Estudio de los virus de peces del banco pesquero Flemish Cap (Terranova)

En el presente estudio se analizaron un total de 250 peces sin síntomas aparentes de enfermedad, pertenecientes a 8 especies: gallineta (Sebastes mentella), fletán negro (Reinhardtius hippoglossoides), platija americana (Hippoglossoides platessoides), coreano (Glyptocephalus cynoglossus), bacalao (Gadus morhua), granadero (Macrourus berglax), perro del norte (Anarhichas lupus) y Antimora rostrata que se capturaron en el transcurso de una campaña de investigación pesquera realizada a bordo del buque oceanográfico Cornide de Saavedra en aguas de Terranova, en el banco pesquero Flemish Cap, durante los meses de junio y julio de 1999.In the present study we have analyzed a total of 250 apparently healthy fish, belonging to 8 different species: deepwater redfish (Sebastes mentella), Greenland halibut (Reinhardtius hippoglossoides), American plaice (Hippoglossoides platessoides), witch flounder (Glyptocephalus cynoglossus), Atlantic cod (Gadus morhua), onion-eye grenadier (Macrourus berglax), Atlantic wolf-fish (Anarhichas lupus) and blue antimora (Antimora rostrata). These fish were caught in the summer of 1999 during a marine research campaign carried out in the Flemish Cap fishery, close to Newfoundland

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories

New insights on the role of paired membrane structures in coronavirus replication

The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the Coronaviridae family members, but across the order Nidovirales. Taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral RNA. However, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. Here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication

Phosphorylation of serine 225 in hepatitis C virus NS5A regulates protein-protein interactions.

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A, however the mechanistic details of the role of this post-translational modification in the virus life cycle remains obscure. We previously reported (Ross-Thriepland et al, 2015) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis 30 revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as Amphiphysin II) and vesicle associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/western blotting, immunofluorescence and proximity ligation assay. Importantly, siRNA-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localisation of replication complexes. IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A

