Virion- and Vesicle-Containing ER Cisternae Are Part of One Continuous Intracellular Membrane Network Animation through a Z series of 1

Virion- and Vesicle-Containing ER Cisternae Are Part of One Continuous Intracellular Membrane Network Animation through a Z series of 1.996 nm thick digital slices (total thickness 198 nm) of a single-axis tomogram (corresponding to Figure?6C), reconstructed from an 250 nm thick section of a DENV-infected Huh-7 cell. GUID:?0D9A9062-C616-45EC-B8CD-2BD0FCBCC685 Movie S3. Virus-Induced Vesicles Are Continuous with the ER and Have Pore-like Openings that Are Tightly Associated with Virus Budding Structures Animation through the central portion of Z series of 1.78 nm thick digital slices (total thickness 178 nm) of a dual-axis tomogram (corresponding to Figures 7FC7I), reconstructed from an 250 nm thick section of a DENV-infected Huh-7 cell. Colored overlay shows a 3D Caerulomycin A surface model of the virus-induced membranes. ER and Golgi membranes are depicted in yellow, inner vesicle membranes in light brown, and virus particles in red. Note the continuity of the ER and vesicle membranes and the putative budding profiles around the NE membrane opposite of the pores. Single virus particles are located within the NE and virions within vesicles near the Golgi stack. (5.3M) GUID:?97364DB2-1133-4365-A3EE-D676650259C4 Movie S4. Three-Dimensional Surface Model of Virus-Induced Vesicles with Pores, Virus Budding Structures, and Single Virions in an Infected Cell Three-dimensional surface rendering of ER and Golgi membranes (yellow), virus-induced vesicles (light brown), and virus particles (red) of the reconstructed 3D volume displayed in Figures 7FC7I and Movie S3. Note the vesicle pores directly connecting the vesicle lumen to the cytosol. (1.1M) GUID:?D79039D2-F820-4460-A92D-7A26EAEA9ECB Summary 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 Mouse monoclonal to alpha Actin 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. family, most notably hepatitis C virus (HCV). In cells either Caerulomycin A made up of a persistently replicating subgenome or infected with HCV, intensive structural rearrangements, designated the membranous web, were found (Gosert et?al., 2003, Miller and Krijnse-Locker, 2008). However, the induced membrane alterations are much more heterogenous, with irregular assemblies of membranous Caerulomycin A vesicles that vary in size and profound alterations of presumably ER-derived membranes. At variance with flaviviruses, the membranous web of HCV contains interspersed lipid droplets that play a major role in the assembly of infectious virus particles (Boulant et?al., 2007, Miyanari et?al., 2007, Shavinskaya et?al., 2007). These droplets appear to be tightly linked to the replication sites, but due to the high complexity of these membrane structures, a detailed analysis will be very difficult. In this respect, the 3D model developed here for DENV may serve as a template to unravel the architecture Caerulomycin A of the HCV replication and assembly compartments. In conclusion, we have resolved the 3D structures of flavivirus-induced membrane rearrangements. Our results provide a possible explanation for the spatial coupling of the different steps of the DENV replication cycle. The next obvious step is usually to integrate available information about the structures and functions of the viral and cellular factors involved in the biogenesis of these replication factories, but also to decipher the temporal relationship orchestrating.