The viral replication organelles within cells studied by electron microscopy

Sachse, Martin; Castro, Isabel Fernández de; Tenorio, Raquel; Risco, Cristina

doi:10.1016/bs.aivir.2019.07.005

Cited by 20 publications

(15 citation statements)

References 149 publications

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“…Inside the spherule, the CHIKV replication complex (RC) associates with the inner face of membranes through nsP1, the viral capping enzyme that contains unique membrane binding capacity and displays membrane-dependent methyl/guanylyltransferase activities (71)(72)(73)(74). Three-dimensional (3D) cryo-electron tomography was successfully applied to resolve the complex spatial organization of replication membranes formed by many positive-stranded RNA viruses (75)(76)(77)(78)(79)(80)(81). Conversely, the 3D architecture and biogenesis of alphavirus spherules remain enigmatic.…”

Section: Building Membrane Spherules To Ensure Viral Replicationmentioning

confidence: 99%

New Insights into Chikungunya Virus Infection and Pathogenesis

et al. 2021

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Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus responsible for major outbreaks of disease since 2004 in the Indian Ocean islands, South east Asia, and the Americas. CHIKV causes debilitating musculoskeletal disorders in humans that are characterized by fever, rash, polyarthralgia, and myalgia. The disease is often self-limiting and nonlethal; however, some patients experience atypical or severe clinical manifestations, as well as a chronic rheumatic syndrome. Unfortunately, no efficient antivirals against CHIKV infection are available so far, highlighting the importance of deepening our knowledge of CHIKV host cell interactions and viral replication strategies. In this review, we discuss recent breakthroughs in the molecular mechanisms that regulate CHIKV infection and lay down the foundations to understand viral pathogenesis. We describe the role of the recently identified host factors co-opted by the virus for infection and pathogenesis, and emphasize the importance of CHIKV nonstructural proteins in both replication complex assembly and host immune response evasion. Expected final online publication date for the Annual Review of Virology, Volume 8 is September 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Section: Building Membrane Spherules To Ensure Viral Replicationmentioning

confidence: 99%

New Insights into Chikungunya Virus Infection and Pathogenesis

et al. 2021

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“…Virus-induced compartmentalization of the cell is not an exclusive replication strategy of HAdVs. Assembly of compartments termed “viral organelles” or “factories”, “inclusion bodies” or “viroplasms” have been described for other viruses as sites for viral genome replication that concomitantly serve to conceal the viral genome from detection by cellular antiviral mechanisms [ 24 , 25 , 26 , 27 , 28 ]. Additional events of the viral replication cycle, such as viral gene transcription and RNA posttranscriptional processing, as well as the assembly of virus progeny, may also be coordinated in virus-induced compartments [ 23 , 28 , 29 , 30 , 31 , 32 ].…”

Section: Introductionmentioning

confidence: 99%

Evidence That the Adenovirus Single-Stranded DNA Binding Protein Mediates the Assembly of Biomolecular Condensates to Form Viral Replication Compartments

Hidalgo

Pimentel

Mojica-Santamaría

et al. 2021

Viruses

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A common viral replication strategy is characterized by the assembly of intracellular compartments that concentrate factors needed for viral replication and simultaneously conceal the viral genome from host-defense mechanisms. Recently, various membrane-less virus-induced compartments and cellular organelles have been shown to represent biomolecular condensates (BMCs) that assemble through liquid-liquid phase separation (LLPS). In the present work, we analyze biophysical properties of intranuclear replication compartments (RCs) induced during human adenovirus (HAdV) infection. The viral ssDNA-binding protein (DBP) is a major component of RCs that contains intrinsically disordered and low complexity proline-rich regions, features shared with proteins that drive phase transitions. Using fluorescence recovery after photobleaching (FRAP) and time-lapse studies in living HAdV-infected cells, we show that DBP-positive RCs display properties of liquid BMCs, which can fuse and divide, and eventually form an intranuclear mesh with less fluid-like features. Moreover, the transient expression of DBP recapitulates the assembly and liquid-like properties of RCs in HAdV-infected cells. These results are of relevance as they indicate that DBP may be a scaffold protein for the assembly of HAdV-RCs and should contribute to future studies on the role of BMCs in virus-host cell interactions.

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“…Flat embedding of cell monolayers combined with plane sectioning of the cells, with the cutting plane parallel to the substrate, has proven a powerful method for accessing information about the ultrastructure of cells grown in two dimensions (2D), especially migrating cells [11]. In the field of virology, flat embedding and plane sectioning of virus infected cells has been used in only a few studies, for example with cells grown on ACLAR embedding films [12] or with cells grown on gridded slides for correlative light-electron microscopy studies [13].…”

Section: Introductionmentioning

confidence: 99%

Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2

et al. 2021

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Despite the development of new diagnostic methods, co-culture, based on sample inoculation of cell monolayers coupled with electron microscopy (EM) observation, remains the gold standard in virology. Indeed, co-culture allows for the study of cell morphology (infected and not infected), the ultrastructure of the inoculated virus, and the different steps of the virus infectious cycle. Most EM methods for studying virus cycles are applied after infected cells are produced in large quantities and detached to obtain a pellet. Here, cell culture was performed in sterilized, collagen-coated single-break strip wells. After one day in culture, cells were infected with SARS-CoV-2. Wells of interest were fixed at different time points, from 2 to 36 h post-infection. Microwave-assisted resin embedding was accomplished directly in the wells in 4 h. Finally, ultra-thin sections were cut directly through the infected-cell monolayers. Our methodology requires, in total, less than four days for preparing and observing cells. Furthermore, by observing undetached infected cell monolayers, we were able to observe new ultrastructural findings, such as cell–cell interactions and baso-apical cellular organization related to the virus infectious cycle. Our innovative methodology thus not only saves time for preparation but also adds precision and new knowledge about viral infection, as shown here for SARS-CoV-2.

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The viral replication organelles within cells studied by electron microscopy

Cited by 20 publications

References 149 publications

New Insights into Chikungunya Virus Infection and Pathogenesis

New Insights into Chikungunya Virus Infection and Pathogenesis

Evidence That the Adenovirus Single-Stranded DNA Binding Protein Mediates the Assembly of Biomolecular Condensates to Form Viral Replication Compartments

Innovative Approach to Fast Electron Microscopy Using the Example of a Culture of Virus-Infected Cells: An Application to SARS-CoV-2

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