An Update on the Intracellular and Intercellular Trafficking of Carmoviruses

[EN] Despite harboring the smallest genomes among plant RNA viruses, carmoviruses have emerged as an ideal model system for studying essential steps of the viral cycle including intracellular and intercellular trafficking. Two small movement proteins, formerly known as double gene block proteins (DGBp1 and DGBp2), have been involved in the movement throughout the plant of some members of carmovirus genera. DGBp1 RNA-binding capability was indispensable for cell-to-cell movement indicating that viral genomes must interact with DGBp1 to be transported. Further investigation on Melon necrotic spot virus (MNSV) DGBp1 subcellular localization and dynamics also supported this idea as this protein showed an actin-dependent movement along microfilaments and accumulated at the cellular periphery. Regarding DGBp2, subcellular localization studies showed that MNSV and Pelargonium flower break virus DGBp2s were inserted into the endoplasmic reticulum (ER) membrane but only MNSV DGBp2 trafficked to plasmodesmata (PD) via the Golgi apparatus through a COPII-dependent pathway. DGBp2 function is still unknown but its localization at PD was a requisite for an efficient cell-to-cell movement. It is also known that MNSV infection can induce a dramatic reorganization of mitochondria resulting in anomalous organelles containing viral RNAs. These putative viral factories were frequently found associated with the ER near the PD leading to the possibility that MNSV movement and replication could be spatially linked. Here, we update the current knowledge of the plant endomembrane system involvement in carmovirus intra-and intercellular movement and the tentative model proposed for MNSV transport within plant cells.This work was funded by grant BIO2014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2014/010 from the Generalitat Valenciana.Navarro Bohigues, JA.; Pallás Benet, V. (2017). An Update on the Intracellular and Intercellular Trafficking of Carmoviruses. Frontiers in Plant Science. 8:1-7. https://doi.org/10.3389/fpls.2017.01801S178Adams, M. J., Lefkowitz, E. J., King, A. M. Q., Harrach, B., Harrison, R. L., Knowles, N. J., … Davison, A. J. (2016). Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016). Archives of Virology, 161(10), 2921-2949. doi:10.1007/s00705-016-2977-6Blake, J. A., Lee, K. W., Morris, T. J., & Elthon, T. E. (2007). Effects of turnip crinkle virus infection on the structure and function of mitochondria and expression of stress proteins in turnips. Physiologia Plantarum, 129(4), 698-706. doi:10.1111/j.1399-3054.2006.00852.xBlanco-Pérez, M., Pérez-Cañamás, M., Ruiz, L., & Hernández, C. (2016). Efficient Translation of Pelargonium line pattern virus RNAs Relies on a TED-Like 3´-Translational Enhancer that Communicates with the Corresponding 5´-Region through a Long-Distance RNA-RNA Interaction. PLOS ONE, 11(4), e0152593. doi:10.1371/journal.pone.0152593Brandizzi, F., Frangne, N., Marc-Martin, S., Hawes, C., Neuhaus, J.-M., & Paris, N. (2002). The Destination for Single-Pass Membrane Proteins Is Influenced Markedly by the Length of the Hydrophobic Domain. The Plant Cell, 14(5), 1077-1092. doi:10.1105/tpc.000620Carrington, J. C., Heaton, L. A., Zuidema, D., Hillman, B. I., & Morris, T. J. (1989). The genome structure of turnip crinkle virus. Virology, 170(1), 219-226. doi:10.1016/0042-6822(89)90369-3Chandra-Shekara, A. C., Navarre, D., Kachroo, A., Kang, H.