Replication of a hepatitis A virus replicon detected by genetic recombination in vivo

Gauss‐Müller, Verena; Kusov, Yuri

doi:10.1099/0022-1317-83-9-2183

Cited by 38 publications

(56 citation statements)

References 33 publications

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“…HAV replication is less efficient in BS-C-1 cells than in liver cells, which are the natural host for HAV replication in vivo and produce larger amounts of infectious virus (35,47) or viral replicon (16,57). To test whether the extent of RNAi depended on the host cell line and the rate of viral replication, the human hepatoma cell line Huh-7 was transfected with the same set of siRNAs and infected with HAV as described above.…”

Section: Resultsmentioning

confidence: 99%

“…Runoff transcripts were characterized by denaturing 0.8% agarose gel and spectrophotometry before transfection into Huh-7 cells. Luciferase activity was measured as described previously (16).…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Total RNA was extracted with TRIzol (Invitrogen), and HAV RNA was reverse transcribed at 50°C for 1 h using 2.5 mM oligo(dT) [15][16][17][18] and 10 U/l SuperScript III (Invitrogen). Real-time PCR with 0.5 M primers specific for HAV 3C (sense, 5Ј-ACTTTGGAAATAGCAGGACT GG-3Ј; antisense, 5Ј-AGATTGATTCGATGAAACC-3Ј) was performed by using SYBR Green supermix (Bio-Rad) in a Bio-Rad iCycler.…”

Section: Cells and Virusesmentioning

confidence: 99%

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Silencing of Hepatitis A Virus Infection by Small Interfering RNAs

Kusov

Palmenberg

Sgro

et al. 2006

J Virol

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Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequencespecific RNAi.Hepatitis A virus (HAV) has a single-stranded RNA genome of 7.5 kb with positive polarity. As a member of the picornavirus family, it is unique in its biological properties, in particular, in its highly protracted replicative cycle in cell cultures and inability to shut off the host cell metabolism. HAV causes a self-limiting infection of the liver with usually mild or no symptoms in young patients, whereas adult patients might suffer from severe symptoms. In immune-compromised or chronically infected patients, fulminant hepatic failure is often associated with a high mortality rate (15, 52). Albeit vaccines against hepatitis A virus can prevent the disease (3, 14, 31), they are futile for fulminant hepatitis. Therefore, therapeutic intervention strategies to prevent progression to a life-threatening liver disease are still in need. Given that the rapid degradation of the HAV genome can ameliorate viral infection and thus prevent fulminant failure, RNA interference (RNAi) based on sequence-specific genome degradation may present a novel and specific therapeutic approach in preventing severe disease progression.Initially demonstrated in Caenorabditis elegans and Drosophila melanogaster, RNAi is now recognized as a valuable tool to silence viral infections (reviewed in references 22, 48, and 50). Among the numerous viruses that have been successfully silenced by RNAi, there are five representatives of the picornavirus family: poliovirus (19,20), human rhinovirus (45), enterovirus 71 (37), foot-and-mouth disease virus (7, 9, 36), and coxsackievirus B3 (1, 40). Recently, we have shown that genome replication of an HAV replicon can be efficiently inhibited by small interfering RNAs (siRNAs) targeting either coding or noncoding regions...

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Section: Resultsmentioning

confidence: 99%

Section: Cells and Virusesmentioning

confidence: 99%

Section: Cells and Virusesmentioning

confidence: 99%

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Silencing of Hepatitis A Virus Infection by Small Interfering RNAs

Kusov

Palmenberg

Sgro

et al. 2006

J Virol

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“…HCV and HAV reporter replicons used for transient-transfection assays were described before: pFKI389Luc-EI/NS3-3=_ET (LucCon1ET) (32), pFKI389Luc-EI/NS3-3=_JFH (LucJFH1) (33), or pT7-18f-Luc (LucHAV) (34,35). The plasmid used for generation of reporter virus JcR2A was described before (36), as was the monocistronic HCV reporter replicon pFK-I389Luc-ubi/NS3-3=/JFH1 (33).…”

mentioning

confidence: 99%

DDX60L Is an Interferon-Stimulated Gene Product Restricting Hepatitis C Virus Replication in Cell Culture

