Director, Center for Hepatitis and Liver Tissue Engineering (2006 - Present)
Honors & Awards
Translational Research Award, American Liver Foundation/Amgen/AASLD (1998-2002)
Regents Scholarship, U.C.S.F. (1984-1993)
Elected member, American Society for Clinical Investigation (2008)
Clinical Scientist Award in Translational Research, Burroughs Wellcome Fund (2005-2010)
Career Award, Burroughs Wellcome Fund (1998-2005)
Physician Postdoctoral Fellowship Award, Howard Hughes Medical Institute (1995-1998)
Summa cum laude, U.C. Berkeley (1984)
Fellowship:Stanford University School of Medicine Registrar (1999) CA
Residency:Stanford University School of Medicine Registrar (1995) CA
Internship:Stanford University School of Medicine Registrar (1994) CA
Medical Education:University of California at San Francisco School of Medicine (1993) CA
Ph.D., U.C.S.F., Biochemistry and Biophysics (1992)
M.D., U.C.S.F., Medicine/MSTP (1993)
Current Research and Scholarly Interests
Dr. Glenn's primary interest is in molecular virology, with a strong emphasis on translating this knowledge into novel antiviral therapies. Current projects aim to better understand the role of prenylation in the life cycles of hepatitis delta virus (HDV) and other viruses-both as a mechanism of intracellular trafficking and trigger of virus assembly, and as a target for a promising antiviral treatment. The function and cell biology of the hepatitis C virus (HCV) non-structural proteins are also being studied. Here the focus is on identifying key determinants of pathogenesis and designing novel antiviral strategies. Other interests include exploitation of hepatic stem cells, development of a small animal model for HCV, and engineered human liver tissues.
Independent Studies (11)
- Directed Investigation
BIOE 392 (Aut, Win, Spr, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Directed Reading in Microbiology and Immunology
MI 299 (Win, Spr)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Graduate Research
MI 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MI 370 (Win, Spr)
- Out-of-Department Graduate Research
BIO 300X (Aut)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
MI 199 (Win, Spr)
- Directed Investigation
Phosphatidylinositol 4,5-Bisphosphate Is an HCV NS5A Ligand and Mediates Replication of the Viral Genome.
2015; 148 (3): 616-625
Phosphoinositides (PIs) bind and regulate localization of proteins via a variety of structural motifs. PI 4,5-bisphosphate (PI[4,5]P2) interacts with and modulates the function of several proteins involved in intracellular vesicular membrane trafficking. We investigated interactions between PI(4,5)P2 and hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and effects on the viral life cycle.We used a combination of quartz crystal microbalance, circular dichroism, molecular genetics, and immunofluorescence to study specific binding of PI(4,5)P2 by the HCV NS5A protein. We evaluated the effects of PI(4,5)P2 on the function of NS5A by expressing wild-type or mutant forms of Bart79I or FL-J6/JFH-5'C19Rluc2AUbi21 RNA in Huh7 cells. We also studied the effects of strategies designed to inhibit PI(4,5)P2 on HCV replication in these cells.The N-terminal amphipathic helix of NS5A bound specifically to PI(4,5)P2, inducing a conformational change that stabilized the interaction between NS5A and TBC1D20, which is required for HCV replication. A pair of positively charged residues within the amphipathic helix (the basic amino acid PI(4,5)P2 pincer domain) was required for PI(4,5)P2 binding and replication of the HCV-RNA genome. A similar motif was found to be conserved across all HCV isolates, as well as amphipathic helices of many pathogens and apolipoproteins.PI(4,5)P2 binds to HCV NS5A to promote replication of the viral RNA genome in hepatocytes. Strategies to disrupt this interaction might be developed to inhibit replication of HCV and other viruses.
View details for DOI 10.1053/j.gastro.2014.11.043
View details for PubMedID 25479136
The interaction between the Hepatitis C proteins NS4B and NS5A is involved in viral replication.
2015; 475: 139-149
Hepatitis C virus (HCV) replicates in membrane associated, highly ordered replication complexes (RCs). These complexes include viral and host proteins necessary for viral RNA genome replication. The interaction network among viral and host proteins underlying the formation of these RCs is yet to be thoroughly characterized. Here, we investigated the association between NS4B and NS5A, two critical RC components. We characterized the interaction between these proteins using fluorescence resonance energy transfer and a mammalian two-hybrid system. Specific tryptophan residues within the C-terminal domain (CTD) of NS4B were shown to mediate this interaction. Domain I of NS5A, was sufficient to mediate its interaction with NS4B. Mutations in the NS4B CTD tryptophan residues abolished viral replication. Moreover, one of these mutations also affected NS5A hyperphosphorylation. These findings provide new insights into the importance of the NS4B-NS5A interaction and serve as a starting point for studying the complex interactions between the replicase subunits.
View details for DOI 10.1016/j.virol.2014.10.021
View details for PubMedID 25462354
Coinfection with hepatitis B and D: Epidemiology, prevalence and disease in patients in Northern California
JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY
2013; 28 (9): 1521-1525
With no report on the overall prevalence and ramifications of hepatitis Delta virus (HDV) infection in the United States for more than two decades, the characteristics of chronic hepatitis B virus (CHB) patients coinfected with HDV, including clinical presentation, rate of hepatitis C virus tri-infection, and HDV viral load, were assessed.At California Pacific Medical Center, a retrospective chart review was conducted on all CHB patients.Of 1191 CHB patients, 499 had been tested for HDV, with 42 (8%) determined to be coinfected; half of these were also hepatitis C virus-infected. Cirrhosis was present in 73% of the coinfected, 80% of the tri-infected, but only 22% of the monoinfected. Twenty-nine patients (69%) were Caucasian non-Hispanic; 10 (24%) were Asians and Pacific Islanders. Of 39 patients for whom HBV-DNA quantification at time of HDV presentation was available, 22 (56%) had undetectable levels; four (10%) had levels > 100 000 IU/mL.HDV affects individuals of all ages and various ethnic groups. Although HBV viral loads are lower, rates of cirrhosis are higher in coinfected patients and higher still in the tri-infected. Our data support revising screening guidelines to advocate for all patients with HBV to be screened for HDV in order to both give the individual patient important information related to the possible need for treatment and to support the public health goal of reducing transmission by educating HDV-negative patients about the need for protection against superinfection and HDV-infected patients about the need to protect against transmission to others.
View details for DOI 10.1111/jgh.12217
View details for Web of Science ID 000323389700017
View details for PubMedID 23574043
The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA.
Nucleic acids research
2013; 41 (6): 3688-3698
Hepatitis C Virus (HCV) and other plus-strand RNA viruses typically require the generation of a small number of negative genomes (20-100× lower than the positive genomes) for replication, making the less-abundant antigenome an attractive target for RNA interference(RNAi)-based therapy. Because of the complementarity of duplex short hairpin RNA/small interfering RNA (shRNA/siRNAs) with both genomic and anti-genomic viral RNA strands, and the potential of both shRNA strands to become part of the targeting complexes, preclinical RNAi studies cannot distinguish which viral strand is actually targeted in infected cells. Here, we addressed the question whether the negative HCV genome was bioaccessible to RNAi. We first screened for the most active shRNA molecules against the most conserved regions in the HCV genome, which were then used to generate asymmetric anti-HCV shRNAs that produce biologically active RNAi specifically directed against the genomic or antigenomic HCV sequences. Using this simple but powerful and effective method to screen for shRNA strand selectivity, we demonstrate that the antigenomic strand of HCV is not a viable RNAi target during HCV replication. These findings provide new insights into HCV biology and have important implications for the design of more effective and safer antiviral RNAi strategies seeking to target HCV and other viruses with similar replicative strategies.