-G., Klessig, D., & Kachroo, P. (2004). Signaling requirements and role of salicylic acid in HRT- and rrt-mediated resistance to turnip crinkle virus in Arabidopsis. The Plant Journal, 40(5), 647-659. doi:10.1111/j.1365-313x.2004.02241.xCohen, Y., Gisel, A., & Zambryski, P. C. (2000). Cell-to-Cell and Systemic Movement of Recombinant Green Fluorescent Protein-Tagged Turnip Crinkle Viruses. Virology, 273(2), 258-266. doi:10.1006/viro.2000.0441Cohen, Y., Qu, F., Gisel, A., Morris, T. J., & Zambryski, P. C. (2000). Nuclear Localization of Turnip Crinkle Virus Movement Protein p8. Virology, 273(2), 276-285. doi:10.1006/viro.2000.0440Gao, F., Kasprzak, W., Stupina, V. A., Shapiro, B. A., & Simon, A. E. (2012). A Ribosome-Binding, 3′ Translational Enhancer Has a T-Shaped Structure and Engages in a Long-Distance RNA-RNA Interaction. Journal of Virology, 86(18), 9828-9842. doi:10.1128/jvi.00677-12García-Castillo, S., Sánchez-Pina, M. A., & Pallás, V. (2003). Spatio-temporal analysis of the RNAs, coat and movement (p7) proteins of Carnation mottle virus in Chenopodium quinoa plants. Journal of General Virology, 84(3), 745-749. doi:10.1099/vir.0.18715-0Genovés, A., Navarro, J. A., & Pallás, V. (2006). Functional analysis of the five melon necrotic spot virus genome-encoded proteins. Journal of General Virology, 87(8), 2371-2380. doi:10.1099/vir.0.81793-0Genovés, A., Navarro, J. A., & Pallás, V. (2009). A self-interacting carmovirus movement protein plays a role in binding of viral RNA during the cell-to-cell movement and shows an actin cytoskeleton dependent location in cell periphery. Virology, 395(1), 133-142. doi:10.1016/j.virol.2009.08.042Genoves, A., Pallas, V., & Navarro, J. A. (2011). Contribution of Topology Determinants of a Viral Movement Protein to Its Membrane Association, Intracellular Traffic, and Viral Cell-to-Cell Movement. Journal of Virology, 85(15), 7797-7809. doi:10.1128/jvi.02465-10Gómez-Aix, C., García-García, M., Aranda, M. A., & Sánchez-Pina, M. A. (2015). Melon necrotic spot virus Replication Occurs in Association with Altered Mitochondria. Molecular Plant-Microbe Interactions®, 28(4), 387-397. doi:10.1094/mpmi-09-14-0274-rGrangeon, R., Jiang, J., & Laliberté, J.-F. (2012). Host endomembrane recruitment for plant RNA virus replication. Current Opinion in Virology, 2(6), 683-690. doi:10.1016/j.coviro.2012.10.003Grangeon, R., Jiang, J., Wan, J., Agbeci, M., Zheng, H., & Laliberté, J.-F. (2013). 6K2-induced vesicles can move cell to cell during turnip mosaic virus infection. Frontiers in Microbiology, 4. doi:10.3389/fmicb.2013.00351Guilley, H., Carrington, J. C., Balàzs, E., Jonard, G., Richards, K., & Morris, T. J. (1985). Nucleotide sequence and genome organization of carnation mottle virus RNA. Nucleic Acids Research, 13(18), 6663-6677. doi:10.1093/nar/13.18.6663Hacker, D. L., Petty, I. T. D., Wei, N., & Morris, T. J. (1992). Turnip crinkle virus genes required for RNA replication and virus movement. Virology, 186(1), 1-8. doi:10.1016/0042-6822(92)90055-tHerrera-Vásquez, J. A., Córdoba-Sellés, M. C., Cebrián, M. C., Alfaro-Fernández, A., & Jordá, C. (2009). Seed transmission ofMelon necrotic spot virusand efficacy of seed-disinfection treatments. Plant Pathology, 58(3), 436-442. doi:10.1111/j.1365-3059.2008.01985.xJiang, J., & Laliberté, J.-F. (2016). Membrane Association for Plant Virus Replication and Movement. Current Research Topics in Plant Virology, 67-85. doi:10.1007/978-3-319-32919-2_3Kaido, M., Tsuno, Y., Mise, K., & Okuno, T. (2009). Endoplasmic reticulum targeting of the Red clover necrotic mosaic virus movement protein is associated with the replication of viral RNA1 but not that of RNA2. Virology, 395(2), 232-242. doi:10.1016/j.virol.2009.09.022Kawakami, S., Watanabe, Y., & Beachy, R. N. (2004). Tobacco mosaic virus infection spreads cell to cell as intact replication complexes. Proceedings of the National Academy of Sciences, 101(16), 6291-6296. doi:10.1073/pnas.0401221101Krczal, G. (1995). Transmission of Pelargonium Flower Break Virus (PFBV) in Irrigation Systems and by Thrips. Plant Disease, 79(2), 163. doi:10.1094/pd-79-0163Lerch-Bader, M., Lundin, C., Kim, H., Nilsson, I., & von Heijne, G. (2008). Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum. Proceedings of the National Academy of Sciences, 105(11), 4127-4132. doi:10.1073/pnas.0711580105Lesemann, D.-E., & Adam, G. (1994). ELECTRON MICROSCOPICAL AND SEROLOGICAL STUDIES ON FOUR ISOMETRICAL PELARGONIUM VIRUSES. Acta Horticulturae, (377), 41-54. doi:10.17660/actahortic.1994.377.3Li, W., Qu, F., & Morris, T. J. (1998). Cell-to-Cell Movement of Turnip Crinkle Virus Is Controlled by Two Small Open Reading Frames That Functionin trans. Virology, 244(2), 405-416. doi:10.1006/viro.1998.9125Liu, C., & Nelson, R. S. (2013). The cell biology of Tobacco mosaic virus replication and movement. Frontiers in Plant Science, 4. doi:10.3389/fpls.2013.00012Marcos, J. F., Vilar, M., Pérez-Payá, E., & Pallás, V. (1999). In VivoDetection, RNA-Binding Properties and Characterization of the RNA-Binding Domain of the p7 Putative Movement Protein from Carnation Mottle Carmovirus (CarMV). Virology, 255(2), 354-365. doi:10.1006/viro.1998.9596Martínez-Gil, L., Johnson, A. E., & Mingarro, I. (2010). Membrane Insertion and Biogenesis of the Turnip Crinkle Virus p9 Movement Protein. Journal of Virology, 84(11), 5520-5527. doi:10.1128/jvi.00125-10Martínez-Gil, L., Saurí, A., Vilar, M., Pallás, V., & Mingarro, I. (2007). Membrane insertion and topology of the p7B movement protein of Melon Necrotic Spot Virus (MNSV). Virology, 367(2), 348-357. doi:10.1016/j.virol.2007.06.006Martínez-Turiño, S., & Hernández, C. (2009). Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors. Journal of General Virology, 90(2), 519-525. doi:10.1099/vir.0.006098-0Martínez-Turiño, S., & Hernández, C. (2011). A membrane-associated movement protein of Pelargonium flower break virus shows RNA-binding activity and contains a biologically relevant leucine zipper-like motif. Virology, 413(2), 310-319. doi:10.1016/j.virol.2011.03.001Martínez-Turiño, S., & Hernández, C. (2012). Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus. Virus Research, 163(2), 580-591. doi:10.1016/j.virusres.2011.12.011Mello, A. F. S., Clark, A. J., & Perry, K. L. (2009). Capsid protein of cowpea chlorotic mottle virus is a determinant for vector transmission by a beetle. Journal of General Virology, 91(2), 545-551. doi:10.1099/vir.0.016402-0Miras, M., Sempere, R. N., Kraft, J. J., Miller, W. A., Aranda, M. A., & Truniger, V. (2013). Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking. New Phytologist, 202(1), 233-246. doi:10.1111/nph.12650Mochizuki, T., Hirai, K., Kanda, A., Ohnishi, J., Ohki, T., & Tsuda, S. (2009). Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain. Virology, 390(2), 239-249. doi:10.1016/j.virol.2009.05.012Morozov, S. Y., & Solovyev, A. G. (2003). Triple gene block: modular design of a multifunctional machine for plant virus movement. Journal of General Virology, 84(6), 1351-1366. doi:10.1099/vir.0.18922-0Mueller, S. J., & Reski, R. (2015). Mitochondrial Dynamics and the ER: The Plant Perspective. Frontiers in Cell and Developmental Biology, 3. doi:10.3389/fcell.2015.00078Navarro, J. A., Genovés, A., Climent, J., Saurí, A., Martínez-Gil, L., Mingarro, I., & Pallás, V. (2006). RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins. Virology, 356(1-2), 57-67. doi:10.1016/j.virol.2006.07.040Nieto, C., Morales, M., Orjeda, G., Clepet, C., Monfort, A., Sturbois, B., … Bendahmane, A. (2006). AneIF4Eallele confers resistance to an uncapped and non-polyadenylated RNA virus in melon. The Plant Journal, 48(3), 452-462. doi:10.1111/j.1365-313x.2006.02885.xOhki, T., Akita, F., Mochizuki, T., Kanda, A., Sasaya, T., & Tsuda, S. (2010). The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus. Virology, 402(1), 129-134. doi:10.1016/j.virol.2010.03.020Panavas, T., Hawkins, C. M., Panaviene, Z., & Nagy, P. D. (2005). The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus. Virology, 338(1), 81-95. doi:10.1016/j.virol.2005.04.025Powers, J. G., Sit, T. L., Qu, F., Morris, T. J., Kim, K.