Immature and Mature Dengue Serotype 1 Virus Structures Provide Insight into the Maturation Process

Abstract

Dengue virus is a major human pathogen that has four serotypes (DENV1 to -4). Here we report the cryoelectron microscopy (cryo-EM) structures of immature and mature DENV1 at 6-and 4.5-Å resolution, respectively. The subnanometer-resolution maps allow accurate placement of all of the surface proteins. Although the immature and mature viruses showed vastly different surface protein organizations, the envelope protein transmembrane (E-TM) regions remain in similar positions. The pivotal role of the E-TM regions leads to the identification of the start and end positions of all surface proteins during maturation. Dengue virus (DENV), the cause of dengue fever, infects 100 million people worldwide every year. It is a member of the Flaviviridae family (1), with four serotypes: DENV1, -2, -3, and -4. Infection with DENV usually causes a self-limiting fever accompanied by rashes and joint pain in patients but might lead to dengue hemorrhagic fever and dengue shock syndrome, which may be fatal.DENV consists of an ϳ500-Å-diameter protein shell embedded in a host-derived lipid membrane and encapsidates an 11-kb single-stranded positive-sense RNA genome. The dengue genome encodes three structural proteins, the core (or capsid), the premembrane (prM), and the envelope (E) protein, that form the virus particle, as well as seven nonstructural proteins (1) that are involved in replication of the virus genome. The newly synthesized immature DENV has a spiky appearance (2) (Fig. 1A) and is typically noninfectious unless it is complexed with certain antibodies (3). Virus maturation occurs during transportation of the virus particle through the trans-Golgi component network (TGN). The acidic environment of these compartments induces structural rearrangement of the virus surface proteins. During this initial maturation process, the furin protease cleaves prM molecules on the virus into pr and M. After leaving the cell, the cleaved pr dissociates from the virus surface, resulting in smooth, fully mature infectious virus particles (4) (Fig. 1B).The E protein is the major structural component of the viral surface. The ectodomain of E protein (5) contains three distinct domains, DI, DII, and DIII (also shown in Fig. 1C), which are connected by flexible links that allow rearrangement of domains during virus assembly, maturation, and infection (5-7). DIII is involved in attachment to host cell receptors, whereas DII is responsible for fusion to the host endosomal membrane during infection (8). The ectodomain is connected to the stem made from amphipathic helices ␣1 and ␣2. The stem, in turn, is anchored to the virus lipid membrane by two transmembrane (TM) ␣-helices, TM1 and TM2.The crystal structure of an E-prM complex (6) shows that pr has a ␤-barrel fold and caps the fusion loop of the E protein, consistent with its function in preventing the newly synthesized virus from fusing back into the cell during maturation. The furin cleavage site lies between the pr molecule and the ectodomain of M protein, which exists as a linear polypeptide ...

Highly Conserved Residues in the Helical Domain of Dengue Virus Type 1 Precursor Membrane Protein Are Involved in Assembly, Precursor Membrane (prM) Protein Cleavage, and Entry

“…A recent cryo-EM study of mature DENV2 virions at high resolution (3.5 Å) revealed that several residues at the N-terminal loop of M protein (prM protein positions 91-110) interact with domain II residues of E protein (34). These interactions were also shown by another cryo-EM study of mature and immature DENV1 virions at 4.5 and 6 Å resolution, respectively (63). Based on these observations and previous structural studies of prM/E proteins and E protein at pre-and postfusion states, a model of maturation was proposed (33, 34, 56, 64 -66).…”

Near-Atomic Resolution Cryo-Electron Microscopic Structure of Dengue Serotype 4 Virus

“…Superposition of the E proteins of DENV4 strain SG/ 06K2270DK1/2005 with DENV2 strain New Guinea C (30) and DENV1 strain PVP159 (19) does not show significant structural differences (Fig. 3A).…”

“…So far, cryo-EM structures of the mature DENV1 (19) and DENV2 (20) are solved to 4.5-Å and 3.6-Å resolutions, respectively. Here we present the structure of mature DENV4 at a 4.1-Å resolution.…”

Recognition determinants of broadly neutralizing human antibodies against dengue viruses

“…In the infected cell, newly synthesized immature DENV virions bud into the ER lumen, where the pH is neutral. These particles contain 180 copies of a heterodimer of E with the precursor membrane glycoprotein prM at their surface 19,20 . As they are subsequently transported to the external medium across the Golgi apparatus, where the pH is acidic, the interaction with prM protects E from undergoing a premature acid--induced fusogenic conformational change 1 .…”