View details for DOI 10.1093/nar/gkt068
View details for PubMedID 23396439
Using Chimeric Mice with Humanized Livers to Predict Human Drug Metabolism and a Drug-Drug Interaction
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
2013; 344 (2): 388-396
Interspecies differences in drug metabolism have made it difficult to use preclinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDIs) that will occur in humans. Although chimeric mice with humanized livers can produce known human metabolites for test substrates, we do not know whether chimeric mice can be used to prospectively predict human drug metabolism or a possible DDI. Therefore, we investigated whether they could provide a more predictive assessment for clemizole, a drug in clinical development for the treatment of hepatitis C virus (HCV) infection. Our results demonstrate, for the first time, that analyses performed in chimeric mice can correctly identify the predominant human drug metabolite before human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, because the predominant human metabolite was found to have synergistic anti-HCV activity. Moreover, studies in chimeric mice also correctly predicted that a DDI would occur in humans when clemizole was coadministered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of preclinical drug assessment.
View details for DOI 10.1124/jpet.112.198697
View details for Web of Science ID 000313745900008
View details for PubMedID 23143674
Structural Map of a MicroRNA-122: Hepatitis C Virus Complex
JOURNAL OF VIROLOGY
2012; 86 (2): 1250-1254
MicroRNA-122 (miR-122) enhances hepatitis C virus (HCV) fitness via targeting two sites in the 5'-untranslated region (UTR) of HCV. We used selective 2'-hydroxyl acylation analyzed by primer extension to resolve the HCV 5'-UTR's RNA secondary structure in the presence of miR-122. Nearly all nucleotides in miR-122 are involved in targeting the second site, beyond classic seed base pairings. These additional interactions enhance HCV replication in cell culture. To our knowledge, this is the first biophysical study of this complex to reveal the importance of 'tail' miR-122 nucleotide interactions.
View details for DOI 10.1128/JVI.06367-11
View details for Web of Science ID 000298674600056
View details for PubMedID 22072754
Simplified RNA secondary structure mapping by automation of SHAPE data analysis
NUCLEIC ACIDS RESEARCH
2011; 39 (22)
SHAPE (Selective 2'-hydroxyl acylation analysed by primer extension) technology has emerged as one of the leading methods of determining RNA secondary structure at the nucleotide level. A significant bottleneck in using SHAPE is the complex and time-consuming data processing that is required. We present here a modified data collection method and a series of algorithms, embodied in a program entitled Fast Analysis of SHAPE traces (FAST), which significantly reduces processing time. We have used this method to resolve the secondary structure of the first ~900 nt of the hepatitis C virus (HCV) genome, including the entire core gene. We have also demonstrated the ability of SHAPE/FAST to detect the binding of a small molecule inhibitor to the HCV internal ribosomal entry site (IRES). In conclusion, FAST allows for high-throughput data processing to match the current high-throughput generation of data possible with SHAPE, reducing the barrier to determining the structure of RNAs of interest.
View details for DOI 10.1093/nar/gkr773
View details for Web of Science ID 000298186000004
View details for PubMedID 21965531
Structural Linkage between Ligand Discrimination and Receptor Activation by Type I Interferons
2011; 146 (4): 621-632
Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.
View details for DOI 10.1016/j.cell.2011.06.048
View details for Web of Science ID 000294043600016
View details for PubMedID 21854986
View details for PubMedCentralID PMC3166218
The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein
2011; 414 (1): 10-18
The hepatitis C virus (HCV) non-structural (NS) 5A protein plays an essential role in the replication of the viral RNA by the membrane-associated replication complex (RC). Recently, a putative NS5A inhibitor, BMS-790052, exhibited the highest potency of any known anti-HCV compound in inhibiting HCV replication in vitro and showed a promising clinical effect in HCV-infected patients. The precise mechanism of action for this new class of potential anti-HCV therapeutics, however, is still unclear. In order to gain further insight into its mode of action, we sought to test the hypothesis that the antiviral effect of BMS-790052 might be mediated by interfering with the functional assembly of the HCV RC. We observed that BMS-790052 indeed altered the subcellular localization and biochemical fractionation of NS5A. Taken together, our data suggest that NS5A inhibitors such as BMS-790052 can suppress viral genome replication by altering the proper localization of NS5A into functional RCs.
View details for DOI 10.1016/j.virol.2011.03.026
View details for Web of Science ID 000290701100002
View details for PubMedID 21513964
View details for PubMedCentralID PMC3096933
Role for ADP Ribosylation Factor 1 in the Regulation of Hepatitis C Virus Replication
JOURNAL OF VIROLOGY
2011; 85 (2): 946-956
We hypothesized that ADP-ribosylation factor 1 (Arf1) plays an important role in the biogenesis and maintenance of infectious hepatitis C virus (HCV). Huh7.5 cells, in which HCV replicates and produces infectious viral particles, were exposed to brefeldin A or golgicide A, pharmacological inhibitors of Arf1 activation. Treatment with these agents caused a reduction in viral RNA levels, the accumulation of infectious particles within the cells, and a reduction in the levels of these particles in the extracellular medium. Fluorescence analyses showed that the viral nonstructural (NS) proteins NS5A and NS3, but not the viral structural protein core, shifted their localization from speckle-like structures in untreated cells to the rims of lipid droplets (LDs) in treated cells. Using pulldown assays, we showed that ectopic overexpression of NS5A in Huh7 cells reduces the levels of GTP-Arf1. Downregulation of Arf1 expression by small interfering RNA (siRNA) decreased both the levels of HCV RNA and the production of infectious viral particles and altered the localization of NS5A to the peripheries of LDs. Together, our data provide novel insights into the role of Arf1 in the regulation of viral RNA replication and the production of infectious HCV.
View details for DOI 10.1128/JVI.00753-10
View details for Web of Science ID 000285554300030
View details for PubMedID 21068255
Hydrophobic nanoparticles improve permeability of cell-encapsulating poly(ethylene glycol) hydrogels while maintaining patternability
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (48): 20709-20714
Cell encapsulating poly(ethylene glycol) hydrogels represent a promising approach for constructing 3D cultures designed to more closely approximate in vivo tissue environment. Improved strategies are needed, however, to optimally balance hydrogel permeability to support metabolic activities of encapsulated cells, while maintaining patternability to restore key aspects of tissue architecture. Herein, we have developed one such strategy incorporating hydrophobic nanoparticles to partially induce looser cross-linking density at the particle-hydrogel interface. Strikingly, our network design significantly increased hydrogel permeability, while only minimally affecting the matrix mechanical strength or prepolymer viscosity. This structural advantage improved viability and functions of encapsulated cells and permitted micron-scale structures to control over spatial distribution of incorporated cells. We expect that this design strategy holds promise for the development of more advanced artificial tissues that can promote high levels of cell metabolic activity and recapitulate key architectural features.
