scholarly journals Compartmentalized replication organelle of flavivirus at the ER and the factors involved

2021 ◽  
Author(s):  
Yali Ci ◽  
Lei Shi
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Membrane ◽  
Rna Viruses ◽  
Viral Proteins ◽  
Host Factors ◽  
Viral Rna ◽  
Host Protein ◽  
Membrane Structures ◽  
Multiple Factors ◽  
Positive Sense

AbstractFlaviviruses are positive-sense single-stranded RNA viruses that pose a considerable threat to human health. Flaviviruses replicate in compartmentalized replication organelles derived from the host endoplasmic reticulum (ER). The characteristic architecture of flavivirus replication organelles includes invaginated vesicle packets and convoluted membrane structures. Multiple factors, including both viral proteins and host factors, contribute to the biogenesis of the flavivirus replication organelle. Several viral nonstructural (NS) proteins with membrane activity induce ER rearrangement to build replication compartments, and other NS proteins constitute the replication complexes (RC) in the compartments. Host protein and lipid factors facilitate the formation of replication organelles. The lipid membrane, proteins and viral RNA together form the functional compartmentalized replication organelle, in which the flaviviruses efficiently synthesize viral RNA. Here, we reviewed recent advances in understanding the structure and biogenesis of flavivirus replication organelles, and we further discuss the function of virus NS proteins and related host factors as well as their roles in building the replication organelle.

scholarly journals Infectious Bronchitis Virus Generates Spherules from Zippered Endoplasmic Reticulum Membranes

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Helena J. Maier ◽  
Philippa C. Hawes ◽  
Eleanor M. Cottam ◽  
Judith Mantell ◽  
Paul Verkade ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Infectious Bronchitis Virus ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Membrane Vesicles ◽  
Viral Rna ◽  
Double Membrane ◽  
Infectious Bronchitis ◽  
Positive Sense ◽  
Infected Cells

ABSTRACTReplication of positive-sense RNA viruses is associated with the rearrangement of cellular membranes. Previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian cell line but also in primary avian cells and in epithelial cells ofex vivotracheal organ cultures. In all cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes.IMPORTANCEAll positive-sense single-stranded RNA viruses induce rearranged cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.

scholarly journals Flavivirus Replication Organelle Biogenesis in the Endoplasmic Reticulum: Comparison with Other Single-Stranded Positive-Sense RNA Viruses

2019 ◽  
Vol 20 (9) ◽  
pp. 2336 ◽  
Author(s):  
Masashi Arakawa ◽  
Eiji Morita
Keyword(s):  
Endoplasmic Reticulum ◽  
Virus Replication ◽  
Host Factors ◽  
Genome Replication ◽  
Organelle Biogenesis ◽  
Membrane Structures ◽  
Positive Sense ◽  
Viral Genome Replication ◽  
Amorphous Structures

Some single-stranded positive-sense RNA [ssRNA(+)] viruses, including Flavivirus, generate specific organelle-like structures in the host endoplasmic reticulum (ER). These structures are called virus replication organelles and consist of two distinct subdomains, the vesicle packets (VPs) and the convoluted membranes (CMs). The VPs are clusters of small vesicle compartments and are considered to be the site of viral genome replication. The CMs are electron-dense amorphous structures observed in proximity to the VPs, but the exact roles of CMs are mostly unknown. Several recent studies have revealed that flaviviruses recruit several host factors that are usually used for the biogenesis of other conventional organelles and usurp their function to generate virus replication organelles. In the current review, we summarize recent studies focusing on the role of host factors in the formation of virus replication organelles and discuss how these intricate membrane structures are organized.

scholarly journals Innate Immune DNA Sensing of Flaviviruses

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 979
Author(s):  
Tongtong Zhu ◽  
Ana Fernandez-Sesma
Keyword(s):  
Innate Immune System ◽  
Rna Viruses ◽  
Innate Immune ◽  
Host Factors ◽  
Dna Sensing ◽  
Antiviral Responses ◽  
Sensory Pathways ◽  
Sensing System ◽  
Positive Sense ◽  
Successful Replication

Flaviviruses are arthropod-borne RNA viruses that have been used extensively to study host antiviral responses. Often selected just to represent standard single-stranded positive-sense RNA viruses in early studies, the Flavivirus genus over time has taught us how truly unique it is in its remarkable ability to target not just the RNA sensory pathways but also the cytosolic DNA sensing system for its successful replication inside the host cell. This review summarizes the main developments on the unexpected antagonistic strategies utilized by different flaviviruses, with RNA genomes, against the host cyclic GAMP synthase (cGAS)/stimulator of interferon genes (STING) cytosolic DNA sensing pathway in mammalian systems. On the basis of the recent advancements on this topic, we hypothesize that the mechanisms of viral sensing and innate immunity are much more fluid than what we had anticipated, and both viral and host factors will continue to be found as important factors contributing to the host innate immune system in the future.

