scholarly journals A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A

2006 ◽  
Vol 87 (1) ◽  
pp. 93-102 ◽  
Author(s):  
Christopher J. McCormick ◽  
David Brown ◽  
Stephen Griffin ◽  
Lisa Challinor ◽  
David J. Rowlands ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Point Mutation ◽  
Active Site ◽  
Natural Infection ◽  
Polymerase Activity ◽  
Encephalomyocarditis Virus ◽  
Structural Proteins ◽  
Hcv Rna ◽  
Viral Transcript

Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

scholarly journals Interaction of Hepatitis C Virus Core Protein with Viral Sense RNA and Suppression of Its Translation

1999 ◽  
Vol 73 (12) ◽  
pp. 9718-9725 ◽  
Author(s):  
Takashi Shimoike ◽  
Shigetaka Mimori ◽  
Hideki Tani ◽  
Yoshiharu Matsuura ◽  
Tatsuo Miyamura
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Firefly Luciferase ◽  
Encephalomyocarditis Virus ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Genomic Rna ◽  
The Core ◽  
Hcv Core Protein

ABSTRACT To clarify the binding properties of hepatitis C virus (HCV) core protein and its viral RNA for the encapsidation, morphogenesis, and replication of HCV, the specific interaction of HCV core protein with its genomic RNA synthesized in vitro was examined in an in vivo system. The positive-sense RNA from the 5′ end to nucleotide (nt) 2327, which covers the 5′ untranslated region (5′UTR) and a part of the coding region of HCV structural proteins, interacted with HCV core protein, while no interaction was observed in the same region of negative-sense RNA and in other regions of viral and antiviral sense RNAs. The internal ribosome entry site (IRES) exists around the 5′UTR of HCV; therefore, the interaction of the core protein with this region of HCV RNA suggests that there is some effect on its cap-independent translation. Cells expressing HCV core protein were transfected with reporter RNAs consisting of nt 1 to 709 of HCV RNA (the 5′UTR of HCV and about two-thirds of the core protein coding regions) followed by a firefly luciferase gene (HCV07Luc RNA). The translation of HCV07Luc RNA was suppressed in cells expressing the core protein, whereas no significant suppression was observed in the case of a reporter RNA possessing the IRES of encephalomyocarditis virus followed by a firefly luciferase. This suppression by the core protein occurred in a dose-dependent manner. The expression of the E1 envelope protein of HCV or β-galactosidase did not suppress the translation of both HCV and EMCV reporter RNAs. We then examined the regions that are important for suppression of translation by the core protein and found that the region from nt 1 to 344 was enough to exert this suppression. These results suggest that the HCV core protein interacts with viral genomic RNA at a specific region to form nucleocapsids and regulates the expression of HCV by interacting with the 5′UTR.

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 Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver

2004 ◽  
Vol 85 (6) ◽  
pp. 1497-1507 ◽  
Author(s):  
Søren U. Nielsen ◽  
Margaret F. Bassendine ◽  
Alastair D. Burt ◽  
Debra J. Bevitt ◽  
Geoffrey L. Toms
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Molecular Mass ◽  
Single Species ◽  
Northern Blotting ◽  
High Titre ◽  
Structural Proteins ◽  
Hcv Rna ◽  
Chronic Hcv

In the absence of satisfactory cell culture systems for hepatitis C virus (HCV), virtually all that is known about the proteins of the virus has been learned by the study of recombinant proteins. Characterization of virus proteins from patients with HCV has been retarded by the low virus titre in blood and limited availability of infected tissue. Here, the authors have identified a primary infection in a liver transplanted into an immunodeficient patient with chronic HCV. The patient required re-transplant and the infected liver, removed 6 weeks after the initial transplant, had a very high titre of HCV, 5×109 International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1·04 g ml−1 with 73 % of virus below 1·08 g ml−1. Full-length HCV RNA was detected by Northern blotting and the ratio between positive- and negative-strand HCV RNA was determined as 60. HCV was partially purified by precipitation with heparin/Mn2+ and a single species of each of the three structural proteins, core, E1 and E2, was detected by Western blotting. The molecular mass of core was 20 kDa, which corresponds to the mature form from recombinant sources. The molecular mass of glycoprotein E1 was 31 kDa before and 21 kDa after deglycosylation with PNGase F or endoglycosidase H. Glycoprotein E2 was 62 kDa before and 36 kDa after deglycosylation, but E2-P7 was not detected. This was in contrast to recombinant sources of E2 which contain E2-P7.

scholarly journals PKR-Dependent Mechanisms of Gene Expression from a Subgenomic Hepatitis C Virus Clone

2002 ◽  
Vol 76 (21) ◽  
pp. 10637-10653 ◽  
Author(s):  
Ana Maria Rivas-Estilla ◽  
Yuri Svitkin ◽  
Marcelo Lopez Lastra ◽  
Maria Hatzoglou ◽  
Averell Sherker ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mrna Translation ◽  
Encephalomyocarditis Virus ◽  
Hcv Rna ◽  
Nptii Gene ◽  
Cell Defense ◽  
Huh7 Cells ◽  
Hcv Ires

ABSTRACT Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2α phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2α phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-α is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2α phosphorylation is induced by IFN-α in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2α phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2α phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3′ untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.

scholarly journals Guanosine inhibits hepatitis C virus replication and increases indel frequencies, associated with altered intracellular nucleotide pools

