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Brett D Lindenbach

Publications and source records attributed to Brett D Lindenbach.

13 recordsLinked to original sources
PubMed2006

Silencing of USP18 potentiates the antiviral activity of interferon against hepatitis C virus infection.

BACKGROUND & AIMS: Modulation of the host innate immune response is an attractive means of inhibiting hepatitis C virus (HCV) replication. Having previously determined that expression of the interferon-sensitive gene (ISG)15 protease USP18 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-alfa therapy, we hypothesized that USP18 might hinder the ability of IFN to inhibit HCV replication. METHODS: The role of USP18 in IFN antiviral activity was examined using an in vitro model of HCV replication that reproduces the full viral life cycle. USP18 was silenced specifically using small inhibitory RNAs (siRNAs), and the dose response of HCV replication and infectious virus production to IFN-alfa was measured. RESULTS: The siRNA knockdown of USP18 in human cells consistently potentiated the ability of IFN to inhibit HCV-RNA replication and infectious virus particle production by a factor of 1-2 log(10). USP18 knockdown also resulted in a number of cellular changes consistent with increased sensitivity to IFN. Decreasing USP18 expression led to increased cellular protein ISGylation in response to exogenous IFN-alfa, prolonged tyrosine phosphorylation of signal transducer and activation of transcription (STAT1), and a general enhancement of IFN-stimulated gene expression. CONCLUSIONS: These data suggest that USP18 modulates the anti-HCV type I IFN response, and is a possible therapeutic target for the treatment of HCV infection.

Antiviral Agents↗
PubMed2006

Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6/JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle.

Animals↗
PubMed2006

Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells.

Cell entry of hepatitis C virus, pseudoparticles (HCVpp) and cell culture grown virus (HCVcc), requires the interaction of viral glycoproteins with CD81 and other as yet unknown cellular factors. One of these is likely to be the scavenger receptor class B type I (SR-BI). To further understand the role of SR-BI, we examined the effect of SR-BI ligands on HCVpp and HCVcc infectivity. Oxidized low-density lipoprotein (oxLDL), but not native LDL, potently inhibited HCVpp and HCVcc cell entry. Pseudoparticles bearing unrelated viral glycoproteins or bovine viral diarrhea virus were not affected. A dose-dependent inhibition was observed for HCVpp bearing diverse viral glycoproteins with an approximate IC50 of 1.5 microg/mL apolipoprotein content, which is within the range of oxLDL reported to be present in human plasma. The ability of lipoprotein components to bind to target cells associated with their antiviral activity, suggesting a mechanism of action which targets a cell surface receptor critical for HCV infection of the host cell. However, binding of soluble E2 to SR-BI or CD81 was not affected by oxLDL, suggesting that oxLDL does not act as a simple receptor blocker. At the same time, oxLDL incubation altered the biophysical properties of HCVpp, suggesting a ternary interaction of oxLDL with both virus and target cells. In conclusion, the SR-BI ligand oxLDL is a potent cell entry inhibitor for a broad range of HCV strains in vitro. These findings suggest that SR-BI is an essential component of the cellular HCV receptor complex.

CD36 Antigens↗
PubMed2006

Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry.

Hepatitis C virus (HCV) is an important human pathogen associated with chronic liver disease. Recently, based on a genotype 2a isolate, tissue culture systems supporting complete replication and infectious virus production have been developed. In this study, we used cell culture-produced infectious HCV to analyze the viral entry pathway into Huh-7.5 cells. Bafilomycin A1 and concanamycin A, inhibitors of vacuolar ATPases, prevented HCV entry when they were present prior to infection and had minimal effect on downstream replication events. HCV entry therefore appears to be pH dependent, requiring an acidified intracellular compartment. For many other enveloped viruses, acidic pH triggers an irreversible conformational change, which promotes virion-endosomal membrane fusion. Such viruses are often inactivated by low pH. In the case of HCV, exposure of virions to acidic pH followed by return to neutral pH did not affect their infectivity. This parallels the observation made for the related pestivirus bovine viral diarrhea virus. Low pH could activate the entry of cell surface-bound HCV but only after prolonged incubation at 37 degrees C. This suggests that there are rate-limiting, postbinding events that are needed to render HCV competent for low-pH-triggered entry. Such events may involve interaction with a cellular coreceptor or other factors but do not require cathepsins B and L, late endosomal proteases that activate Ebola virus and reovirus for entry.

Cathepsin B↗
PubMed2005

Unravelling hepatitis C virus replication from genome to function.

Since the discovery of the hepatitis C virus over 15 years ago, scientists have raced to develop diagnostics, study the virus and find new therapies. Yet virtually every attempt to dissect this pathogen has met with roadblocks that impeded progress. Its replication was restricted to humans or experimentally infected chimpanzees, and efficient growth of the virus in cell culture failed until very recently. Nevertheless hard-fought progress has been made and the first wave of antiviral drugs is entering clinical trials.