View details for DOI 10.1073/pnas.1005211107
View details for Web of Science ID 000284762400026
View details for PubMedID 21071674
The Future of HCV Therapy: NS4B as an Antiviral Target
2010; 2 (11): 2481-2492
Chronic hepatitis C virus (HCV) infection is a major worldwide cause of liver disease, including cirrhosis and hepatocellular carcinoma. It is estimated that more than 170 million individuals are infected with HCV, with three to four million new cases each year. The current standard of care, combination treatment with interferon and ribavirin, eradicates the virus in only about 50% of chronically infected patients. Notably, neither of these drugs directly target HCV. Many new antiviral therapies that specifically target hepatitis C (e.g. NS3 protease or NS5B polymerase inhibitors) are therefore in development, with a significant number having advanced into clinical trials. The nonstructural 4B (NS4B) protein, is among the least characterized of the HCV structural and nonstructural proteins and has been subjected to few pharmacological studies. NS4B is an integral membrane protein with at least four predicted transmembrane (TM) domains. A variety of functions have been postulated for NS4B, such as the ability to induce the membranous web replication platform, RNA binding and NTPase activity. This review summarizes potential targets within the nonstructural protein NS4B, with a focus on novel classes of NS4B inhibitors.
View details for DOI 10.3390/v2112481
View details for Web of Science ID 000284582500005
View details for PubMedID 21157574
Prevalence, Correlates, and Viral Dynamics of Hepatitis Delta among Injection Drug Users
JOURNAL OF INFECTIOUS DISEASES
2010; 202 (6): 845-852
Most hepatitis delta virus (HDV) prevalence estimates from the United States are >10 years old, and HDV has shown significant temporal variation in other populations. HDV-hepatitis B virus (HBV) dual infection progresses rapidly, has more complications, and has a different treatment regimen than HBV infection alone. Accurate estimates of prevalence and risk factors are important to help clinicians decide who to screen.Injection drug users in Baltimore, Maryland, who were positive for HBV serologic markers were tested for hepatitis delta antibody (HDAb) at 2 time periods: 1988-1989 (194 participants) and 2005-2006 (258 participants). Those who were HDAb positive in 2005-2006, plus a random sample of HDAb negative, HBV-positive participants were tested for HDV RNA, HBV DNA, and HCV RNA. Characteristics associated with HDV exposure and viremia were identified.HDV prevalence declined from 15% in 1988-1989 to 11% in 2005-2006. Among those with chronic HBV infection, prevalence increased from 29% (14 of 48 participants) to 50% (19 of 38 participants) (P=.05). Visiting a "shooting gallery" (a location where people gather to inject illegal drugs) was a strong correlate of HDAb positivity (relative risk, 3.08; P=.01). Eight (32%) of those who were HDAb positive had HDV viremia. Viremic participants had elevated liver enzyme levels and more emergency room visits.The temporal increase in HDV prevalence among those with chronic HBV infection is troubling; understanding this change should be a priority to prevent the burden from increasing.
View details for DOI 10.1086/655808
View details for Web of Science ID 000281091200004
View details for PubMedID 20701536
Transdifferentiation of adipose-derived stem cells into hepatocytes: a new approach
2010; 30 (6): 913-922
Several studies have demonstrated techniques in differentiating human adipose-derived stem cells (hADSCs) into hepatocytes. Unfortunately, transdifferentiation is inefficient, and the function of these induced hepatocyte-like cells (which we termed 'iHeps') is low compared with that of real hepatocytes.We aimed to identify transcriptional deficiencies in iHeps that are critical to hepatocyte development, which may provide insights into improving the efficiency of transdifferentiation.hADSCs were differentiated into iHeps, and iHeps were assayed for hepatocyte-like activity. iHeps were then screened for expression of several growth factors, receptors and transcription factors (TFs) critical to liver development using reverse transcription-polymerase chain reaction (RT-PCR). Deficient TFs were transduced into hADSCs and hepatocyte function was reassessed after hepatic differentiation.Differentiation of hADSCs into iHeps resulted in the upregulation of hepatic proteins. However, the levels of expression of hepatocyte-specific proteins in these iHeps were well below those of Huh 7.5 hepatoma cells, used in comparison. Five developmental TFs were notably absent on the RT-PCR screen. Lentiviral transduction of these TFs into hADSCs followed by culture in hepatocyte induction medium resulted in increased albumin expression compared with untransduced hADSCs treated in a parallel fashion.These five missing TFs are known to regulate hepatocyte differentiation and some are required to establish the competence of the foregut endoderm. Presumably due to their mesenchymal lineage, hADSCs do not express these endodermal TFs and are not fully competent to respond to critical developmental signals. Supplementation of these TFs may induce competency and enhance the differentiation of hADSCs into hepatocytes.
View details for DOI 10.1111/j.1478-3231.2010.02231.x
View details for Web of Science ID 000278311800019
View details for PubMedID 20353420
A small molecule inhibits HCV replication and alters NS4B's subcellular distribution
2010; 87 (1): 1-8
Hepatitis C Virus (HCV) is a leading cause of liver disease and represents a significant public health challenge. Treatments for this disease are inadequate and improved antiviral therapies are necessary. Several such antivirals are in development, most of which target the well-characterized NS3 protease or the NS5B polymerase. In contrast, the nonstructural 4B (NS4B) protein, though essential for HCV RNA replication, has been the subject of few pharmacological studies. One of the functions ascribed to this protein is the ability to form intracellular membrane-associated foci (MAF), which are believed to be related to the sites of viral replication. Here, we report the identification of a small molecule that inhibits HCV replication and disrupts the organization of these MAF. Genetic analysis links the compound's mode of action to the NS4B gene product, and transient transfections of NS4B-GFP demonstrate that treatment with this compound can lead to the formation of novel elongated assemblies of NS4B. Furthermore, an in vitro dynamic light scattering assay provides evidence that the second amphipathic helix of NS4B may be the target of the drug. Our results demonstrate that this molecule represents a new potential class of HCV inhibitors and also provides us with a useful tool for studying the HCV life cycle.
View details for DOI 10.1016/j.antiviral.2010.03.013
View details for Web of Science ID 000279452800001
View details for PubMedID 20363257
View details for PubMedCentralID PMC3909674
The hepatitis C virus (HCV) NS4B RNA binding inhibitor clemizole is highly synergistic with HCV protease inhibitors.
journal of infectious diseases
2010; 202 (1): 65-74
We recently identified a compound, clemizole hydrochloride, that inhibits NS4B's RNA binding and hepatitis C virus (HCV) replication. Although significant, clemizole's antiviral effect is moderate (50% effective concentration of 8 microM against an HCV genotype 2a clone). We hypothesized that the combination of clemizole with other anti-HCV agents can increase the antiviral effect over that achieved with each drug alone and could also decrease the emergence of viral resistance.Luciferase reporter-linked HCV replication assays were used to study the antiviral effects of drug combinations that included clemizole. Data were analyzed using Loewe additivity and Bliss independence models for synergy, and resistance studies were performed using HCV colony formation assays.Clemizole's antiviral effect was highly synergistic with the HCV protease inhibitors SCH503034 and VX950, without toxicity. In contrast, combinations of clemizole with either interferon, ribavirin, or the nucleoside (NM283) and nonnucleoside (HCV796) HCV polymerase inhibitors were additive. Furthermore, combination of clemizole with SCH503034 decreased the frequency of drug-resistant mutants, compared with treatment with either drug alone. Finally, no cross-resistance to clemizole of SCH503034-resistant mutants (or vice versa) was observed.Clemizole can yield high-level synergy with the protease inhibitor class. Inclusion of clemizole in future anti-HCV cocktails can represent an attractive paradigm for increasing current virologic response rates.