2020 ◽  
Author(s):  
Rosario Sabariegos ◽  
Ana M. Ortega-Prieto ◽  
Luis Díaz-Martínez ◽  
Ana Grande-Pérez ◽  
Isabel Gallego ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
De Novo ◽  
Polymerase Activity ◽  
Progeny Virus ◽  
Inhibition Mechanism ◽  
Hcv Rna ◽  
Template Switching ◽  
Human Hepatoma Cells

AbstractIn the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua and not the other standard nucleosides inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di- and tri-phosphate (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTP or NDP modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes.Author summaryRibonucleoside metabolism is essential for replication of RNA viruses. In this article we describe the antiviral activity of the natural ribonucleoside guanosine (Gua). We demonstrate that hepatitis C virus (HCV) replication is inhibited in the presence of increasing concentrations of this ribonucleoside and that this inhibition does not occur as a consequence of a direct inhibition of HCV polymerase. Cells exposed to increasing concentrations of Gua show imbalances in the intracellular concentrations of nucleoside-diphosphates and triphosphates and as the virus is passaged in these cells, it accumulates mutations that reduce its infectivity and decimate its normal spreading capacity.

scholarly journals Characterization of liver histopathology in a transgenic mouse model expressing genotype 1a hepatitis C virus core and envelope proteins 1 and 2

2005 ◽  
Vol 86 (8) ◽  
pp. 2185-2196 ◽  
Author(s):  
Turaya Naas ◽  
Masoud Ghorbani ◽  
Ikuri Alvarez-Maya ◽  
Michael Lapner ◽  
Rashmi Kothary ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mouse Model ◽  
Protein Expression ◽  
Transgenic Mouse ◽  
Transgenic Mouse Model ◽  
Structural Proteins ◽  
Hcv Rna ◽  
Hcv Structural Proteins

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and hepatocellular carcinoma worldwide. The purpose of this study was to determine how the HCV structural proteins affect the dynamic structural and functional properties of hepatocytes and measure the extra-hepatic manifestations induced by these viral proteins. A transgenic mouse model was established by expressing core, E1 and E2 proteins downstream of a CMV promoter. HCV RNA was detected using RT-PCR in transgenic mouse model tissues, such as liver, kidney, spleen and heart. Expression of the transgene was analysed by real-time PCR to quantify viral RNA in different tissues at different ages. Immunofluorescence analysis revealed the expression of core, E1 and E2 proteins predominantly in hepatocytes. Lower levels of protein expression were detected in spleen and kidneys. HCV RNA and viral protein expression increased in the liver with age. Histological analysis of liver cells demonstrated steatosis in transgenic mice older than 3 months, which was more progressed with age. Electron microscopy analysis revealed alterations in nuclei, mitochondria and endoplasmic reticulum. HCV structural proteins induce a severe hepatopathy in the transgenic mouse model. These mice became more prone to liver and lymphoid tumour development and hepatocellular carcinoma. In this model, the extra-hepatic effects of HCV, which included swelling of renal tubular cells, were mild. It is likely that the HCV structural proteins mediate some of the histological alterations in hepatocytes by interfering with lipid transport and liver metabolism.

scholarly journals Mechanism of Hepatitis C Virus RNA Polymerase Inhibition with Dihydroxypyrimidines

2009 ◽  
Vol 54 (3) ◽  
pp. 977-983 ◽  
Author(s):  
Megan H. Powdrill ◽  
Jerome Deval ◽  
Frank Narjes ◽  
Raffaele De Francesco ◽  
Matthias Götte
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Hcv Rna ◽  
Virus Rna ◽  
Nucleotide Incorporation ◽  
Biochemical Mechanisms ◽  
Elongation Phase ◽  
Specificity Domain

ABSTRACT We studied the biochemical mechanisms associated with inhibition and resistance to a 4,5-dihydroxypyrimidine carboxylate that inhibits the hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B. On the basis of the structure of the pharmacophore, it has been suggested that these compounds may act as pyrophosphate (PPi) mimics. We monitored nucleotide incorporation events during the elongation phase and showed that the polymerase activity of wild-type NS5B was inhibited by the dihydroxypyrimidine at a 50% inhibitory concentration (IC50) of 0.73 μM. Enzymes with the G152E or P156L mutation, either of which confers resistance to this compound, showed four- to fivefold increases in IC50s. The inhibitor was competitive with respect to nucleotide incorporation. It was likewise effective at preventing the PPi-mediated excision of an incorporated chain terminator in a competitive fashion. In the absence of the dihydroxypyrimidine, the reaction was not significantly affected by the G152E or P156L mutation. These data suggest that the resistance associated with these two mutations is unlikely due to an altered interaction with the pyrophosphate-mimicking domain of the compound but, rather, is due to altered interactions with its specificity domain at a region distant from the active site. Together, our findings provide strong experimental evidence that supports the notion that the members of this class of compounds can act as PPi mimics that have the potential to mechanistically complement established nucleoside and nonnucleoside analogue inhibitors.

scholarly journals The 3′-Untranslated Region of Hepatitis C Virus RNA Enhances Translation from an Internal Ribosomal Entry Site

1998 ◽  
Vol 72 (11) ◽  
pp. 8789-8796 ◽  
Author(s):  
Takayoshi Ito ◽  
Stanley M. Tahara ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Untranslated Region ◽  
Rna Stability ◽  
Encephalomyocarditis Virus ◽  
Hcv Rna ◽  
Globin Mrna ◽  
Entry Site ◽  
Polypyrimidine Tract Binding Protein ◽  
Virus Rna

ABSTRACT Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3′-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5′-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3′-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5′-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3′ end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.

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