scholarly journals Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

2018 ◽  
Vol 92 (8) ◽  
Author(s):  
Maija K. Pietilä ◽  
Martijn J. van Hemert ◽  
Tero Ahola
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rna Viruses ◽  
Viral Rna ◽  
Cellular Membranes ◽  
Positive Strand ◽  
Membrane Markers ◽  
Highly Active ◽  
Replicase Proteins ◽  
Positive Strand Rna

ABSTRACTPositive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi membranes, and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER), and late endosome markers were retained in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins, and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication, as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the cofractionation of the RC-carrying plasma membrane and ER suggests that SFV recruits ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity, demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules, but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs.IMPORTANCESimilar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories, but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA andin vitroreplication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes were enriched with the replication complexes, demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have developed here a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.

scholarly journals Network-Guided Discovery of Influenza Virus Replication Host Factors

2018 ◽  
Author(s):  
Emily E. Ackerman ◽  
Eiryo Kawakami ◽  
Manami Katoh ◽  
Tokiko Watanabe ◽  
Shinji Watanabe ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Drug Targets ◽  
Antiviral Drug ◽  
Viral Proteins ◽  
Host Factors ◽  
Host Protein ◽  
Ppi Network ◽  
Host Proteins ◽  
Influenza Virus Replication

ABSTRACTThe position of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of pathways regulated by viruses (where proteins themselves do not interact with viral proteins) or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining a human PPI network with virus-host protein interaction data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Network analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow. We have isolated a subnetwork of the human PPI network which connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes. Selecting proteins based on network topology within the subnetwork, we performed an siRNA screen to determine if the subnetwork was enriched for virus replication host factors and if network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins – more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCEIntegrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e. proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

scholarly journals Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Philip V'kovski ◽  
Markus Gerber ◽  
Jenna Kelly ◽  
Stephanie Pfaender ◽  
Nadine Ebert ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Host Factors ◽  
Viral Rna ◽  
Host Proteins ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Starting Point ◽  
Positive Strand Rna

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.

scholarly journals Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

2016 ◽  
Vol 90 (7) ◽  
pp. 3676-3683 ◽  
Author(s):  
James R. Short ◽  
Jeffrey A. Speir ◽  
Radhika Gopal ◽  
Logan M. Pankratz ◽  
Jason Lanman ◽  
...  
Keyword(s):  
Host Defense ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Rna Viruses ◽  
Rna Replication ◽  
Viral Rna ◽  
Flock House Virus ◽  
Double Stranded Rna ◽  
Content Type ◽  
Positive Sense

ABSTRACTViruses that generate double-stranded RNA (dsRNA) during replication must overcome host defense systems designed to detect this infection intermediate. All positive-sense RNA viruses studied to date modify host membranes to help facilitate the sequestration of dsRNA from host defenses and concentrate replication factors to enhance RNA production. Flock House virus (FHV) is an attractive model for the study of these processes since it is well characterized and infectsDrosophilacells, which are known to have a highly effective RNA silencing system. During infection, FHV modifies the outer membrane of host mitochondria to form numerous membrane invaginations, called spherules, that are ∼50 nm in diameter and known to be the site of viral RNA replication. While previous studies have outlined basic structural features of these invaginations, very little is known about the mechanism underlying their formation. Here we describe the optimization of an experimental system for the analysis of FHV host membrane modifications using crude mitochondrial preparations from infectedDrosophilacells. These preparations can be programmed to synthesize both single- and double-stranded FHV RNA. The system was used to demonstrate that dsRNA is protected from nuclease digestion by virus-induced membrane invaginations and that spherules play an important role in stimulating RNA replication. Finally, we show that spherules generated during FHV infection appear to be dynamic as evidenced by their ability to form or disperse based on the presence or absence of RNA synthesis.IMPORTANCEIt is well established that positive-sense RNA viruses induce significant membrane rearrangements in infected cells. However, the molecular mechanisms underlying these rearrangements, particularly membrane invagination and spherule formation, remain essentially unknown. How the formation of spherules enhances viral RNA synthesis is also not understood, although it is assumed to be partly a result of evading host defense pathways. To help interrogate some of these issues, we optimized a cell-free replication system consisting of mitochondria isolated from Flock House virus-infectedDrosophilacells for use in biochemical and structural studies. Our data suggest that spherules generated during Flock House virus replication are dynamic, protect double-stranded RNA, and enhance RNA replication in general. Cryo-electron microscopy suggests that the samples are amenable to detailed structural analyses of spherules engaged in RNA synthesis. This system thus provides a foundation for understanding the molecular mechanisms underlying spherule formation, maintenance, and function during positive-sense viral RNA replication.

