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1.
Virology ; 567: 1-14, 2022 02.
Article in English | MEDLINE | ID: covidwho-1628759

ABSTRACT

The coronavirus nucleocapsid (N) protein comprises two RNA-binding domains connected by a central spacer, which contains a serine- and arginine-rich (SR) region. The SR region engages the largest subunit of the viral replicase-transcriptase, nonstructural protein 3 (nsp3), in an interaction that is essential for efficient initiation of infection by genomic RNA. We carried out an extensive genetic analysis of the SR region of the N protein of mouse hepatitis virus in order to more precisely define its role in RNA synthesis. We further examined the N-nsp3 interaction through construction of nsp3 mutants and by creation of an interspecies N protein chimera. Our results indicate a role for the central spacer as an interaction hub of the N molecule that is partially regulated by phosphorylation. These findings are discussed in relation to the recent discovery that nsp3 forms a molecular pore in the double-membrane vesicles that sequester the coronavirus replicase-transcriptase.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Intracellular Membranes/metabolism , Viral Replicase Complex Proteins/metabolism , Amino Acid Motifs , Animals , Cell Line , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Mice , Murine hepatitis virus , Mutation , Protein Binding , Protein Domains , RNA, Viral/biosynthesis , Viral Replicase Complex Proteins/chemistry , Viral Replicase Complex Proteins/genetics , Viral Replication Compartments/metabolism
2.
Viruses ; 13(12)2021 12 17.
Article in English | MEDLINE | ID: covidwho-1580424

ABSTRACT

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.


Subject(s)
Coronavirus/metabolism , Intracellular Membranes/metabolism , RNA, Viral/biosynthesis , Animals , Cell Line , Coronavirus/classification , Coronavirus/growth & development , Cytoplasm/metabolism , Humans , Infectious bronchitis virus/growth & development , Infectious bronchitis virus/metabolism , RNA, Double-Stranded/metabolism , Viral Replication Compartments/metabolism
3.
Cell Rep ; 37(8): 110049, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1509642

ABSTRACT

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.


Subject(s)
Autophagy/physiology , Hepacivirus/physiology , SARS-CoV-2/physiology , Viral Replication Compartments/metabolism , Autophagosomes/metabolism , Carrier Proteins/metabolism , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class III Phosphatidylinositol 3-Kinases/metabolism , Humans , Phosphatidylinositol Phosphates/metabolism , RNA, Viral/biosynthesis , Viral Nonstructural Proteins/metabolism , Virus Replication
4.
Cell Host Microbe ; 28(6): 853-866.e5, 2020 12 09.
Article in English | MEDLINE | ID: covidwho-1385263

ABSTRACT

Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.


Subject(s)
COVID-19/genetics , Endoplasmic Reticulum/ultrastructure , SARS-CoV-2/ultrastructure , Viral Replication Compartments/ultrastructure , COVID-19/diagnostic imaging , COVID-19/pathology , COVID-19/virology , Cell Death/genetics , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/virology , Humans , Microscopy, Electron , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Viral Replication Compartments/metabolism , Virus Replication/genetics
5.
J Med Virol ; 93(7): 4616-4619, 2021 07.
Article in English | MEDLINE | ID: covidwho-1263086

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) has been identified to be a mutation hot spot, with the P323L mutation being commonly observed in viral genomes isolated from North America. RdRp forms a complex with nonstructural proteins nsp7 and nsp8 to form the minimal replication/transcription machinery required for genome replication. As mutations in RdRp may affect formation of the RdRp-nsp7-nsp8 supercomplex, we analyzed viral genomes to identify mutations in nsp7 and nsp8 protein sequences. Based on in silico analysis of predicted structures of the supercomplex comprising of native and mutated proteins, we demonstrate that specific mutations in nsp7 and nsp8 proteins may have a role in stabilization of the replication/transcription complex.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/genetics , SARS-CoV-2/physiology , Viral Nonstructural Proteins/genetics , Viral Replication Compartments/chemistry , Amino Acid Sequence , Computer Simulation , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Genome, Viral , Humans , Models, Molecular , Mutation , Protein Stability , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Replication Compartments/metabolism
7.
Virology ; 556: 9-22, 2021 04.
Article in English | MEDLINE | ID: covidwho-985483

ABSTRACT

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.


Subject(s)
Coronavirus Infections/virology , Endoplasmic Reticulum/metabolism , Murine hepatitis virus/physiology , Viral Replication Compartments/metabolism , Animals , Cell Line , Electron Microscope Tomography , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Lysosomes/metabolism , Lysosomes/ultrastructure , Lysosomes/virology , Mice , Viral Proteins/metabolism , Viral Replication Compartments/ultrastructure , Virion/metabolism , Virus Assembly , Virus Replication
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