Dengue virus-induced autophagosomes and changes in endomembrane ultrastructure imaged by electron tomography and whole-mount grid-cell culture techniques

Journal of Electron Microscopy 59(6): 503–511 (2010) doi: 10.1093/jmicro/dfq063 .................................................................................................................................................................................................................................................. Biological: Full-length Dengue virus-induced autophagosomes and changes in endomembrane ultrastructure imaged by electron tomography and whole-mount grid-cell culture techniques 1 Electron Microscopy Group, National Institute of Virology (ICMR), 20A Dr Ambedkar Road, Pune 411001, India and 2National Institute of Immunohematology (ICMR), 13th Floor KEM Hospital, Parel, Mumbai, India *To whom correspondence should be addressed. E-mail: ............................................................................................................................................................................................ Abstract The biogenesis events and formation of dengue virus (DENV) in the infected host cells remain incompletely understood. In the present study, we examined the ultrastructural changes associated with DENV-2 replication in three susceptible host cells, C6/36, Vero and SK Hep1, a cell line of human endothelial origin, using transmission electron microscopy, wholemount grid-cell culture techniques and electron tomography (ET). The prominent feature in C6/36 cells was the formation of large perinuclear vacuoles with mature DENV particles, and on-grid whole-mount examination of the infected Vero cells showed different forms of DENV core structures associated with cellular membranes within 48 h after infection. Distinct multivesicular structures and prominent autophagic vesicles were seen in the infected SK Hep1 cells when compared with the other two cell lines. ET showed the three-dimensional organization of these vesicles as a continuous system. This is the first report of ET-based analysis of DENV-2 replication in a human endothelial cell line. These results further emphasizes the strong role played by intracellular host membranes–virus interactions in the biogenesis of DENV and strongly argues for the possibility of targeting compounds to block such structure formation as key anti-dengue agents. ............................................................................................................................................................................................ Keywords electron microscopy, dengue, electron tomography, on-grid cell culture ............................................................................................................................................................................................ Received 28 January 2010, accepted 14 July 2010, online 12 August 2010 ............................................................................................................................................................................................ Introduction The earliest electron microscopic observation of dengue viruses (DENV) probably dates back to 1948 by Sabin and Schleisinger on mouse brainderived virus particles and later in 1954 by Reagan and Brueckner in cell culture [1]. These studies, although limited by many technical hurdles like specimen processing and imaging artefacts, pioneered the use of electron microscopy (EM) in DENV research. Subsequently, over the last five decades, the ultrastructure and morphogenesis of DENV have been studied extensively and many facets of DENV replication in host cell and particle organization of the virion have begun to emerge [2,3]. Despite much research on DENV morphology, a review of the present knowledge on morphogenesis events of DENV replication and assembly in the host cell remain, to a great extent, incompletely understood. The backbone of these lacunae resides with the fact that synchronized time-lapse imaging of DENVinfected cells to monitor the progress of the virus replication events using EM-based techniques have .................................................................................................................................................................................................................................................. © The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: Shobha Gangodkar1, Preksha Jain1, Nishikant Dixit1, Kanjaksha Ghosh2 and Atanu Basu1,* 504 J O U R N A L O F E L E C T R O N M I C R O S C O P Y, Vol. 59, No. 6, 2010 Materials and methods Cells and virus Vero E6 cells were grown in minimum essential medium (MEM; HiMedia Ltd, India) supplemented with 10% foetal bovine serum (GIBCO, USA), 1% L-glutamine, penicillin and streptomycin (HiMedia Ltd, India). Cells were grown in six-well tissue culture plates (Nunc, USA) in a culture volume of 1 ml and under 5% CO2 at 37°C. The C6/36 cells were taken from the cell repository of the National Institute of Virology (NIV) in Pune and grown in Mitsuhasi and Maraorosch insect cell growth medium (HiMedia Ltd, India) supplemented with penicillin G, streptomycin, yeastolate and 10% tryptose phosphate broth at 29°C. The third cell line we used in this study was the SK Hep1, a cell line of human endothelial origin [6] and susceptible to DENV (unpublished data). The SK Hep1 cells were obtained from the National Center for Cell Sciences in Pune and grown in MEM supplemented with 10% foetal bovine serum, 1% L-glutamine, streptomycin and penicillin solution (HiMedia Ltd, India). The virus used in the present study was the Tr1751 strain of DENV-2 virus obtained from the NIV virus repository. Virus infection Vero E6, C6/36 and SK Hep1 cells were grown to a confluency of >80% as observed by inverted phasecontrast microscopy (Eclipse T2000, Nikon Corp., Japan) and exposed to 1 × 103 LD50 dose of DENV2 for 1 h with gentle shaking at 37°C and under 5% CO2. The virus-containing media was washed off with two changes of media and the cells were grown in complete media at 37°C and 5% CO2. Culturing cells on EM grids Vero E6 and C6/36 cells were cultured directly on EM grids using the earlier method of Hyatt et al. [7] with modifications. Briefly, 400-mesh nickel EM grids (Ted Pella, Switzerland) were provided with formvar support, carbon coated and sterilized by floatation (coated side down) over 3% glutaraldehyde under ultraviolet light. The grids were then removed, rinsed thoroughly with sterile deionized water and used for culturing cells. The surface of the formvar support was further conditioned for cell adhesion by treatment with poly-L-lysine (Sigma Chemicals, USA) or alcian blue (Sigma Chemicals, USA). Cells grown to more than 80% confluency were made into a single-cell suspension, seeded directly on the coated grids and cultured with complete media under 5% CO2 and at 37°C. Cells were stained with propidium iodide to image the n (...truncated)