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Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus
- Source :
- PLoS Pathogens, PLoS Pathogens, Vol 15, Iss 1, p e1007530 (2019)
- Publication Year :
- 2019
- Publisher :
- Public Library of Science (PLoS), 2019.
-
Abstract
- Tombusviruses depend on subversions of multiple host factors and retarget cellular pathways to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) recruit the cellular Vps34 phosphatidylinositol 3-kinase (PI3K) into the large viral replication compartment. The kinase function of Vps34 is critical for TBSV replication, suggesting that PI(3)P phosphoinositide is utilized by TBSV for building of the replication compartment. We also observed increased expression of Vps34 and the higher abundance of PI(3)P in the presence of the tombusviral replication proteins, which likely leads to more efficient tombusvirus replication. Accordingly, overexpression of PI(3)P phosphatase in yeast or plants inhibited TBSV replication on the peroxisomal membranes and CIRV replication on the mitochondrial membranes. Moreover, the purified PI(3)P phosphatase reduced TBSV replicase assembly in a cell-free system. Detection of PI(3)P with antibody or a bioprobe revealed the enrichment of PI(3)P in the replication compartment. Vps34 is directly recruited into the replication compartment through interaction with p33 replication protein. Gene deletion analysis in surrogate yeast host unraveled that TBSV replication requires the vesicle transport function of Vps34. In the absence of Vps34, TBSV cannot efficiently recruit the Rab5-positive early endosomes, which provide PE-rich membranes for membrane biogenesis of the TBSV replication compartment. We found that Vps34 and PI(3)P needed for the stability of the p33 replication protein, which is degraded by the 26S proteasome when PI(3)P abundance was decreased by an inhibitor of Vps34. In summary, Vps34 and PI(3)P are needed for providing the optimal microenvironment for the replication of the peroxisomal TBSV and the mitochondrial CIRV.<br />Author summary Replication of RNA viruses infecting various eukaryotic organisms is the central step in the infection process that leads to generation of progeny viruses. The replication process requires the assembly of numerous viral replicase complexes within the large replication compartment, whose formation is not well understood. Using tombusviruses and the model host yeast, the authors discovered that a highly conserved cellular lipid kinase, Vps34 phosphatidylinositol 3-kinase (PI3K), is critical for the formation of the viral replication compartment. Expression of PI3K mutants and the PI(3)P phosphatase revealed that the PI(3)P phosphoinositide produced by Vps34 is crucial for tombusvirus replication. Tombusviruses co-opt Vps34 through interaction with the viral replication protein into the replication compartment. In vitro reconstitution of the tombusvirus replicase revealed the need for Vps34 and PI(3)P for the full-activity of the viral replicase. Chemical inhibition of Vps34 in yeast or plants showed that PI(3)P is important for the replication of several plant viruses within the Tombusviridae family and the insect-infecting Nodamuravirus. These results open up the possibility that the cellular Vps34 PI3K could be a target for new antiviral strategies.
- Subjects :
- Leaves
Confocal Microscopy
Tombusvirus
Plant Science
Phosphatidylinositols
Virus Replication
Biochemistry
Electrophoretic Blotting
Phosphatidylinositol Phosphates
Medicine and Health Sciences
Biology (General)
Gel Electrophoresis
Microscopy
0303 health sciences
biology
Chemistry
Plant Anatomy
030302 biochemistry & molecular biology
Fungal Diseases
Eukaryota
Light Microscopy
Mitochondria
3. Good health
Cell biology
Infectious Diseases
Mitochondrial Membranes
RNA, Viral
Cellular Structures and Organelles
Research Article
QH301-705.5
Endosome
Immunology
Molecular Probe Techniques
RNA-dependent RNA polymerase
Research and Analysis Methods
Microbiology
Electrophoretic Techniques
Viral Proteins
03 medical and health sciences
Virology
Peroxisomes
Genetics
RNA Viruses
Molecular Biology Techniques
Protein Interactions
Molecular Biology
030304 developmental biology
fungi
Organisms
Fungi
Biology and Life Sciences
Proteins
RNA
RNA virus
Cell Biology
RC581-607
RNA-Dependent RNA Polymerase
biology.organism_classification
Class III Phosphatidylinositol 3-Kinases
Viral Replication
Yeast
Yeast Infections
Viral replication
Membrane biogenesis
Parasitology
Immunologic diseases. Allergy
Northern Blot
Tomato bushy stunt virus
Confocal Laser Microscopy
Subjects
Details
- ISSN :
- 15537374
- Volume :
- 15
- Database :
- OpenAIRE
- Journal :
- PLOS Pathogens
- Accession number :
- edsair.doi.dedup.....8186982431baea3877df9f1a83c2a078