterodimers (Fig. 4 and fig. S1). The hydrophobic fusion loop in each of the three E proteins within one spike is covered and surrounded by loops of the pr peptides and the carbohydrate moieties associated with Asn69 in pr, thus making the surface of the immature particle more hydrophilic (Fig. 4B) and protecting the E protein from premature fusion.

Fig. 4.

Pseudo-atomic structure of the neutral-pH immature dengue virus. (A) Stereoview of one spike of the immature virus as seen from outside the virus, colored as in Fig. 3A. The background to each heterodimer is colored red, green, or blue in accordance with the color code used in Fig. 2 and table S3. (B) Stereoview showing the fusion loop of the E molecule and the protecting pr loops and glycans (21). The arrow indicates the direction of viewing from outside the virus. (C) Stereoview of the fitted pr-E Cα backbones with the Cα trace of the extended M polypeptide (black) running along the edge of each E protein. The Cα backbones of the pr peptides are in bold. The approximate site of furin cleavage is marked with a black arrow in the blue molecule.

The cryoEM density representing the surface spikes was set to zero at all points within 3.0 Å of every atom in the fitted x-ray structure. There then remained traces of density that ran along the edge of each E molecule toward the lipid membrane (fig. S1C and fig. S2, A to C). These density traces were positioned similarly on each E molecule, in agreement with density in the crystal structure that had been presumed to be the N-terminal region of the M protein (fig. S2D) (16). The trace of the M protein suggested that the pr polypeptide chain is extended linearly along the surface of the E protein, mostly on the inside of the spike (fig. S1C). The position of the furin cleavage site could be reasonably well inferred by building an extended polypeptide chain into the density traces representing the junction of the pr peptide with the M protein (Fig. 4C and movie S1). Docking of the known structure of furin (18) onto the cleavage site showed that furin would be sterically hindered from binding to any of the three prM-E heterodimers within a spike, thereby demonstrating why furin is unable to cleave the pr polypeptide in the immature virus at neutral pH (4).

The accompanying paper (4) shows that low-pH immature virus particles (Fig. 2B) have a structure in which the arrangement of the E proteins is essentially the same as that of the mature virus (Fig. 2D). However, the interface between the DII domains in the E dimer is in part the same surface where the extended polypeptide of the M protein binds in the neutral-pH immature virus (fig. S3A). Thus, if the M protein were in the conformation as found in the immature virus at neutral pH, it would sterically block the formation of the E dimer. Hence, the conformation of M must be different in the low-pH immature virus, consistent with its apparent flexibility in the crystal structure of the heterodimer and also consistent with the changed position of the M protein's transmembrane helices during maturation (fig. S3) (15). Indeed, the low-pH conformation, unlike the neutral-pH conformation of the M protein, has been found to be accessible to furin cleavage (4).

The large conformational change that occurs when the immature virus changes from the neutral-pH to the low-pH form was found to be reversible for dengue virus (Fig. 2, A and B), as long as prM was still intact (4). However, once prM had been cleaved (Fig. 2C), there was no further conformational change when the pH was returned to neutral (Fig. 2, C and D); instead, the cleaved pr peptide was released (4). Apparently the extended polypeptide of the M protein, along the side of the E protein (Fig. 4C and fig. S2), is essential for maintaining the reversibility of the conformational change (Fig. 2, A and B). An analogy might be the effect of a drawstring that opens and closes a curtain. Once the string (i.e., the M protein) is cut (i.e., furin cleavage), there can be no further movement. Mutating a conserved histidine residue to alanine in the M protein (His99 → Ala) in Japanese encephalitis virus inhibits the formation of prM-E heterodimers (19). The corresponding residue, His98 in dengue virus, is located approximately in the center of the extended M protein, opposite the hydrophobic surfaces of helices αA and αB in the E protein. Thus, a change of pH might alter the interactions between M and E, leading to the transition between the “spiky” and “smooth” virus conformations.

On one hand, pr remains bound to E when the immature low-pH virus is returned to neutral pH, thus protecting the immature virus against fusion. On the other hand, after cleavage of M, pr is released from E to activate the virus when returning the pH to neutral. The average area of contact between a pr peptide and an E protein in a spike of the immature virus at neutral pH is slightly larger than the area of contact in the immature virus at low pH (table S4). Thus, not only is each pr peptide tethered covalently to the M protein, but also the pr peptide probably has a slightly greater affinity for the trimeric E protein spike of immature virus at neutral pH relative to the dimeric smooth surface of mature virus. In contrast, His244 of the E protein is highly conserved and is situated in the prM-E interface opposite the completely conserved Asp63 of the pr peptide (Fig. 3C), causing the affinity of the pr peptide for the E protein to decrease when the pH is raised to neutral. This would allow the pr peptide to be released at neutral pH from the dimeric E structure, but only when the pr peptide has been cleaved. As evolution is highly conservative of structure (20), the maturation process described here for dengue virus is likely to have structural homologies in other enveloped viruses.

Supporting Online Material

www.sciencemag.org/cgi/content/full/319/5871/1830/DC1

Materials and Methods

Figs. S1 to S3

Tables S1 to S4

Movie S1

References

References and Notes

View Abstract

Navigate This Article