-H., & Lommel, S. A. (2008). A Versatile Assay for the Identification of RNA Silencing Suppressors Based on Complementation of Viral Movement. Molecular Plant-Microbe Interactions®, 21(7), 879-890. doi:10.1094/mpmi-21-7-0879Qu, F., Ren, T., & Morris, T. J. (2003). The Coat Protein of Turnip Crinkle Virus Suppresses Posttranscriptional Gene Silencing at an Early Initiation Step. Journal of Virology, 77(1), 511-522. doi:10.1128/jvi.77.1.511-522.2003Riviere, C. J., & Rochon, D. M. (1990). Nucleotide sequence and genomic organization of melon necrotic spot virus. Journal of General Virology, 71(9), 1887-1896. doi:10.1099/0022-1317-71-9-1887Romero-Brey, I., & Bartenschlager, R. (2014). Membranous Replication Factories Induced by Plus-Strand RNA Viruses. Viruses, 6(7), 2826-2857. doi:10.3390/v6072826Russo, M., & Martelli, G. P. (1982). Ultrastructure of turnip crinkle- and saguaro cactus virus-infected tissues. Virology, 118(1), 109-116. doi:10.1016/0042-6822(82)90324-5Saurí, A., Saksena, S., Salgado, J., Johnson, A. E., & Mingarro, I. (2005). Double-spanning Plant Viral Movement Protein Integration into the Endoplasmic Reticulum Membrane Is Signal Recognition Particle-dependent, Translocon-mediated, and Concerted. Journal of Biological Chemistry, 280(27), 25907-25912. doi:10.1074/jbc.m412476200Serra-Soriano, M., Antonio Navarro, J., & Pallás, V. (2016). Dissecting the multifunctional role of the N-terminal domain of theMelon necrotic spot viruscoat protein in RNA packaging, viral movement and interference with antiviral plant defence. Molecular Plant Pathology, 18(6), 837-849. doi:10.1111/mpp.12448Serra-Soriano, M., Pallás, V., & Navarro, J. A. (2014). A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements. The Plant Journal, 77(6), 863-879. doi:10.1111/tpj.12435Shi, Y., Ryabov, E. V., van Wezel, R., Li, C., Jin, M., Wang, W., … Hong, Y. (2009). Suppression of local RNA silencing is not sufficient to promote cell-to-cell movement ofTurnip crinkle virusinNicotiana benthamiana. Plant Signaling & Behavior, 4(1), 15-22. doi:10.4161/psb.4.1.7573Teakle, D. S. (1980). FUNGI. Vectors of Plant Pathogens, 417-438. doi:10.1016/b978-0-12-326450-3.50021-8Thomas, C. L., Leh, V., Lederer, C., & Maule, A. J. (2003). Turnip crinkle virus coat protein mediates suppression of RNA silencing in nicotiana benthamiana. Virology, 306(1), 33-41. doi:10.1016/s0042-6822(02)00018-1Tilsner, J., Linnik, O., Louveaux, M., Roberts, I. M., Chapman, S. N., & Oparka, K. J. (2013). 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Hepatitis C virus, cholesterol and lipoproteins--impact for the viral life cycle and pathogenesis of liver disease.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, including chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatitis C infection associates with lipid and lipoprotein metabolism disorders such as hepatic steatosis, hypobetalipoproteinemia, and hypocholesterolemia. Furthermore, virus production is dependent on hepatic very-low-density lipoprotein (VLDL) assembly, and circulating virions are physically associated with lipoproteins in complexes termed lipoviral particles. Evidence has indicated several functional roles for the formation of these complexes, including co-opting of lipoprotein receptors for attachment and entry, concealing epitopes to facilitate immune escape, and hijacking host factors for HCV maturation and secretion. Here, we review the evidence surrounding pathogenesis of the hepatitis C infection regarding lipoprotein engagement, cholesterol and triglyceride regulation, and the molecular mechanisms underlying these effects

Membranous Replication Factories Induced by Plus-Strand RNA Viruses

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles