Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Are trans-complementation systems suitable for hepatitis C virus life cycle studies?

ABSTRACT: Complementation is a naturally occurring genetic mechanism that has been studied for a number of plus-strand RNA viruses. Although trans-complementation is well documented for Flaviviridae family viruses, the first such system for hepatitis C virus (HCV) was only described in 2005. Since then, the development of a number of HCV trans-complementation models has improved our knowledge of HCV protein functions and interactions, genome replication and viral particle assembly. These models have also been used to produce defective viruses and so improvements are necessary for vaccine assays. This review provides an update on HCV trans-complementation systems, the viral mechanisms studied therewith and the production and characterization of trans-encapsidated particles.

SUBMITTER: Fournier C

PROVIDER: S-EPMC7167126 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Are trans-complementation systems suitable for hepatitis C virus life cycle studies?

Fournier C C Duverlie G G Castelain S S

Journal of viral hepatitis 20130214 4

Complementation is a naturally occurring genetic mechanism that has been studied for a number of plus-strand RNA viruses. Although trans-complementation is well documented for Flaviviridae family viruses, the first such system for hepatitis C virus (HCV) was only described in 2005. Since then, the development of a number of HCV trans-complementation models has improved our knowledge of HCV protein functions and interactions, genome replication and viral particle assembly. These models have also ...[more]

PMID: 23490366

Similar Datasets

Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A.

Project description:Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.

| S-EPMC538567 | biostudies-literature

Life cycle and morphogenesis of the hepatitis E virus.

Project description:Hepatitis E virus (HEV) is transmitted primarily via contaminated water and food by the fecal oral route and causes epidemics in developing countries. In industrialized countries, zoonotic transmission of HEV is prevalent. In addition, HEV is the major cause of acute hepatitis in healthy adults and can cause chronic hepatitis in immunocompromised patients, with pregnant HEV-infected women having increased mortality rates of approximately 25%. HEV was once an understudied and neglected virus. However, in recent years, the safety of blood products with respect to HEV has increasingly been considered to be a public health problem. The establishment of HEV infection models has enabled significant progress to be made in understanding its life cycle. HEV infects cells via a receptor (complex) that has yet to be identified. The HEV replication cycle is initiated immediately after the (+) stranded RNA genome is released into the cell cytosol. Subsequently, infectious viral particles are released by the ESCRT complex as quasi-enveloped viruses (eHEVs) into the serum, whereas feces and urine contain only nonenveloped infectious viral progeny. The uncoating of the viral envelope takes place in the biliary tract, resulting in the generation of a nonenveloped virus that is more resistant to environmental stress and possesses a higher infectivity than that of eHEV. This review summarizes the current knowledge regarding the HEV life cycle, viral morphogenesis, established model systems and vaccine development.

| S-EPMC6265337 | biostudies-other

Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle.

Project description:Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.

| S-EPMC4882586 | biostudies-other

Investigating the hepatitis B virus life cycle using engineered reporter hepatitis B viruses.

Project description:Chronic infection with hepatitis B virus (HBV) increases the risk of developing fibrosis, cirrhosis or hepatocellular carcinoma. Current therapies are limited to type-I interferons and/or nucleos(t)ide analogues; however, these are only partially effective. The development of novel anti-HBV agents for new treatment strategies has been hampered by the lack of a suitable system that allows the in vitro replication of HBV. Studies of virus infection/replication at the molecular level using wild-type HBV are labor-intensive and time-consuming. To overcome these problems, we previously constructed a recombinant reporter HBV bearing the NanoLuc gene and showed its usefulness in identifying factors that affect HBV proliferation. Because this system mimics the early stage of the HBV life cycle faithfully, we conducted a quantitative analysis of HBV infectivity to several human hepatocyte cell lines as well as the effect of dimethyl sulfoxide and HBV protein X on the early stage of HBV proliferation using this system. Furthermore, we developed a system to produce a reporter HBV expressing a pol gene. These reporter HBV may provide an opportunity to enhance our understanding of the HBV life cycle and aid strategies for the development of new anti-HBV agents.

| S-EPMC5765299 | biostudies-literature

N6-Methyladenosine Modification of Hepatitis B Virus RNA Differentially Regulates The Viral Life Cycle

Project description:N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within epsilon (stem loop structure is the site for m6A modification. The epsilon stem loop is located in the 3’ terminus of all HBV mRNAs and both termini (5’ and 3’) of the pgRNA. Mutational analysis of the identified m6A site in the 5’ epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A of the 3’ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory functions in pgRNA. Overall, this study reveals how m6A regulates HBV gene expression and reverse transcription, leading to a new level of understanding of the HBV life cycle. .

