Mutagenesis of the DI/DIII Linker in Dengue Virus Envelope Protein Impairs Viral Particle Assembly

Abstract

The dengue virus (DV) envelope (E) protein is important in mediating viral entry and assembly of progeny virus during cellular infection. Domains I and III (DI and DIII, respectively) of the DV E protein are connected by a highly conserved but poorly ordered region, the DI/DIII linker. Although the flexibility of the DI/DIII linker is thought to be important for accommodating the structural rearrangements undergone by the E protein during viral entry, the function of the linker in the DV infectious cycle is not well understood. In this study, we performed site-directed mutagenesis on conserved residues in the DI/DIII linker of the DV2 E protein and showed that the resulting mutations had little or no effect on the entry process but greatly affected virus assembly. Biochemical fractionation and immunofluorescence microscopy experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the DI/DIII linker mutants express the DV structural proteins at the sites of particle assembly near the ER but fail to form infectious particles. This defect is not due to disruption of E's interaction with prM and pr in immature and mature virions, respectively. Serial passaging of the DV2 mutant E-Y299F led to the identification of a mutation in the membrane-proximal stem region of E that fully compensates for the assembly defect of this DI/DIII linker mutant. Together, our results suggest a critical and previously unidentified role for the E protein DI/DIII linker region during the DV2 assembly process. Dengue virus (DV) is a significant human pathogen and the cause of dengue fever and dengue hemorrhagic fever. The four dengue virus serotypes (DV1, DV2, DV3, and DV4) are members of the Flaviviridae family and have a positive-sense RNA genome encoding a single polyprotein. This polyprotein is processed by host-and DV-encoded proteases into 10 proteins: three structural proteins-core (C), premembrane (prM), and envelope (E)-and seven nonstructural proteins-NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. Following RNA replication and translation, the viral RNA is encapsidated by C to form the nucleocapsid that buds at the endoplasmic reticulum (ER) membrane to associate with the prM and E proteins and form an immature DV virion (25). This immature virion then transits through the secretory pathway, where the virion is matured through cleavage of prM into the membrane (M) protein by furin in the trans-Golgi (21, 22). The mature virion then exits the cell and enters new cells via endocytosis. The acidic environment in the host endosome serves as the physiological trigger for a major conformational change in the E protein that leads to the insertion of its fusion loops in the host endosomal membrane (2, 16). This results in fusion of the viral membrane with the host membrane and delivery of the viral genome to the cytoplasm.The DV E protein belongs to the class II fusion proteins, which share a tertiary structure (11). The DV E protein is anchored in the membrane by two transmembrane domains (28)....

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

“…This genetic complementation results in the production of pseudoinfectious virus-like particles capable of only a single round of infection, referred to herein as reporter virus particles (RVPs). RVPs have been used extensively to study flavivirus biology (43)(44)(45)(46)(47)(48) and virus-antibody interactions (35,36,(49)(50)(51) and as tools to screen antiviral compounds (19,52,53). The mechanism of flavivirus RNA packaging is not well understood and appears to be relatively nonselective.…”

Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination

“…DIII is also believed to contain the receptor-binding sites to the host cell (Erb et al, 2010;Mukhopadhyay et al, 2005) and has been implicated in determining host range, tropism and virulence (Lindenbach et al, 2007). A hinge region formed by the four strands that span between the different DI and DII coding segments provides the flexibility for E conformational changes during virus maturation (Butrapet et al, 2011;Monath et al, 2002), while a linker of 11 aa connects DI to DIII and is fundamental for proper E folding (de Wispelaere & Yang, 2012). …”

The Small Molecules AZD0530 and Dasatinib Inhibit Dengue Virus RNA Replication via Fyn Kinase

“…An advantage to this system is that it allows us to distinguish a viral assembly defect from a viral RNA replication defect since the VLPs are formed independently of viral RNA replication. Plasmid pcDNA3.1-D2.VLP (21), which encodes a codon-optimized cassette directing expression of the prM-E proteins of DV2, was transfected into Huh7 cells, and the cells were then treated with DMSO, 10 M AZD0530, or 5 M dasatinib at 4 h posttransfection. At 24 h posttreatment, VLPs were concentrated from the supernatants by PEG precipitation and purified by ultracentrifugation on a sucrose cushion.…”

“…The strategy to mutate the pRS-D2 plasmid by exploiting yeast recombination was as described before (21). The mutation NS4B-T108I (nucleotide change T7148 to C) was introduced directly through amplification of the compensatory virus cDNA using the primers D2-5339 FW and D2-8006 RV.…”

“…Y1876). Yeast DNA was extracted as previously described (21) and used to transform Stbl2 cells (Life Technologies, catalog no. 10268-019) for amplification.…”