scholarly journals Picornaviruses: A View from 3A

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 456
Author(s):  
Terry Jackson ◽  
Graham J. Belsham
Keyword(s):  
Viral Replication ◽  
Structural Proteins ◽  
Cellular Functions ◽  
Host Proteins ◽  
Replication Complex ◽  
Wide Range ◽  
Viral Replication Complex ◽  
Protein Shell ◽  
Human And Animal Diseases

Picornaviruses are comprised of a positive-sense RNA genome surrounded by a protein shell (or capsid). They are ubiquitous in vertebrates and cause a wide range of important human and animal diseases. The genome encodes a single large polyprotein that is processed to structural (capsid) and non-structural proteins. The non-structural proteins have key functions within the viral replication complex. Some, such as 3Dpol (the RNA dependent RNA polymerase) have conserved functions and participate directly in replicating the viral genome, whereas others, such as 3A, have accessory roles. The 3A proteins are highly divergent across the Picornaviridae and have specific roles both within and outside of the replication complex, which differ between the different genera. These roles include subverting host proteins to generate replication organelles and inhibition of cellular functions (such as protein secretion) to influence virus replication efficiency and the host response to infection. In addition, 3A proteins are associated with the determination of host range. However, recent observations have challenged some of the roles assigned to 3A and suggest that other viral proteins may carry them out. In this review, we revisit the roles of 3A in the picornavirus life cycle. The 3AB precursor and mature 3A have distinct functions during viral replication and, therefore, we have also included discussion of some of the roles assigned to 3AB.

scholarly journals Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Mun-Teng Wong ◽  
Steve S. Chen
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Ternary Complex ◽  
Choline Kinase ◽  
Replication Complex ◽  
Viral Replication Complex ◽  
Phosphatidylinositol 4

ABSTRACT In this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα-specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα-NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5A-stimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication. IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the “membranous web” structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation.

scholarly journals Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex

2002 ◽  
Vol 76 (12) ◽  
pp. 5974-5984 ◽  
Author(s):  
Denise Egger ◽  
Benno Wölk ◽  
Rainer Gosert ◽  
Leonardo Bianchi ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Replication Complex ◽  
Hepatitis C Virus Proteins ◽  
Infected Cells ◽  
Viral Replication Complex ◽  
Membrane Budding ◽  
Matrix Expression ◽  
Distinct Membrane

ABSTRACT Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.

scholarly journals Sphingosine Kinase 2 associates with the nsP3 of chikungunya virus and is required for replication

2020 ◽  
Author(s):  
Opeoluwa O. Oyewole ◽  
St Patrick Reid
Keyword(s):  
Viral Replication ◽  
Chikungunya Virus ◽  
Protein Production ◽  
Sphingosine Kinase ◽  
Lipid Kinase ◽  
Replication Complex ◽  
Sphingosine Kinase 2 ◽  
Sphingosine 1 Phosphate ◽  
Viral Replication Complex ◽  
Genetic Targeting

AbstractSphingosine kinase 2 (SK2) is a lipid kinase that catalyzes the production of sphingosine-1-phosphate (S1P) from sphingosine. Previously, we have shown that SK2 is recruited to the viral replication complex (VRC) early during chikungunya virus (CHIKV) infection. In the present study, we demonstrate that SK2 is required for viral replication and protein production. Treatment with a SK2 inhibitor significantly impaired the function of a CHIKV replicon. Similarly, compound treatment or genetic targeting resulted in impaired viral protein production. Mechanistically, we demonstrate that CHIKV nsP3 binds to SK2. Association of nsP3 with SK2 was mediated, in part, through the FGDF motifs within the hypervariable domain (HVD) of nsP3. In a competition assay, SK2 competed with G3BP for binding to nsP3. Collectively, these results extend our previous findings and identify SK2 as a CHIKV host factor recruited by nsP3.

scholarly journals Spatial and temporal control of norovirus protease activity is determined by polyprotein processing and intermolecular interactions within the viral replication complex

2017 ◽  
Author(s):  
Edward Emmott ◽  
Alexis de Rougemont ◽  
Jürgen Haas ◽  
Ian Goodfellow
Keyword(s):  
Viral Replication ◽  
Protein Interactions ◽  
Full Range ◽  
Antiviral Drug ◽  
Viral Gastroenteritis ◽  
Protein Protein Interactions ◽  
Replication Complex ◽  
Viral Replication Complex ◽  
Stable Forms

AbstractNorovirus infections are a major cause of acute viral gastroenteritis and a significant burden to human health globally. A vital process for norovirus replication is the processing of the nonstructural polyprotein, by an internal protease, into the necessary viral components required to form the viral replication complex. This cleavage occurs at different rates resulting in the accumulation of stable precursor forms. In this report, we characterized how precursor forms of the norovirus protease accumulate during infection. Using stable forms of the protease precursors we demonstrated that these are all proteolytically activein vitro, but that when expressed in cells, activity is determined by both substrate and protease localization. Whilst all precursors could cleave a replication complex-associated substrate, only a subset of precursors lacking NS4 were capable of efficiently cleaving a cytoplasmic substrate. For the first time, the full range of protein-protein interactions between murine and human norovirus proteins were mapped by LUMIER assay, with conserved interactions between replication complex members, modifying the localization of a subset of precursors. Finally, we demonstrate that re-targeting of a poorly cleaved artificial cytoplasmic substrate to the replication complex is sufficient to permit efficient cleavage in the context of norovirus infection. This offers a model for how norovirus can regulate the timing of substrate cleavage throughout the replication cycle. The norovirus protease represents a key target in the search for effective antiviral treatments for norovirus infection. An improved understanding of protease function and regulation, as well as identification of interactions between the other non-structural proteins, offers new avenues for antiviral drug design.

scholarly journals Plum Pox Virus6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection

2016 ◽  
Vol 90 (10) ◽  
pp. 5119-5131 ◽  
Author(s):  
Hongguang Cui ◽  
Aiming Wang
Keyword(s):  
Viral Replication ◽  
Functional Role ◽  
Early Infection ◽  
Plum Pox Virus ◽  
Cdna Clones ◽  
Cleavage Sites ◽  
Replication Complex ◽  
Viral Replication Complex ◽  
Infection Stage

ABSTRACTThe potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a CanadianPlum pox virus(PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. Thetransexpression of 6K1 or thecisexpression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage.IMPORTANCEPotyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature proteins, with the majority of them having been at least partially functionally characterized. However, the functional role of a small protein named 6K1 remains obscure. In this study, we showed that deletion of 6K1 or a short motif/region of 6K1 in the full-length cDNA clones of plum pox virus abolishes viral replication and that mutation of the N- or C-terminal cleavage sites of 6K1 to prevent its release from the polyprotein greatly attenuates or completely inhibits viral replication, suggesting its important role in potyviral infection. We report that 6K1 forms punctate structures and targets the replication vesicles in PPV-infected plant leaf cells at the early infection stage. Our data reveal that 6K1 is an important viral protein of the potyviral replication complex.

scholarly journals Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex

2015 ◽  
Vol 4 (1) ◽  
pp. 1-9 ◽  
Author(s):  
St Patrick Reid ◽  
Sarah R Tritsch ◽  
Krishna Kota ◽  
Chih-Yuan Chiang ◽  
Lian Dong ◽  
...  
Keyword(s):  
Viral Replication ◽  
Chikungunya Virus ◽  
Sphingosine Kinase ◽  
Host Factor ◽  
Replication Complex ◽  
Sphingosine Kinase 2 ◽  
Viral Replication Complex
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