scholarly journals Function, Architecture, and Biogenesis of Reovirus Replication Neoorganelles

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 288 ◽  
Author(s):  
Raquel Tenorio ◽  
Isabel Fernández de Castro ◽  
Jonathan J. Knowlton ◽  
Paula F. Zamora ◽  
Danica M. Sutherland ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Host Cells ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Particle Assembly ◽  
The Matrix ◽  
Viral Genome Replication ◽  
Pathogenic Viruses ◽  
Infected Cells ◽  
Reovirus Replication

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA–protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses.

scholarly journals Reovirus σNS and μNS Proteins Remodel the Endoplasmic Reticulum to Build Replication Neo-Organelles

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Raquel Tenorio ◽  
Isabel Fernández de Castro ◽  
Jonathan J. Knowlton ◽  
Paula F. Zamora ◽  
Christopher H. Lee ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Viral Infection ◽  
Viral Genome ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Genome Replication ◽  
Particle Assembly ◽  
Viral Genome Replication ◽  
Reovirus Replication ◽  
Cellular Compartments

ABSTRACTLike most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles.IMPORTANCEViruses modify cellular structures to build replication organelles. These organelles serve as sites of viral genome replication and particle morphogenesis and are essential for viral infection. However, how these organelles are constructed is not well understood. We found that the replication organelles of mammalian reoviruses are formed by collections of membranous tubules and vesicles derived from extensive remodeling of the peripheral endoplasmic reticulum (ER). We also observed that ER tubulation and vesiculation are triggered by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles and provide functions for two enigmatic reovirus replication proteins. Most importantly, this research uncovers a new mechanism by which viruses form factories for particle assembly.

scholarly journals Reovirus σNS and μNS Proteins Form Cytoplasmic Inclusion Structures in the Absence of Viral Infection

2003 ◽  
Vol 77 (10) ◽  
pp. 5948-5963 ◽  
Author(s):  
Michelle M. Becker ◽  
Timothy R. Peters ◽  
Terence S. Dermody
Keyword(s):  
Viral Infection ◽  
Cytoplasmic Inclusion ◽  
Temperature Sensitive ◽  
Genome Replication ◽  
Structural Protein ◽  
Reovirus Infection ◽  
Viral Genome Replication ◽  
Infected Cells ◽  
Reovirus Replication ◽  
Viral Inclusions

ABSTRACT Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, σNS and μNS, and structural protein σ3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed σNS, μNS, and σ3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, σNS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed σNS is functional as it complements the defect in temperature-sensitive, σNS-mutant virus tsE320. In both transfected and infected cells, μNS is found in punctate cytoplasmic structures and σ3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of μNS and σ3 is not altered when the proteins are expressed together or with σNS. However, when expressed with μNS, σNS colocalizes with μNS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both σNS and μNS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, σNS interacts with μNS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that σNS and μNS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.

scholarly journals Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Paula F. Zamora ◽  
Liya Hu ◽  
Jonathan J. Knowlton ◽  
Roni M. Lahr ◽  
Rodolfo A. Moreno ◽  
...  
Keyword(s):  
Virus Replication ◽  
Viral Genome ◽  
Nonstructural Protein ◽  
Dsrna Virus ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Content Type ◽  
The Family ◽  
Viral Genome Replication

ABSTRACTViral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, usingin vitroand cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases the viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication.IMPORTANCEFollowing infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the familyReoviridaeencode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different viruses in the familyReoviridaediverge in primary sequence, they are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Usingin vitroand cell culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the familyReoviridaeassort and replicate their genomes.

scholarly journals Reovirus Forms Neo-Organelles for Progeny Particle Assembly within Reorganized Cell Membranes

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Isabel Fernández de Castro ◽  
Paula F. Zamora ◽  
Laura Ooms ◽  
José Jesús Fernández ◽  
Caroline M.-H. Lai ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Viral Replication ◽  
Cell Membranes ◽  
Light And Electron Microscopy ◽  
Host Cells ◽  
Pharmacological Intervention ◽  
Genome Replication ◽  
Particle Assembly ◽  
Intermediate Compartment

