scholarly journals Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

2009 ◽  
Vol 83 (17) ◽  
pp. 8379-8395 ◽  
Author(s):  
Tung Phan ◽  
Rudolf K. F. Beran ◽  
Christopher Peters ◽  
Ivo C. Lorenz ◽  
Brett D. Lindenbach
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Particle ◽  
Rna Binding ◽  
Virus Assembly ◽  
Genetic Selection ◽  
Nonstructural Proteins ◽  
Particle Assembly ◽  
Enzyme Complexes

ABSTRACT The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.

scholarly journals The Acidic Domain of Hepatitis C Virus NS4A Contributes to RNA Replication and Virus Particle Assembly

2010 ◽  
Vol 85 (3) ◽  
pp. 1193-1204 ◽  
Author(s):  
T. Phan ◽  
A. Kohlway ◽  
P. Dimberu ◽  
A. M. Pyle ◽  
B. D. Lindenbach
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Particle ◽  
Rna Replication ◽  
Particle Assembly ◽  
Acidic Domain

Role of lipid metabolism in hepatitis C virus assembly and entry

2010 ◽  
Vol 102 (1) ◽  
pp. 63-74 ◽  
Author(s):  
Costin‐Ioan Popescu ◽  
Jean Dubuisson
Keyword(s):  
Hepatitis C Virus ◽  
Lipid Metabolism ◽  
Hepatitis C ◽  
Virus Assembly

scholarly journals Hepatitis C Virus NS2 Protein Serves as a Scaffold for Virus Assembly by Interacting with both Structural and Nonstructural Proteins

2010 ◽  
Vol 85 (1) ◽  
pp. 86-97 ◽  
Author(s):  
Y. Ma ◽  
M. Anantpadma ◽  
J. M. Timpe ◽  
S. Shanmugam ◽  
S. M. Singh ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Assembly ◽  
Nonstructural Proteins

scholarly journals Palmitoylation of Hepatitis C Virus NS2 Regulates Its Subcellular Localization and NS2-NS3 Autocleavage

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
Ming-Jhan Wu ◽  
Saravanabalaji Shanmugam ◽  
Christoph Welsch ◽  
MinKyung Yi
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Envelope Protein ◽  
Virus Assembly ◽  
Rna Replication ◽  
Encephalomyocarditis Virus ◽  
Hcv Rna ◽  
Particle Assembly ◽  
Infectious Particle ◽  
Multiple Functions

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly. IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.

scholarly journals Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection

2016 ◽  
Vol 90 (21) ◽  
pp. 9632-9643 ◽  
Author(s):  
Zaili Yang ◽  
Xiaoning Wang ◽  
Xiumei Chi ◽  
Fanfan Zhao ◽  
Jinxu Guo ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Apolipoprotein E ◽  
Virus Assembly ◽  
Hcv Infection ◽  
Apoe Level ◽  
New Perspective ◽  
Apoe Expression

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic liver disease, infecting approximately 170 million people worldwide. HCV assembly is tightly associated with the lipoprotein pathway. Exchangeable apolipoprotein E (apoE) is incorporated on infectious HCV virions and is important for infectious HCV virion morphogenesis and entry. Moreover, the virion apoE level is positively correlated with its ability to escape E2 antibody neutralization. However, the role of apoE exchange in the HCV life cycle is unclear. In this study, the relationship between apoE expression and cell permissiveness to HCV infection was assessed by infecting apoE knockdown and derived apoE rescue cell lines with HCV. Exchange of apoE between lipoproteins and HCV lipoviral particles (LVPs) was evaluated by immunoprecipitation, infectivity testing, and viral genome quantification. Cell and heparin column binding assays were applied to determine the attachment efficiency of LVPs with different levels of incorporated apoE. The results showed that cell permissiveness for HCV infection was determined by exogenous apoE-associated lipoproteins. Furthermore, apoE exchange did occur between HCV LVPs and lipoproteins, which was important to maintain a high apoE level on LVPs. Lipid-free apoE was capable of enhancing HCV infectivity for apoE knockdown cells but not apoE rescue cells. A higher apoE level on LVPs conferred more efficient LVP attachment to both the cell surface and heparin beads. This study revealed that exogenous apoE-incorporating lipoproteins from uninfected hepatocytes safeguarded the apoE level of LVPs for more efficient attachment during HCV infection. IMPORTANCE In this study, a neglected but important role of apoE exchange in HCV LVP infectivity after virus assembly and release was identified. The data indicated that apoE expression level in uninfected cells is important for high permissiveness to HCV infection. Secreted apoE-associated lipoprotein specifically enhances infection of HCV LVPs. apoE exchange between HCV LVP and lipoproteins is important to maintain an adequate apoE level on LVPs for their efficient attachment to cell surface. These data defined for the first time an extracellular role of exchangeable apoE in HCV infection and suggested that exchangeable apolipoproteins reach a natural equilibrium between HCV LVPs and lipoprotein particles, which provides a new perspective to the understanding of the heterogeneity of HCV LVPs in composition.

scholarly journals Systems biology analysis of hepatitis C virus infection reveals the role of copy number increases in regions of chromosome 1q in hepatocellular carcinoma metabolism

2016 ◽  
Vol 12 (5) ◽  
pp. 1496-1506 ◽  
Author(s):  
Ibrahim E. Elsemman ◽  
Adil Mardinoglu ◽  
Saeed Shoaie ◽  
Taysir H. Soliman ◽  
Jens Nielsen
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Copy Number ◽  
Virus Assembly ◽  
Hepatitis C Virus Infection ◽  
Chromosome 1Q ◽  
Host Target

We reconstructed hepatitis C virus assembly reactions to find host-target metabolites impeding this reaction.

scholarly journals Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex

2009 ◽  
Vol 83 (13) ◽  
pp. 6554-6565 ◽  
Author(s):  
Zhe Liu ◽  
Feng Yang ◽  
Jason M. Robotham ◽  
Hengli Tang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Critical Role ◽  
Cyclophilin A ◽  
Hcv Rna ◽  
Nonstructural Proteins ◽  
Ppiase Activity ◽  
Replication Complex ◽  
And Function

ABSTRACT Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine's anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA's PPIase activity in the proper assembly and function of the HCV RC.

Sign in / Sign up

Export Citation Format

Share Document