View details for DOI 10.1086/653080
View details for PubMedID 20486856
Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems
2010; 6 (2)
We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.
View details for DOI 10.1371/journal.ppat.1000764
View details for Web of Science ID 000275295900016
View details for PubMedID 20169186
Identification of a Class of HCV Inhibitors Directed Against the Nonstructural Protein NS4B
SCIENCE TRANSLATIONAL MEDICINE
2010; 2 (15)
New classes of drugs are needed to combat hepatitis C virus (HCV), an important worldwide cause of liver disease. We describe an activity of a key domain, an amphipathic helix we termed 4BAH2, within a specific HCV nonstructural protein, NS4B. In addition to its proposed role in viral replication, we validate 4BAH2 as essential for HCV genome replication and identify first-generation small-molecule inhibitors of 4BAH2 that specifically prevent HCV replication within cells. Mechanistic studies reveal that the inhibitors target 4BAH2 function by preventing either 4BAH2 oligomerization or 4BAH2 membrane association. 4BAH2 inhibitors represent an additional class of compounds with potential to effectively treat HCV.
View details for DOI 10.1126/scitranslmed.3000331
View details for Web of Science ID 000277264100002
View details for PubMedID 20371471
View details for PubMedCentralID PMC3025298
Mixing the right hepatitis C inhibitor cocktail.
Trends in molecular medicine
Therapy for hepatitis C virus (HCV) infection is on the cusp of a new era. Until now, standard-of-care therapy has involved interferon (IFN) and ribavirin. With the first successful Phase III trials of specific targeted antiviral therapy for HCV (STAT-C) compounds, as well as three trials in progress giving the first glimpse of IFN-free combinations of STAT-C agents, this review looks ahead to the new classes of anti-HCV agents currently in clinical development. Successful pharmacologic control of HIV and TB frames the discussion, as well as consideration of the mutation frequency of HCV replication. Maximizing synergy between agents and minimizing cumulative toxicity will be critical to the design of future IFN-free STAT-C regimens.
View details for DOI 10.1016/j.molmed.2010.10.005
View details for PubMedID 21106440
Mechanism of an Amphipathic alpha-Helical Peptide's Antiviral Activity Involves Size-Dependent Virus Particle Lysis
ACS CHEMICAL BIOLOGY
2009; 4 (12): 1061-1067
The N-terminal region of the hepatitis C virus (HCV) nonstructural protein NS5A contains an amphipathic alpha-helix that is necessary and sufficient for NS5A membrane association. A synthetic peptide (AH) comprising this amphipathic helix is able to lyse lipid vesicles that serve as a model system for virus particles. Based on quartz crystal microbalance-dissipation (QCM-D) experiments, the degree of vesicle rupturing was found to be inversely related to vesicle size, with maximal activity in the size range of several medically important viruses. In order to confirm and further study vesicle rupture, dynamic light scattering (DLS) and atomic force microscopy (AFM) experiments were also performed. The size dependence of vesicle rupturing helps explain the peptide's observed effect on the infectivity of a wide range of viruses. Further, in vitro studies demonstrated that AH peptide treatment significantly decreased the infectivity of HCV particles. Thus, the AH peptide might be used to rupture HCV particles extra-corporally (for HCV prevention) and within infected individuals (for HCV therapy).
View details for DOI 10.1021/cb900149b
View details for Web of Science ID 000272845900010
View details for PubMedID 19928982
The reliable targeting of specific drug release profiles by integrating arrays of different albumin-encapsulated microsphere types
2009; 30 (34): 6648-6654
Biodegradable polymer microspheres have been successfully utilized as a medium for controlled protein or peptide-based drug release. Because the release kinetics has been typically controlled by modulating physical or chemical properties of the medium, these parameters must be optimized to obtain a specific release profile. However, due to the complexity of the release mechanism and the complicated interplay between various design parameters of the release medium, detailed prediction of the resulting release profile is a challenge. Herein we suggest a simple method to target specific release profiles more efficiently by integrating release profiles for an array of different microsphere types. This scheme is based on our observation that the resulting release profile from a mixture of different samples can be predicted as the linear summation of the individually measured release profiles of each sample. Hence, by employing a linear equation at each time point and formulating them as a matrix equation, we could determine how much of each microsphere type to include in a mixture in order to have a specific release profile. In accordance with this method, several targeted release profiles were successfully obtained. We expect that the proposed method will allow us to overcome limitations in controlling complicated release mechanisms so that drug delivery systems can be reliably designed to satisfy clinical demands.
View details for DOI 10.1016/j.biomaterials.2009.08.035
View details for Web of Science ID 000271347900010
View details for PubMedID 19775742
The Anti-Hepatitis C Agent Nitazoxanide Induces Phosphorylation of Eukaryotic Initiation Factor 2 alpha Via Protein Kinase Activated by Double-Stranded RNA Activation
2009; 137 (5): 1827-1835
New therapies are needed to treat patients infected with hepatitis C virus (HCV), a major worldwide cause of chronic liver disease. Nitazoxanide (NTZ), originally used to treat cryptosporidiosis infection, recently was shown to have unexpected antiviral activity in the HCV replicon system and in chronically infected patients. A pilot clinical study suggested that NTZ can augment the antiviral effect of interferon (IFN), although the molecular basis for its effect was unknown.We analyzed the effects of NTZ on the regulation of eukaryotic initiation factor-2alpha (eIF2alpha) and its IFN-induced kinase, protein kinase activated by double-stranded RNA (PKR), in cells that support HCV RNA replication and in vitro biochemical assays.NTZ increased eIF2alpha phosphorylation, a modification known to mediate host cell antiviral defenses. The addition of IFN to cell cultures increased NTZ-induced eIF2alpha phosphorylation. NTZ also increased PKR phosphorylation. In vitro, NTZ promoted PKR autophosphorylation, a key step in activating PKR's kinase activity for eIF2alpha. Finally, NTZ-induced eIF2alpha phosphorylation was reduced in the presence of specific inhibitors of PKR autophosphorylation.An important mechanism of NTZ's action involves activation of PKR, a key kinase that regulates the cell's innate antiviral response. These observations could explain the clinical antiviral effect of NTZ. NTZ might represent a new class of small molecules capable of potentiating and recapitulating important antiviral effects of IFN.