Grünvogel

Esser-Nobis

Reustle

et al. 2015

J Virol

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All major types of interferon (IFN) efficiently inhibit hepatitis C virus (HCV) replication in vitro and in vivo.Remarkably, HCV replication is not sensitive to IFN-␥ in the hepatoma cell line Huh6, despite an intact signaling pathway. We performed transcriptome analyses between Huh6 and Huh-7 cells to identify effector genes of the IFN-␥ response and thereby identified the DExD/H box helicase DEAD box polypeptide 60-like (DDX60L) as a restriction factor of HCV replication. DDX60L and its homolog DEAD box polypeptide 60 (DDX60) were both induced upon viral infection and IFN treatment in primary human hepatocytes. However, exclusively DDX60L knockdown increased HCV replication in Huh-7 cells and rescued HCV replication from type II IFN as well as type I and III IFN treatment, suggesting that DDX60L is an important effector protein of the innate immune response against HCV. In contrast, we found no impact of DDX60L on replication of hepatitis A virus. DDX60L protein was detectable only upon strong ectopic overexpression, displayed a broad cytoplasmic distribution, but caused cytopathic effects under these conditions. DDX60L knockdown did not alter interferon-stimulated gene (ISG) induction after IFN treatment but inhibited HCV replication upon ectopic expression, suggesting that it is a direct effector of the innate immune response. It most likely inhibits viral RNA replication, since we found neither impact of DDX60L on translation or stability of HCV subgenomic replicons nor additional impact on assembly of infectious virus. Similar to DDX60, DDX60L had a moderate impact on RIG-I dependent activation of innate immunity, suggesting additional functions in the sensing of viral RNA. IMPORTANCEInterferons induce a plethora of interferon-stimulated genes (ISGs), which are our first line of defense against viral infections. In addition, IFNs have been used in antiviral therapy, in particular against the human pathogen hepatitis C virus (HCV); still, their mechanism of action is not well understood, since diverse, overlapping sets of antagonistic effector ISGs target viruses with different biologies. Our work identifies DDX60L as a novel factor that inhibits replication of HCV. DDX60L expression is regulated similarly to that of its homolog DDX60, but our data suggest that it has distinct functions, since we found no contribution of DDX60 in combatting HCV replication. The identification of novel components of the innate immune response contributes to a comprehensive understanding of the complex mechanisms governing antiviral defense.

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“…However, so far the only functional HAV replicon replicates only in Huh7 cells [Gauss-Mü ller and Kusov, 2002;Yi and Lemon, 2002]. …”

Section: Discussionmentioning

confidence: 99%

Importance of amino acid 216 in nonstructural protein 2B for replication of hepatitis A virus in cell culture and in vivo

Graff

Emerson

2003

Journal of Medical Virology

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Clinical isolates of hepatitis A virus (HAV) replicate inefficiently in cell culture unless mutations are acquired throughout the genome. An Ala-to-Val substitution in the nonstructural protein 2B (2B-216) was known to have a major impact on replication in cell culture. Analysis of chimeric viruses confirmed that the 2B-A[216]V change was critical for efficient replication and that Leu or Ile could substitute for Val. Viruses containing Val, Ile, or Leu at 2B-216 all replicated with similar kinetics in cell culture, whereas the virus containing Ala at this position grew 10- to 20-fold less efficiently. In contrast, in vivo, virus with either Ala or Val at 2B-216 replicated equally efficiently when tested in a chimpanzee and in tamarins, and each amino acid was stably maintained. Attempts to complement wild-type 2B in trans with adapted 2B provided by co-infection with a second viable HAV mutant failed to enhance replication of the virus containing the wild-type 2B sequence.

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Replication of a hepatitis A virus replicon detected by genetic recombination in vivo

Cited by 38 publications

References 33 publications

Silencing of Hepatitis A Virus Infection by Small Interfering RNAs

Silencing of Hepatitis A Virus Infection by Small Interfering RNAs

DDX60L Is an Interferon-Stimulated Gene Product Restricting Hepatitis C Virus Replication in Cell Culture

Importance of amino acid 216 in nonstructural protein 2B for replication of hepatitis A virus in cell culture and in vivo

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