ABSTRACTMost viruses that replicate in the cytoplasm of host cells form neo-organelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral replication proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Despite the importance of inclusion complexes in viral replication, there are key gaps in the knowledge of how these organelles form and mediate their functions. Reoviruses are nonenveloped, double-stranded RNA (dsRNA) viruses that serve as tractable experimental models for studies of dsRNA virus replication and pathogenesis. Following reovirus entry into cells, replication occurs in large cytoplasmic structures termed inclusions that fill with progeny virions. Reovirus inclusions are nucleated by viral nonstructural proteins, which in turn recruit viral structural proteins for genome replication and particle assembly. Components of reovirus inclusions are poorly understood, but these structures are generally thought to be devoid of membranes. We used transmission electron microscopy and three-dimensional image reconstructions to visualize reovirus inclusions in infected cells. These studies revealed that reovirus inclusions form within a membranous network. Viral inclusions contain filled and empty viral particles and microtubules and appose mitochondria and rough endoplasmic reticulum (RER). Immunofluorescence confocal microscopy analysis demonstrated that markers of the ER and ER-Golgi intermediate compartment (ERGIC) codistribute with inclusions during infection, as does dsRNA. dsRNA colocalizes with the viral protein σNS and an ERGIC marker inside inclusions. These findings suggest that cell membranes within reovirus inclusions form a scaffold to coordinate viral replication and assembly.IMPORTANCEViruses alter the architecture of host cells to form an intracellular environment conducive to viral replication. This step in viral infection requires the concerted action of viral and host components and is potentially vulnerable to pharmacological intervention. Reoviruses form large cytoplasmic replication sites called inclusions, which have been described as membrane-free structures. Despite the importance of inclusions in the reovirus replication cycle, little is known about their formation and composition. We used light and electron microscopy to demonstrate that reovirus inclusions are membrane-containing structures and that the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment interact closely with these viral organelles. These findings enhance our understanding of the cellular machinery usurped by viruses to form inclusion organelles and complete an infectious cycle. This information, in turn, may foster the development of antiviral drugs that impede this essential viral replication step.

scholarly journals Acyl-CoA Thioesterases; a rheostat that controls activated fatty acids modulates dengue virus serotype 2 replication

2021 ◽  
Author(s):  
Laura A. St Clair ◽  
Stephanie A. Mills ◽  
Elena Lian ◽  
Paul S. Soma ◽  
Aritra Nag ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Viral Replication ◽  
Protein Translation ◽  
Fatty Acyl ◽  
Host Cells ◽  
Genome Replication ◽  
Type I ◽  
Loss Of Function ◽  
Particle Assembly ◽  
Virus Serotype

During infection with dengue viruses (DENVs), the lipid landscape within host cells is significantly altered to assemble membrane platforms that support viral replication and particle assembly. Fatty acyl-CoAs are key intermediates in the biosynthesis of complex lipids that form these membranes. They also function as key signaling lipids in the cell. Here, we carried out loss of function studies on acyl-CoA thioesterases (ACOTs), a family of enzymes that hydrolyze fatty acyl-CoAs to free fatty acids and coenzyme A, to understand their influence on the lifecycle of DENVs. Loss of function of the type I ACOTs 1 (cytoplasmic) and 2 (mitochondrial) together significantly increased DENV serotype 2 (DENV2) viral replication and infectious particle release. However, isolated knockdown of mitochondrial ACOT2 significantly decreased DENV2 protein translation, genome replication, and infectious virus release. Furthermore, loss of ACOT7 function, a mitochondrial type II ACOT, similarly suppressed DENV2. As ACOT1 and ACOT2 are splice variants, these data suggest that location (cytosol and mitochondria, respectively) rather than function of these proteins may account for the differences in DENV2 infection phenotype. Additionally, loss of mitochondrial ACOT2 and ACOT7 expression also altered the expression of several ACOTs located in multiple organelle compartments within the cell highlighting a complex relationship between ACOTs in the DENV2 virus lifecycle.