View details for DOI 10.1053/j.gastro.2009.07.056
View details for Web of Science ID 000271500700040
View details for PubMedID 19664635
The Effect of Donor Race on the Survival of Black Americans Undergoing Liver Transplantation for Chronic Hepatitis C
2009; 15 (9): 1126-1132
The purpose of this study was to determine the effect of donor race on the outcome of black patients with chronic hepatitis C infection who undergo liver transplantation. The records for deceased donor liver transplants that occurred in the United States between January 1998 and December 2007 were obtained from the United Network for Organ Sharing. 26,212 records contained sufficient data to be included in the analysis. Of these, 11,989 (45.7%) records were for patients positive for hepatitis C virus (HCV) and 1292 (4.9%) were for patients both HCV-positive and black. Black recipients with white donors were found to have significantly worse outcomes than all other recipient-donor race combinations (P < 0.001). The crude 5-year survival rate for black recipients who had a black donor was 14% higher than the 5-year survival rate for black recipients who had a white donor. Multivariate regression analysis determined that a graft from a race-unmatched donor was an independent risk factor for graft failure (hazard ratio = 1.41, 95% confidence interval = 1.11-1.79) among HCV-positive black recipients but not among HCV-negative black recipients after adjustments for donor age, recipient age, cold ischemia time, serum creatinine, serum bilirubin, diabetes mellitus, body mass index, and donor cytomegalovirus status. The observation that race-unmatched grafts are a risk factor in HCV-positive black recipients, but not in HCV-negative black recipients, suggests an alteration of the graft-host relationship by HCV. In conclusion, our results suggest that HCV-positive black recipients who undergo liver transplantation can have increased graft survival if their donors are black, with survival rates approaching those of white liver transplant recipients.
View details for DOI 10.1002/lt.21835
View details for Web of Science ID 000269596700018
View details for PubMedID 19718638
The Evolution of the Major Hepatitis C Genotypes Correlates with Clinical Response to Interferon Therapy
2009; 4 (8)
Patients chronically infected with hepatitis C virus (HCV) require significantly different durations of therapy and achieve substantially different sustained virologic response rates to interferon-based therapies, depending on the HCV genotype with which they are infected. There currently exists no systematic framework that explains these genotype-specific response rates. Since humans are the only known natural hosts for HCV-a virus that is at least hundreds of years old-one possibility is that over the time frame of this relationship, HCV accumulated adaptive mutations that confer increasing resistance to the human immune system. Given that interferon therapy functions by triggering an immune response, we hypothesized that clinical response rates are a reflection of viral evolutionary adaptations to the immune system.We have performed the first phylogenetic analysis to include all available full-length HCV genomic sequences (n = 345). This resulted in a new cladogram of HCV. This tree establishes for the first time the relative evolutionary ages of the major HCV genotypes. The outcome data from prospective clinical trials that studied interferon and ribavirin therapy was then mapped onto this new tree. This mapping revealed a correlation between genotype-specific responses to therapy and respective genotype age. This correlation allows us to predict that genotypes 5 and 6, for which there currently are no published prospective trials, will likely have intermediate response rates, similar to genotype 3. Ancestral protein sequence reconstruction was also performed, which identified the HCV proteins E2 and NS5A as potential determinants of genotype-specific clinical outcome. Biochemical studies have independently identified these same two proteins as having genotype-specific abilities to inhibit the innate immune factor double-stranded RNA-dependent protein kinase (PKR).An evolutionary analysis of all available HCV genomes supports the hypothesis that immune selection was a significant driving force in the divergence of the major HCV genotypes and that viral factors that acquired the ability to inhibit the immune response may play a role in determining genotype-specific response rates to interferon therapy.
View details for DOI 10.1371/journal.pone.0006579
View details for Web of Science ID 000268935600008
View details for PubMedID 19668364
Alpha-Helical Peptide-Induced Vesicle Rupture Revealing New Insight into the Vesicle Fusion Process As Monitored in Situ by Quartz Crystal Microbalance-Dissipation and Reflectometry
2009; 81 (12): 4752-4761
We have used simultaneous quartz crystal microbalance-dissipation (QCM-D) monitoring and four-detector optical reflectometry to monitor in situ the structural transformation of intact vesicles to a lipid bilayer on a gold surface. The structural transformation of lipid vesicles to a bilayer was achieved by introducing a particular amphipathic, alpha-helical (AH) peptide. The combined experimental apparatus allows us to simultaneously follow the acoustic and optical property changes of the vesicle rupturing process upon interaction with AH peptides. While QCM-D and reflectometry have similar sensitivities in terms of mass and thickness resolution, there are unique advantages in operating these techniques simultaneously on the same substrate. These advantages permit us to (1) follow the complex interaction between AH peptides and intact vesicles with both acoustic and optical mass measurements, (2) calculate the amount of dynamically coupled water during the interaction between AH peptides and intact vesicles, (3) demonstrate that the unexpectedly large increase of both adsorbed mass and the film's energy dissipation is mainly caused by swelling of the vesicles during the binding interaction with AH peptides, and (4) permit us to understand the structural transformation from intact vesicles to a bilayer via the AH peptide interaction by monitoring viscoelastic properties, acoustic mass, optical mass, and thickness changes of both the binding and destabilization processes. From the deduced "hydration signature" we followed the complex transformation of lipid assemblies. On the basis of this information, a mechanism of this structural transformation is proposed that provides new insight into the process of vesicle fusion on solid substrates.
View details for DOI 10.1021/ac900242s
View details for Web of Science ID 000266969700012
View details for PubMedID 19459601
Viral infection of human progenitor and liver-derived cells encapsulated in three-dimensional PEG-based hydrogel
2009; 4 (1)
We have studied the encapsulation of human progenitor cells into 3D PEG hydrogels. Replication-incompetent lentivirus promoter reporter vectors were found to efficiently detect the in vivo expression of human hepatic genes in hydrogel-encapsulated liver progenitor cells. Similarly, hydrogel-encapsulated cells could be efficiently infected with hepatitis C virus, and progeny infectious virus could be recovered from the media supernatants of the hydrogels. Provocatively, the diameters of these virus particles range from approximately 50 to 100 nm, while the calculated mesh size of the 8 k hydrogel is 44.6 +/- 1.7 A. To reconcile how viral particles can penetrate the hydrogels to infect the encapsulated cells, we propose that microfractures/defects of the hydrogel result in a functional pore size of up to 20 fold greater than predicted by theoretical mesh calculations. These results suggest a new model of hydrogel structure, and have exciting implications for tissue engineering and hepatitis virus studies.
View details for DOI 10.1088/1748-6041/4/1/011001
View details for Web of Science ID 000263071200001
View details for PubMedID 18981544
Potential for Hepatitis C Virus Resistance to Nitazoxanide or Tizoxanide
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
2008; 52 (11): 4069-4071
Nitazoxanide and its primary metabolite, tizoxanide, inhibit hepatitis C virus (HCV) replication in HCV replicon systems. To study the potential for resistance, we subjected Huh7 cells harboring HCV replicons to serial passage in 250 muM G418 and increasing concentrations of nitazoxanide or tizoxanide. Passage of the replicon-containing cell lines in either compound resulted in increases in the 50% effective concentrations (EC(50)s) (7- to 13-fold), EC(90)s (14- to 36-fold), and 50% cytotoxic concentrations (2- to 4-fold) of both compounds. Serial passage in either compound did not alter the susceptibility of HCV replicons to ribavirin or 2'-C-methylcytidine. Interestingly, serial passage in nitazoxanide or tizoxanide resulted in increased sensitivity to alpha interferon 2b: EC(50)s and EC(90)s were reduced three- and eightfold, respectively. Replicons isolated from these cell lines had no greater ability to confer tizoxanide resistance, or increased susceptibility to alpha interferon, than replicons isolated from the parental cell line that had not previously been exposed to nitazoxanide or tizoxanide. These findings are indicative of a cell-mediated activity differing from that of other anti-HCV drugs but complementary with interferon and are consistent with the enhanced response rates observed clinically when nitazoxanide is combined with pegylated interferon therapy. Finally, unlike data for other compounds in advanced clinical development for HCV, these data are consistent with resistance in HCV replicon-containing cell lines conferred by changes in the host and not by mutations in the virus.