2019-01-01 | GSE114486 | GEO

Small-molecule effectors of hepatitis B virus capsid assembly give insight into virus life cycle.

Project description:The relationship between the physical chemistry and biology of self-assembly is poorly understood, but it will be critical to quantitatively understand infection and for the design of antivirals that target virus genesis. Here we take advantage of heteroaryldihydropyrimidines (HAPs), which affect hepatitis B virus (HBV) assembly, to gain insight and correlate in vitro assembly with HBV replication in culture. Based on a low-resolution crystal structure of a capsid-HAP complex, a closely related series of HAPs were designed and synthesized. These differentially strengthen the association between neighboring capsid proteins, alter the kinetics of assembly, and give rise to aberrant structures incompatible with a functional capsid. The chemical nature of the HAP variants correlated well with the structure of the HAP binding pocket. The thermodynamics and kinetics of in vitro assembly had strong and predictable effects on product morphology. However, only the kinetics of in vitro assembly had a strong correlation with inhibition of HBV replication in HepG2.2.15 cells; there was at best a weak correlation between assembly thermodynamics and replication. The correlation between assembly kinetics and virus suppression implies a competition between successful assembly and misassembly, small molecule induced or otherwise. This is a predictive and testable model for the mechanism of action of assembly effectors.

| S-EPMC2566253 | biostudies-literature

Trans-complementation of an NS2 defect in a late step in hepatitis C virus (HCV) particle assembly and maturation.

Project description:Recent studies using cell culture infection systems that recapitulate the entire life cycle of hepatitis C virus (HCV) indicate that several nonstructural viral proteins, including NS2, NS3, and NS5A, are involved in the process of viral assembly and release. Other recent work suggests that Ser-168 of NS2 is a target of CK2 kinase-mediated phosphorylation, and that this controls the stability of the genotype 1a NS2 protein. Here, we show that Ser-168 is a critical determinant in the production of infectious virus particles. Substitution of Ser-168 with Ala (or Gly) ablated production of infectious virus by cells transfected with a chimeric viral RNA (HJ3-5) containing core-NS2 sequences from the genotype 1a H77 virus within the background of genotype 2a JFH1 virus. An S168A substitution also impaired production of virus by cells transfected with JFH1 RNA. This mutation did not alter polyprotein processing or genome replication. This defect in virus production could be rescued by expression of wt NS2 in trans from an alphavirus replicon. The trans-complementing activities of NS2 from genotypes 1a and 2a demonstrated strong preferences for rescue of the homologous genotype. Importantly, the S168A mutation did not alter the association of core or NS5A proteins with host cell lipid droplets, nor prevent the assembly of core into particles with sedimentation and buoyant density properties similar to infectious virus, indicating that NS2 acts subsequent to the involvement of core, NS5A, and NS3 in particle assembly. Second-site mutations in NS2 as well as in NS5A can rescue the defect in virus production imposed by the S168G mutation. In aggregate, these results indicate that NS2 functions in trans, in a late-post assembly maturation step, perhaps in concert with NS5A, to confer infectivity to the HCV particle.

| S-EPMC2669722 | biostudies-other

Completion of the entire hepatitis C virus life cycle in genetically humanized mice.

Project description:More than 130 million people worldwide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective against HCV infection, and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry, we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice. Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo. Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viraemia over several weeks. In mice lacking the essential cellular co-factor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes in vivo. Persistently infected mice produce de novo infectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing evidence for the completion of the entire HCV life cycle in inbred mice. This genetically humanized mouse model opens new opportunities to dissect genetically HCV infection in vivo and provides an important preclinical platform for testing and prioritizing drug candidates and may also have utility for evaluating vaccine efficacy.

| S-EPMC3858853 | biostudies-literature

Reliance of host cholesterol metabolic pathways for the life cycle of hepatitis C virus.

Project description:Hepatitis C virus (HCV), a single-stranded positive-sense RNA virus of the Flaviviridae family, infects more than 170 million people worldwide and is the leading cause of liver failure in the United States. A unique feature of HCV is that the viral life cycle depends on cholesterol metabolism in host cells. This review summarizes the cholesterol metabolic pathways that are required for the replication, secretion, and entry of HCV. The potential application of drugs that alter host cholesterol metabolism in treating HCV infection is also discussed.

| S-EPMC1959368 | biostudies-other

Genotype-specific complementation of hepatitis delta virus RNA replication by hepatitis delta antigen.

Project description:Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.

| S-EPMC109725 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data