scholarly journals Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells

2008 ◽  
Vol 82 (20) ◽  
pp. 10088-10101 ◽  
Author(s):  
Rodion Gorchakov ◽  
Natalia Garmashova ◽  
Elena Frolova ◽  
Ilya Frolov
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Protein Complexes ◽  
Nonstructural Proteins ◽  
Cell Nuclei ◽  
High Concentration ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Rna Complexes

ABSTRACT Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

scholarly journals SARS-CoV-2: from its discovery to genome structure, transcription, and replication

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ayslan Castro Brant ◽  
Wei Tian ◽  
Vladimir Majerciak ◽  
Wei Yang ◽  
Zhi-Ming Zheng
Keyword(s):  
Viral Genome ◽  
Genome Structure ◽  
Membrane Vesicles ◽  
Regulatory Sequence ◽  
Atypical Pneumonia ◽  
Genome Replication ◽  
Long Distance ◽  
Viral Genome Replication ◽  
Infected Cells ◽  
Rna Interaction

AbstractSARS-CoV-2 is an extremely contagious respiratory virus causing adult atypical pneumonia COVID-19 with severe acute respiratory syndrome (SARS). SARS-CoV-2 has a single-stranded, positive-sense RNA (+RNA) genome of ~ 29.9 kb and exhibits significant genetic shift from different isolates. After entering the susceptible cells expressing both ACE2 and TMPRSS2, the SARS-CoV-2 genome directly functions as an mRNA to translate two polyproteins from the ORF1a and ORF1b region, which are cleaved by two viral proteases into sixteen non-structural proteins (nsp1-16) to initiate viral genome replication and transcription. The SARS-CoV-2 genome also encodes four structural (S, E, M and N) and up to six accessory (3a, 6, 7a, 7b, 8, and 9b) proteins, but their translation requires newly synthesized individual subgenomic RNAs (sgRNA) in the infected cells. Synthesis of the full-length viral genomic RNA (gRNA) and sgRNAs are conducted inside double-membrane vesicles (DMVs) by the viral replication and transcription complex (RTC), which comprises nsp7, nsp8, nsp9, nsp12, nsp13 and a short RNA primer. To produce sgRNAs, RTC starts RNA synthesis from the highly structured gRNA 3' end and switches template at various transcription regulatory sequence (TRSB) sites along the gRNA body probably mediated by a long-distance RNA–RNA interaction. The TRS motif in the gRNA 5' leader (TRSL) is responsible for the RNA–RNA interaction with the TRSB upstream of each ORF and skipping of the viral genome in between them to produce individual sgRNAs. Abundance of individual sgRNAs and viral gRNA synthesized in the infected cells depend on the location and read-through efficiency of each TRSB. Although more studies are needed, the unprecedented COVID-19 pandemic has taught the world a painful lesson that is to invest and proactively prepare future emergence of other types of coronaviruses and any other possible biological horrors.

scholarly journals Molecular Insights into the Flavivirus Replication Complex

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 956
Author(s):  
Kaïn van den Elsen ◽  
Jun Ping Quek ◽  
Dahai Luo
Keyword(s):  
Unmet Need ◽  
Genome Replication ◽  
Replication Process ◽  
Particle Assembly ◽  
Virus Family ◽  
Replication Complex ◽  
Molecular Events ◽  
Virus Rna ◽  
Viral Genome Replication ◽  
Molecular Aspects

Flaviviruses are vector-borne RNA viruses, many of which are clinically relevant human viral pathogens, such as dengue, Zika, Japanese encephalitis, West Nile and yellow fever viruses. Millions of people are infected with these viruses around the world each year. Vaccines are only available for some members of this large virus family, and there are no effective antiviral drugs to treat flavivirus infections. The unmet need for vaccines and therapies against these flaviviral infections drives research towards a better understanding of the epidemiology, biology and immunology of flaviviruses. In this review, we discuss the basic biology of the flavivirus replication process and focus on the molecular aspects of viral genome replication. Within the virus-induced intracellular membranous compartments, flaviviral RNA genome replication takes place, starting from viral poly protein expression and processing to the assembly of the virus RNA replication complex, followed by the delivery of the progeny viral RNA to the viral particle assembly sites. We attempt to update the latest understanding of the key molecular events during this process and highlight knowledge gaps for future studies.