View details for DOI 10.1128/AAC.00078-08
View details for Web of Science ID 000260305600035
View details for PubMedID 18710916
Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis
2008; 26 (9): 1019-1027
More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3' terminus of the negative strand of the viral genome with a dissociation constant (Kd) of approximately 3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.
View details for DOI 10.1038/nbt.1490
View details for Web of Science ID 000259074700025
View details for PubMedID 18758449
Isolation and transcriptional profiling of purified hepatic cells derived from human embryonic stem cells
2008; 26 (8): 2032-2041
The differentiation of human embryonic stem cells (hESCs) into functional hepatocytes provides a powerful in vitro model system for studying the molecular mechanisms governing liver development. Furthermore, a well-characterized renewable supply of hepatocytes differentiated from hESCs could be used for in vitro assays of drug metabolism and toxicology, screening of potential antiviral agents, and cell-based therapies to treat liver disease. In this study, we describe a protocol for the differentiation of hESCs toward hepatic cells with complex cellular morphologies. Putative hepatic cells were identified and isolated using a lentiviral vector, containing the alpha-fetoprotein promoter driving enhanced green fluorescent protein expression (AFP:eGFP). Whole-genome transcriptional profiling was performed on triplicate samples of AFP:eGFP+ and AFP:eGFP- cell populations using the recently released Affymetrix Exon Array ST 1.0 (Santa Clara, CA, http://www.affymetrix.com). Statistical analysis of the transcriptional profiles demonstrated that the AFP:eGFP+ population is highly enriched for genes characteristic of hepatic cells. These data provide a unique insight into the complex process of hepatocyte differentiation, point to signaling pathways that may be manipulated to more efficiently direct the differentiation of hESCs toward mature hepatocytes, and identify molecular markers that may be used for further dissection of hepatic cell differentiation from hESCs. Disclosure of potential conflicts of interest is found at the end of this article.
View details for DOI 10.1634/stemcells.2007-0964
View details for Web of Science ID 000258297500011
View details for PubMedID 18535157
Isolation of human fetal liver progenitors and their enhanced proliferation by three-dimensional coculture with endothelial cells
TISSUE ENGINEERING PART A
2008; 14 (6): 995-1006
Liver progenitor cells, characterized by the coexpression of biliary and hepatocyte lineage markers and the ability to form colonies in culture, were isolated by flow cytometry from primary human fetal livers. These prospectively isolated liver progenitor cells supported hepatitis D virus infection, expressed, and produced albumin and alpha-fetoprotein, as tracked by albumin- and alpha-fetoprotein-driven lentiviral promoter reporter constructs and measured by ELISA, respectively. Coculture in three-dimensional (3D) fibrin gel with endothelial cells resulted in the formation of vascular structures by the endothelial cells and increased proliferation of liver progenitors. The enhanced proliferation of liver progenitors that was observed when liver progenitors and endothelial cells were cultured in direct contact was not achieved when liver progenitors and endothelial cells were cultured on adjacent but separate matrices and when they were cultured across transwell membranes. In conclusion, coculture of liver progenitors and endothelial cells in three-dimensional matrix resulted in enhanced liver progenitor proliferation and function. This coculture methodology offers a novel coculture system that could be applied for the development of engineered liver tissues.
View details for DOI 10.1089/ten.tea.2007.0087
View details for Web of Science ID 000256969800005
View details for PubMedID 19230124
The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without ha-ras co-transfection
2008; 47 (3): 827-835
Hepatitis C virus (HCV) is an important cause of chronic liver disease and is complicated by hepatocellular carcinoma (HCC). Mechanisms whereby the virus promotes cellular transformation are poorly understood. We hypothesized that the guanosine triphosphatase activity encoded in the HCV NS4B protein's nucleotide binding motif (NBM) might play a role in the transformation process. Here we report that NS4B can transform NIH-3T3 cells, leading to tumor formation in vivo. This transformation was independent of co-transfection with activated Ha-ras. Detailed analyses of NS4B mutants revealed that this transforming activity could be progressively inhibited and completely abrogated by increasing genetic impairment of the NS4B nucleotide binding motif.NS4B has in vitro and in vivo tumorigenic potential, and the NS4B transforming activity is indeed mediated by its NBM. Moreover, our results suggest that pharmacological inhibition of the latter might inhibit not only HCV replication but also the associated HCC.
View details for DOI 10.1002/hep.22108
View details for Web of Science ID 000253698900009
View details for PubMedID 18081150
TBC1D20 is a Rab1 GTPase-activating protein that mediates hepatitis C virus replication
JOURNAL OF BIOLOGICAL CHEMISTRY
2007; 282 (50): 36354-36361
Like other viruses, productive hepatitis C virus (HCV) infection depends on certain critical host factors. We have recently shown that an interaction between HCV nonstructural protein NS5A and a host protein, TBC1D20, is necessary for efficient HCV replication. TBC1D20 contains a TBC (Tre-2, Bub2, and Cdc16) domain present in most known Rab GTPase-activating proteins (GAPs). The latter are master regulators of vesicular membrane transport, as they control the activity of membrane-associated Rab proteins. To better understand the role of the NS5A-TBC1D20 interaction in the HCV life cycle, we used a biochemical screen to identify the TBC1D20 Rab substrate. TBC1D20 was found to be the first known GAP for Rab1, which is implicated in the regulation of anterograde traffic between the endoplasmic reticulum and the Golgi complex. Mutation of amino acids implicated in Rab GTPase activation by other TBC domain-containing GAPs abrogated the ability of TBC1D20 to activate Rab1 GTPase. Overexpression of TBC1D20 blocked the transport of exogenous vesicular stomatitis virus G protein from the endoplasmic reticulum, validating the involvement of TBC1D20 in this pathway. Rab1 depletion significantly decreased HCV RNA levels, suggesting a role for Rab1 in HCV replication. These results highlight a novel mechanism by which viruses can hijack host cell machinery and suggest an attractive model whereby the NS5A-TBC1D20 interaction may promote viral membrane-associated RNA replication.
View details for DOI 10.1074/jbc.M705221200
View details for Web of Science ID 000251458300026
View details for PubMedID 17901050
Current treatment options in gastroenterology
2007; 10 (6): 456-463
The hepatitis delta virus (HDV) is a defective RNA virus that requires the help of hepatitis B virus (HBV) for virion assembly and penetration into hepatocytes. Thus, it can only cause disease in individuals who carry HBV. HDV infection is present worldwide, predominantly in tropical and subtropical areas. However, with increased HBV control, HDV infection has significantly declined, although stably persisting, in developed countries. Most HDV-infected individuals develop chronic hepatitis D, and in approximately 80% of those cases, chronic hepatitis D progresses to cirrhosis within 5 to 10 years. The only approved therapy for chronic hepatitis D is interferon-alpha. Although transplantation offers a safe therapeutic option for managing end-stage HDV disease, novel therapeutic approaches are urgently needed.
View details for PubMedID 18221606
Creation of lipid partitions by deposition of amphipathic viral peptides
2007; 23 (21): 10855-10863
Phospholipid vesicles exhibit a natural characteristic to fuse and reform into a continuous single bilayer membrane on hydrophilic solid substrates such as glass, mica, and silica. The resulting solid-supported bilayer mimics physiological tendencies such as lipid flip-flop and lateral mobility. The lateral mobility of fluorescently labeled lipids fused into solid-supported bilayers is found to change upon deposition on the membrane surface of an amphipathic alpha-helical peptide (AH) derived from the hepatitis C virus (HCV) NS5A protein. The binding of the AH peptide to a phospholipid bilayer, with the helical axis parallel to the bilayer, leads to immobilization of the bilayer. We used AFM to better understand the mechanistic details of this specific interaction, and determined that the diminished fluidity of the bilayer is due to membrane thinning. Utilizing this specific interaction between AH peptides and lipid molecules, we demonstrate a novel process for the creation of lipid partition by employing AH peptides as agents to immobilize lipid molecules, thus creating a patterned solid support with partition-defined areas of freely mobile lipid bilayers. This architecture could have a wide range of applications in novel sensing, biotechnology, high-throughput screening, and biomimetic strategies.
View details for DOI 10.1021/1000640h
View details for Web of Science ID 000249948700058
View details for PubMedID 17803321
A Rab-GAP TBC domain protein binds hepatitis C virus NS5A and mediates viral replication
JOURNAL OF VIROLOGY
2007; 81 (20): 11096-11105
Hepatitis C virus (HCV) is an important cause of liver disease worldwide. Current therapies are inadequate for most patients. Using a two-hybrid screen, we isolated a novel cellular binding partner interacting with the N terminus of HCV nonstructural protein NS5A. This partner contains a TBC Rab-GAP (GTPase-activating protein) homology domain found in all known Rab-activating proteins. As the first described interaction between such a Rab-GAP and a viral protein, this finding suggests a new mechanism whereby viruses may subvert host cell machinery for mediating the endocytosis, trafficking, and sorting of their own proteins. Moreover, depleting the expression of this partner severely impairs HCV RNA replication with no obvious effect on cell viability. These results suggest that pharmacologic disruption of this NS5A-interacting partner can be contemplated as a potential new antiviral strategy against a pathogen affecting nearly 3% of the world's population.
View details for DOI 10.1128/JVI.01249-07
View details for Web of Science ID 000250019400025
View details for PubMedID 17686842
Employing two different quartz crystal microbalance models to study changes in viscoelastic behavior upon transformation of lipid vesicles to a bilayer on a gold surface
2007; 79 (18): 7027-7035
By analyzing the viscoelastic properties of two distinct layers, a layer of "soft" vesicles and a "rigid" bilayer, we have created a model system to permit the study of film behavior in the region of nonlinear mass and frequency change (non-Sauerbrey). The structural transformation of lipid vesicles to a bilayer is shown to be accompanied by significant changes in their physical properties. After the adsorption and saturation of intact vesicles on gold surfaces, the adsorbed vesicle layer exhibits a soft, water-rich, viscoelastic state. The AH peptide, a vesicle-destabilizing agent, is then added to trigger the formation of a much thinner (approximately 5 nm), compact, and rigid bilayer. In this study, we used the quartz crystal microbalance with dissipation technique. Large non-Sauerbrey frequency and energy dissipation changes characterize the viscoelastic nature of adsorbed intact vesicle films thicker than approximately 10 nm. Once the transformation is complete, the frequency changes along with zero energy dissipation for sufficiently thin films (t approximately 5 nm) were effectively modeled with the Sauerbrey equation. Furthermore, we checked the validity of the Voigt-Voinova model in which the quartz substrate is treated as a Voigt element, which is beyond the Sauerbrey description. The calculations treating the film as having a constant viscosity agreed well with the Voigt-Voinova model. These results were compared to calculations done using the electromechanical (EM) model, which does not require a series expansion. The Voigt-Voinova results were in excellent agreement with the EM model, providing evidence that the expansion used in their study is quite accurate.
View details for Web of Science ID 000249527700016
View details for PubMedID 17685547
- Employing an amphipathic viral peptide to create a lipid bilayer on Au and TiO2 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 2007; 129 (33): 10050-?
Binding dynamics of hepatitis C virus' NS5A amphipathic peptide to cell and model membranes
JOURNAL OF VIROLOGY
2007; 81 (12): 6682-6689
Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.
View details for DOI 10.1128/JVI.02783-06
View details for Web of Science ID 000246987500050
View details for PubMedID 17428867
View details for PubMedCentralID PMC1900085
- The power of silence: Application of small interfering RNAs to gastrointestinal diseases GASTROENTEROLOGY 2007; 132 (7): 2291-2295
Molecular virology of the hepatitis C virus: Implication for novel therapies
INFECTIOUS DISEASE CLINICS OF NORTH AMERICA
2006; 20 (1): 81-?
With the advent of second-generation agents that for the first time specifically target individual HCV proteins, HCV-specific therapy has arrived. The study of HCV molecular virology has helped make this possible and is helping us to identify additional new antiviral targets that will be targeted by third-generation drugs. Key to these efforts is the development of high-efficiency HCV replicons. The future effective pharmacologic control of HCV will likely consist of a cocktail of simultaneously administered virus-specific agents with independent targets. This should minimize the emergence of resistance against any single agent. The way we treat HCV should change dramatically over the next few years.
View details for DOI 10.1016/j.idc.2006.01.001
View details for Web of Science ID 000236520400006
View details for PubMedID 16527650
Prenylation of HDAg and antiviral drug development
HEPATITIS DELTA VIRUS
2006; 307: 133-149
Hepatitis delta virus (HDV) is an important cause of acute and chronic liver disease. Current medical therapies are unable to effectively eradicate HDV infections. Research into the molecular virology of the HDV life cycle has revealed a fascinating collection of biology. These insights are now beginning to be translated into new potential treatment strategies. For example, an essential step in the virus assembly process involves the post-translational lipid modification of a specific HDV protein, namely prenylation of large delta antigen. Preventing prenylation abolishes virus particle formation. Drugs capable of specifically inhibiting prenylation have been developed for use in humans. These agents represent a new class of antiviral agents, with HDV as a first target. Here, a brief review of the HDV life cycle emphasizing the role of prenylation is presented along with implications for drug development and therapy.
View details for Web of Science ID 000238999800007
View details for PubMedID 16903224
Novel therapies for hepatitis C virus based on lessons from virology.
Clinical gastroenterology and hepatology
2005; 3 (10): S86-8
As we improve our understanding of the molecular virology of hepatitis C virus (HCV), a variety of new potential antiviral strategies are emerging. Moving beyond interferon and ribavirin, these new strategies for the first time are aimed at HCV-specific targets. It is expected, by analogy with other infections, that effective pharmacologic control of HCV will be achieved best by using a cocktail of such virus-specific agents, each designed against an independent target. As a result, the way we treat HCV should change dramatically over the next few years.
View details for PubMedID 16234068
Molecular virology of the hepatitis C virus: implication for novel therapies.
Clinics in liver disease
2005; 9 (3): 353-?
