Composition and Three-Dimensional Architecture of the Dengue Virus Replication and Assembly Sites

Abstract

Positive-strand RNA viruses are known to rearrange cellular membranes to facilitate viral genome replication. The biogenesis and three-dimensional organization of these membranes and the link between replication and virus assembly sites is not fully clear. Using electron microscopy, we find Dengue virus (DENV)-induced vesicles, convoluted membranes, and virus particles to be endoplasmic reticulum (ER)-derived, and we detect double-stranded RNA, a presumed marker of RNA replication, inside virus-induced vesicles. Electron tomography (ET) shows DENV-induced membrane structures to be part of one ER-derived network. Furthermore, ET reveals vesicle pores that could enable release of newly synthesized viral RNA and reveals budding of DENV particles on ER membranes directly apposed to vesicle pores. Thus, DENV modifies ER membrane structure to promote replication and efficient encapsidation of the genome into progeny virus. This architecture of DENV replication and assembly sites could explain the coordination of distinct steps of the flavivirus replication cycle.

Post-translational regulation and modifications of flavivirus structural proteins

“…The ER membrane of infected cells is believed to support the translation of the flavivirus polyprotein, heterodimerization of newly formed prM and E, and formation of virions and prM/E particles by budding into the lumen (Roby et al, 2012;Welsch et al, 2009). Although this is the classical model of flavivirus assembly, it may not be universal, as at least one report concerning WNV-Sarafend has demonstrated unusual virion budding at the plasma membrane (Ng et al, 1994).…”

“…Determination of the precise structure of the flavivirus nucleocapsid remains elusive despite documentation of assembly of spherical nucleocapsid-like particles in vitro following incubation of recombinant C with nucleic acids (Kiermayr et al, 2004;Ló pez et al, 2009;Teoh et al, 2014), as well as the visualization of purported nucleocapsids adjacent to VP pores in electron micrographs of DENVinfected cells (Welsch et al, 2009). Regardless of the nature of flavivirus nucleocapsids, their assembly appears to be dependent on the association of C with the ER membrane and lipid droplets, presumably to optimally position the protein between the VP pore where nascent genomic RNA exists and the ER sites of prM/E envelope generation (Samsa et al, 2009;Welsch et al, 2009;Westaway et al, 1997).…”

“…Regardless of the nature of flavivirus nucleocapsids, their assembly appears to be dependent on the association of C with the ER membrane and lipid droplets, presumably to optimally position the protein between the VP pore where nascent genomic RNA exists and the ER sites of prM/E envelope generation (Samsa et al, 2009;Welsch et al, 2009;Westaway et al, 1997).…”

“…However, the efficiency of C dimerization impacts on the overall integrity of the assembled particle in a thermal stability assay (Patkar et al, 2007), suggesting that some interaction between C and prM/E does still occur in the virion. Recent evidence describing the ability of DENV C to act as a histone mimic and cause nucleic acid condensation (Colpitts et al, 2011) supports the hypothesis that the flavivirus nucleocapsid is not an ordered icosahedral shell surrounding the genome but rather the RNA condenses around C dimers.Determination of the precise structure of the flavivirus nucleocapsid remains elusive despite documentation of assembly of spherical nucleocapsid-like particles in vitro following incubation of recombinant C with nucleic acids (Kiermayr et al, 2004;Ló pez et al, 2009;Teoh et al, 2014), as well as the visualization of purported nucleocapsids adjacent to VP pores in electron micrographs of DENVinfected cells (Welsch et al, 2009). Regardless of the nature of flavivirus nucleocapsids, their assembly appears to be dependent on the association of C with the ER membrane and lipid droplets, presumably to optimally position the protein between the VP pore where nascent genomic RNA …”

Hepatitis c virus and host cell lipids: An intimate connection

“…81,108 The origin of these membranes varies depending on the virus and they might be derived from e.g., the ER (Dengue virus), 109 the outer mitochondrial membrane (Flock house virus), 110 or the plasma membrane (alphaviruses). 111,112 It is assumed that the specific protein and lipid composition of these membranes play distinct roles in the replication cycle of a given virus.…”