scholarly journals Heat Shock Protein 90 Ensures the Integrity of Rubella Virus p150 Protein and Supports Viral Replication

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Masafumi Sakata ◽  
Hiroshi Katoh ◽  
Noriyuki Otsuki ◽  
Kiyoko Okamoto ◽  
Yuichiro Nakatsu ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Molecular Chaperone ◽  
Rubella Virus ◽  
Heat Shock Protein 90 ◽  
Host Factor ◽  
Host Factors ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Infected Cells

ABSTRACT Two viral nonstructural proteins, p150 and p90, are expressed in rubella virus (RUBV)-infected cells and mediate viral genome replication, presumably using various host machineries. Molecular chaperones are critical host factors for the maintenance of cellular proteostasis, and certain viral proteins use this chaperone system. The RUBV p150 and p90 proteins are generated from a precursor polyprotein, p200, via processing by the protease activity of its p150 region. This processing is essential for RUBV genome replication. Here we show that heat shock protein 90 (HSP90), a molecular chaperone, is an important host factor for RUBV genome replication. The treatment of RUBV-infected cells with the HSP90 inhibitors 17-allylamino-17-desmethoxygeldanamycin (17-AAG) and ganetespib suppressed RUBV genome replication. HSP90α physically interacted with p150, but not p90. Further analyses into the mechanism of action of the HSP90 inhibitors revealed that HSP90 activity contributes to p150 functional integrity and promotes p200 processing. Collectively, our data demonstrate that RUBV p150 is a client of the HSP90 molecular chaperone and that HSP90 functions as a key host factor for RUBV replication. IMPORTANCE Accumulating evidence indicates that RNA viruses use numerous host factors during replication of their genomes. However, the host factors involved in rubella virus (RUBV) genome replication are largely unknown. In this study, we demonstrate that the HSP90 molecular chaperone is needed for the efficient replication of the RUBV genome. Further, we reveal that HSP90 interacts with RUBV nonstructural protein p150 and its precursor polyprotein, p200. HSP90 contributes to the stability of p150 and the processing of p200 via its protease domain in the p150 region. We conclude that the cellular molecular chaperone HSP90 is a key host factor for functional maturation of nonstructural proteins for RUBV genome replication. These findings provide novel insight into this host-virus interaction.

scholarly journals Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

2021 ◽  
Vol 12 ◽  
Author(s):  
Lisett Liblekas ◽  
Alla Piirsoo ◽  
Annika Laanemets ◽  
Eva-Maria Tombak ◽  
Airiin Laaneväli ◽  
...  
Keyword(s):  
Life Cycle ◽  
Viral Genome ◽  
Maintenance Phase ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Genome Replication ◽  
Viral Genomes ◽  
Viral Genome Replication ◽  
Infected Cells ◽  
Cell Genome

The life-cycle of human papillomaviruses (HPVs) includes three distinct phases of the viral genome replication. First, the viral genome is amplified in the infected cells, and this amplification is often accompanied by the oligomerization of the viral genomes. Second stage includes the replication of viral genomes in concert with the host cell genome. The viral genome is further amplified during the third stage of the viral-life cycle, which takes place only in the differentiated keratinocytes. We have previously shown that the HPV18 genomes utilize at least two distinct replication mechanisms during the initial amplification. One of these mechanisms is a well-described bidirectional replication via theta type of replication intermediates. The nature of another replication mechanism utilized by HPV18 involves most likely recombination-dependent replication. In this paper, we show that the usage of different replication mechanisms is a property shared also by other HPV types, namely HPV11 and HPV5. We further show that the emergence of the recombination dependent replication coincides with the oligomerization of the viral genomes and is dependent on the replicative DNA polymerases. We also show that the oligomeric genomes of HPV18 replicate almost exclusively using recombination dependent mechanism, whereas monomeric HPV31 genomes replicate bi-directionally during the maintenance phase of the viral life-cycle.

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