The study of hepatitis C virus (HCV) molecular virology is helping to shape the future of our anti-HCV strategies by identifying new antiviral targets. With the advent of agents that specifically target individual HCV proteins, HCV-specific therapy has arrived. Key to these efforts is the development of high-efficiency HCV replicons. The future effective pharmacologic control of HCV will likely consist of a cocktail of simultaneously administered virus-specific agents with independent targets. This should minimize the emergence of resistance against any single agent. The way we treat HCV should change dramatically over the next few years.
View details for PubMedID 16023970
Hepatitis C virus core protein associates with detergent-resistant membranes distinct from classical plasma membrane rafts
JOURNAL OF VIROLOGY
2004; 78 (21): 12047-12053
A subpopulation of hepatitis C virus (HCV) core protein in cells harboring full-length HCV replicons is biochemically associated with detergent-resistant membranes (DRMs) in a manner similar to that of markers of classical lipid rafts. Core protein does not, however, colocalize in immunofluorescence studies with classical plasma membrane raft markers, such as caveolin-1 and the B subunit of cholera toxin, suggesting that core protein is bound to cytoplasmic raft microdomains distinct from caveolin-based rafts. Furthermore, while both the structural core protein and the nonstructural protein NS5A associate with membranes, they do not colocalize in the DRMs. Finally, the ability of core protein to localize to the DRMs did not require other elements of the HCV polyprotein. These results may have broad implications for the HCV life cycle and suggest that the HCV core may be a valuable probe for host cell biology.
View details for DOI 10.1128/JVI.78.21.12047-12053.2004
View details for Web of Science ID 000224540900057
View details for PubMedID 15479844
An n-terminal amphipathic helix in hepatitis C virus (HCV) NS4B mediates membrane association, correct localization of replication complex proteins, and HCV RNA replication
JOURNAL OF VIROLOGY
2004; 78 (20): 11393-11400
Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.
View details for DOI 10.1128/JVI.78.20.11393-11400.2004
View details for Web of Science ID 000224229000058
View details for PubMedID 15452261
A nucleotide binding motif in hepatitis C virus (HCV) NS4B mediates HCV RNA replication
JOURNAL OF VIROLOGY
2004; 78 (20): 11288-11295
Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the virus's nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral strategies.
View details for Web of Science ID 000224229000045
View details for PubMedID 15452248
Overdependence on the host-an Achilles' heel of HCV?
2004; 39 (6): 1734-1735
View details for PubMedID 15185316
Testing antivirals against hepatitis delta virus: farnesyl transferase inhibitors.
Methods in molecular medicine
2004; 96: 539-553
View details for PubMedID 14762290
Prenylation inhibitors: a novel class of antiviral agents
JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY
2003; 52 (6): 883-886
Prenylation is a site-specific lipid modification of proteins. Although first described for a variety of cellular proteins, it has become apparent that viruses can also make use of this post-translational modification provided by their host cells. Depriving a virus access to prenylation can have dramatic effects on the targeted virus's life cycle. Selective pharmacological inhibitors of prenylating enzymes have been developed and shown to have potent antiviral effects in both in vitro and in vivo systems. Because prenylation inhibitors target a host cell function, are available in oral form and are surprisingly well tolerated in human trials, these compounds represent an attractive new class of antiviral agents with potential for broad-spectrum activity. After a brief outline of host cell prenylation pathways, we review below the development of prenylation inhibition as an antiviral strategy applied to a prototype target, hepatitis delta virus (HDV), and discuss the potential application of prenylation inhibitors to a broad range of other viruses.
View details for DOI 10.1093/jac/dkg490
View details for Web of Science ID 000187227000001
View details for PubMedID 14613953
Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication
JOURNAL OF VIROLOGY
2003; 77 (10): 6055-6061
We identified an N-terminal amphipathic helix (AH) in one of hepatitis C virus (HCV)'s nonstructural proteins, NS5A. This AH is necessary and sufficient for membrane localization and is conserved across isolates. Genetically disrupting the AH impairs HCV replication. Moreover, an AH peptide-mimic inhibits the membrane association of NS5A in a dose-dependent manner. These results have exciting implications for the HCV life cycle and novel antiviral strategies.
View details for DOI 10.1128/JVI.77.10.6055-6061.2003
View details for Web of Science ID 000182631100051
View details for PubMedID 12719597
A prenylation inhibitor prevents production of infectious hepatitis delta virus particles
JOURNAL OF VIROLOGY
2002; 76 (20): 10465-10472
Hepatitis delta virus (HDV) causes both acute and chronic liver disease throughout the world. Effective medical therapy is lacking. Previous work has shown that the assembly of HDV virus-like particles (VLPs) could be abolished by BZA-5B, a compound with farnesyltransferase inhibitory activity. Here we show that FTI-277, another farnesyltransferase inhibitor, prevented the production of complete, infectious HDV virions of two different genotypes. Thus, in spite of the added complexity and assembly determinants of infectious HDV virions compared to VLPs, the former are also sensitive to pharmacological prenylation inhibition. Moreover, production of HDV genotype III virions, which is associated with particularly severe clinical disease, was as sensitive to prenylation inhibition as was that of HDV genotype I virions. Farnesyltransferase inhibitors thus represent an attractive potential class of novel antiviral agents for use against HDV, including the genotypes associated with most severe disease.
View details for DOI 10.1128/JVI.76.20.10465-10472.2002
View details for Web of Science ID 000178319600041
View details for PubMedID 12239323
Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses
2000; 6 (3): 327-331
Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.
View details for Web of Science ID 000085580500044
View details for PubMedID 10700236
- Shutting the door on hepatitis delta virus (HDV): Sensitivity to prenylation inhibition prompts new therapeutic strategy Viral Hepatitis Reviews 1999; 53: 13-26
Use of a prenylation inhibitor as a novel antiviral agent
JOURNAL OF VIROLOGY
1998; 72 (11): 9303-9306
No specific therapy exists for hepatitis delta virus (HDV), which can cause severe liver disease. Molecular genetic studies have implicated the prenylation site of large delta antigen as a critical determinant of HDV particle assembly. We have established a cell culture model which produces HDV-like particles, and we show that delta antigen prenylation can be pharmacologically inhibited by the prenylation inhibitor BZA-5B. Furthermore, BZA-5B specifically abolishes particle production in a dose-dependent manner. These results demonstrate that the use of such a prenylation inhibitor-based antiviral therapy may be feasible and identify a novel class of potential antiviral agents.
View details for Web of Science ID 000076373700098
View details for PubMedID 9765479
- Finding the iron in the melting pot - Practical use of a new genetic assay for hereditary hemochromatosis WESTERN JOURNAL OF MEDICINE 1998; 168 (6): 525-527
IDENTIFICATION OF A PRENYLATION SITE IN DELTA-VIRUS LARGE ANTIGEN
1992; 256 (5061): 1331-1333
During replication, hepatitis delta virus (HDV) switches from production of small to large delta antigen. Both antigen isoforms have an HDV genome binding domain and are packaged into hepatitis B virus (HBV)-derived envelopes but differ at their carboxy termini. The large antigen was shown to contain a terminal CXXX box and undergo prenylation. The large, but not the small, antigen formed secreted particles when expressed singly with HBV surface antigen. Mutation of Cys211 in the CXXX box of the large antigen abolished both prenylation and particle formation, suggesting that this site is important for virion morphogenesis.
View details for Web of Science ID A1992HW13500037
View details for PubMedID 1598578