scholarly journals Analysis of viral RNA-host protein interactomes enables rapid antiviral drug discovery

2021 ◽  
Author(s):  
Shaojun Zhang ◽  
Wenze Huang ◽  
Lili Ren ◽  
Xiaohui Ju ◽  
Mingli Gong ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Antiviral Drug ◽  
Antiviral Agent ◽  
Viral Rna ◽  
Host Protein ◽  
Host Responses ◽  
Interaction Patterns ◽  
Global Public Health

RNA viruses including SARS-CoV-2, Ebola virus (EBOV), and Zika virus (ZIKV) constitute a major threat to global public health and society. The interactions between viral genomes and host proteins are essential in the life cycle of RNA viruses and thus provide targets for drug development. However, viral RNA-host protein interactions have remained poorly characterized. Here we applied ChIRP-MS to profile the interactomes of human proteins and the RNA genomes of SARS-CoV-2, EBOV, and ZIKV in infected cells. Integrated interactome analyses revealed interaction patterns that reflect both common and virus-specific host responses, and enabled rapid drug screening to target the vulnerable host factors. We identified Enasidenib as a SARS-CoV-2 specific antiviral agent, and Trifluoperazine and Cepharanthine as broad spectrum antivirals against all three RNA viruses.

scholarly journals Regulation of RNA Virus Processes by Viral Genome Structure

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 68
Author(s):  
K. Andrew White
Keyword(s):  
Viral Genome ◽  
Rna Viruses ◽  
Rna Virus ◽  
Genome Structure ◽  
Viral Proteins ◽  
Viral Rna ◽  
Rna Sequences ◽  
Virus Reproduction ◽  
The Family ◽  
Rna Genome

The genomes of RNA viruses contain a variety of RNA sequences and structures that regulate different steps in virus reproduction. Events that are controlled by RNA elements include (i) the translation of viral proteins, (ii) the replication of viral RNA genomes, and (iii) the transcription of viral subgenomic mRNAs. Studies of members of the family Tombusviridae, which possess plus-strand RNA genomes, have revealed novel ways in which the RNA genome structure is utilized to control different viral processes. Recent advances in our understanding of RNA-based viral regulation in select tombusvirids will be presented.

scholarly journals A Cell-free Infection System to Study Translation, Replication and Phage-particle Production During Infection of E. coli by Bacteriophage Qβ

2021 ◽  
Author(s):  
Pernille Skov Rasmussen ◽  
Charlotte Rohde Knudsen
Keyword(s):  
Self Assembly ◽  
Phage Particle ◽  
Particle Production ◽  
Rna Viruses ◽  
Viral Proteins ◽  
Viral Rna ◽  
Translation Regulation ◽  
Custom Made ◽  
A Cell

Abstract Background Viruses infect all kingdoms of life, and new species are continuously being discovered. The single-stranded (+)-RNA viruses comprise the largest group of viruses, which includes pathogens such as Dengue virus, Corona virus and West Nile virus. Also, the simple bacteriophage Qβ belongs to this group of viruses. Studies of the mechanism of Qβ infection can increase our general understanding of the single-stranded (+)-RNA viruses, which can be exploited in the pursuit to treat and prevent of diseases caused by pathogenic (+)-RNA viruses.MethodsIn this study, we have analysed the production of infectious Qβ phage particles in three different cell-free infection systems upon addition of the Qβ genome as a template. The cell-free infection systems were based on cell-free protein expression systems: two commercial systems and one custom-made system. We studied the course of infection by analysing the production of viral RNA, proteins and phage particles produced in the cell-free reactions. The replication of the viral RNA was determined by RT-PCR, while the translation of the viral proteins was examined by radiolabelling, and the production of infectious phage particles was evaluated by double-layered plaque assays. ResultsBacteriophage Qβ was found to replicate in two of the three tested cell-free infection systems. Specifically, the viral RNA was replicated, the viral proteins were translated, and infectious phage particles were produced in the cell-free infection systems. The pattern of translation regulation of the viral proteins appeared similar to in vivo infection. Infectious Qβ phage particles were produced at yields of 25.4 × 104 PFU/μL reaction and 2.5 × 103 PFU/μL reaction in the commercial and custom-made system, respectively. Importantly, intact Qβ phage particles were shown not to replicate in the cell-free infection systems under the tested conditions. ConclusionCell-free infection systems can support replication of viral RNA, translation of viral proteins and self-assembly of infectious Qβ phage particles. We provide opportunities for further optimisation of the phage particle yield. Cell-free infection systems can be used in the future to study newly discovered viruses, the development of antiviral and antibacterial drugs, and in biotechnology.

Sign in / Sign up

Export Citation Format

Share Document