scholarly journals Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses

2012 ◽  
Vol 125 (20) ◽  
pp. 4761-4769 ◽  
Author(s):  
K. Triantafilou ◽  
E. Vakakis ◽  
S. Kar ◽  
E. Richer ◽  
G. L. Evans ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Direct Interaction ◽  
Intermediate Form ◽  
Positive Strand ◽  
Replicative Intermediate ◽  
Positive Strand Rna

scholarly journals Ultrastructural Features of Membranous Replication Organelles Induced by Positive-Stranded RNA Viruses

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2407
Author(s):  
Van Nguyen-Dinh ◽  
Eva Herker
Keyword(s):  
Rna Viruses ◽  
Scientific Progress ◽  
Membrane System ◽  
Building Blocks ◽  
Surveillance Systems ◽  
Animal Cells ◽  
Cell Organelles ◽  
Positive Strand ◽  
Successful Infection ◽  
Positive Strand Rna

All intracellular pathogens critically depend on host cell organelles and metabolites for successful infection and replication. One hallmark of positive-strand RNA viruses is to induce alterations of the (endo)membrane system in order to shield their double-stranded RNA replication intermediates from detection by the host cell’s surveillance systems. This spatial seclusion also allows for accruing host and viral factors and building blocks required for efficient replication of the genome and prevents access of antiviral effectors. Even though the principle is iterated by almost all positive-strand RNA viruses infecting plants and animals, the specific structure and the organellar source of membranes differs. Here, we discuss the characteristic ultrastructural features of the virus-induced membranous replication organelles in plant and animal cells and the scientific progress gained by advanced microscopy methods.

scholarly journals SELECTIVE METAGENOMIC ANALYSIS OF POSITIVE-STRAND RNA VIRUSES IN WASTEWATER

2015 ◽  
Vol 71 (7) ◽  
pp. III_339-III_349
Author(s):  
Shinobu KAZAMA ◽  
Yoshifumi MASAGO ◽  
Satoshi NUMAZAWA ◽  
Tatsuo OMURA
Keyword(s):  
Rna Viruses ◽  
Metagenomic Analysis ◽  
Positive Strand ◽  
Positive Strand Rna

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.

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