Animals↗
PubMed2005

Complete replication of hepatitis C virus in cell culture.

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.

Antibodies, Monoclonal↗
PubMed2005

Identification of microRNAs of the herpesvirus family.

Epstein-Barr virus (EBV or HHV4), a member of the human herpesvirus (HHV) family, has recently been shown to encode microRNAs (miRNAs). In contrast to most eukaryotic miRNAs, these viral miRNAs do not have close homologs in other viral genomes or in the genome of the human host. To identify other miRNA genes in pathogenic viruses, we combined a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types. We cloned ten miRNAs in the Kaposi sarcoma-associated virus (KSHV or HHV8), nine miRNAs in the mouse gammaherpesvirus 68 (MHV68) and nine miRNAs in the human cytomegalovirus (HCMV or HHV5). These miRNA genes are expressed individually or in clusters from either polymerase (pol) II or pol III promoters, and share no substantial sequence homology with one another or with the known human miRNAs. Generally, we predicted miRNAs in several large DNA viruses, and we could neither predict nor experimentally identify miRNAs in the genomes of small RNA viruses or retroviruses.

Chromosome Mapping↗
PubMed2004

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Amino Acid Sequence↗
PubMed2003

Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus.

Positive-strand RNA viruses are the largest virus class and include many pathogens such as hepatitis C virus and the severe acute respiratory syndrome coronavirus (SARS). Brome mosaic virus (BMV) is a representative positive-strand RNA virus whose RNA replication, gene expression, and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. By using traditional yeast genetics, host genes have been identified that function in controlling BMV translation, selecting BMV RNAs as replication templates, activating the replication complex, maintaining a lipid composition required for membrane-associated RNA replication, and other steps. To more globally and systematically identify such host factors, we used engineered BMV derivatives to assay viral RNA replication in each strain of an ordered, genome-wide set of yeast single-gene deletion mutants. Each deletion strain was transformed to express BMV replicase proteins and a BMV RNA replication template with the capsid gene replaced by a luciferase reporter. Luciferase expression, which is dependent on viral RNA replication and RNA-dependent mRNA synthesis, was measured in intact yeast cells. Approximately 4500 yeast deletion strains ( approximately 80% of yeast genes) were screened in duplicate and selected strains analyzed further. This functional genomics approach revealed nearly 100 genes whose absence inhibited or stimulated BMV RNA replication and/or gene expression by 3- to >25-fold. Several of these genes were shown previously to function in BMV replication, validating the approach. Newly identified genes include some in RNA, protein, or membrane modification pathways and genes of unknown function. The results further illuminate virus and cell pathways. Further refinement of virus screening likely will reveal contributions from additional host genes.

Bromovirus↗
PubMed2003

Evasive maneuvers by hepatitis C virus.

The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use of HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

Comment↗
PubMed2002

RNAi targeting an animal virus: news from the front.

Although many eukaryotic organisms exhibit RNA interference, its role as an antiviral pathway is established only for plants. A recent paper demonstrates that it also acts in animals, and reveals a viral suppressor of this process.

Animals↗
PubMed2002

Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication.

Replication of flock house virus (FHV) RNA1 and production of subgenomic RNA3 in the yeast Saccharomyces cerevisiae provide a useful tool for the dissection of FHV molecular biology and host-encoded functions involved in RNA replication. The replication template activity of RNA1 can be separated from its coding potential by supplying the RNA1-encoded replication factor protein A in trans. We constructed a trans-replication system in yeast to examine cis-acting elements in RNA1 that control RNA3 production, as well as RNA1 and RNA2 replication. Two cis elements controlling RNA3 production were found. A proximal subgenomic control element was located just upstream of the RNA3 start site (nucleotides [nt] 2282 to 2777). A short distal element also controlling RNA3 production (distal subgenomic control element) was identified 1.5 kb upstream, at nt 1229 to 1239. Base pairing between these distal and proximal elements was shown to be essential for RNA3 production by covariation analysis and in vivo selection of RNA3-expressing replicons from plasmid libraries containing random sequences in the distal element. Two distinct RNA1 replication elements (RE) were mapped within the 3' quarter of RNA1: the intRE (nt 2322 to 2501) and the 3'RE (nt 2735 to 3011). The 3'RE significantly overlaps the RNA3 region in RNA1, and this information was applied to produce improved RNA3-based vectors for foreign-gene expression. In addition, replication of an RNA2 derivative was dependent on RNA1 templates capable of forming the long-distance interaction that controls RNA3 production.

Base Pairing↗