Understanding How Hepatitis C Virus Builds Its Unctuous Home

Hepatitis C virus genome replication occurs in endoplasmic reticulum-derived membrane compartments, but it is unknown how these structures arise. In this issue of Cell Host & Microbe, Reiss and colleagues (2011) show that the virus recruits a specific lipid kinase to replication sites, stimulates its kinase activity, and alters the phospholipid profile of replication compartments

Flunarizine arrests hepatitis C virus membrane fusion.

Written with support from a Senior Research Fellowship from the Wellcome Trust (grant no.: 101908/Z/13/Z) to Y.M. and from grant R01 GM102869 from the National Institutes of Health to Y.M.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1002/hep.2822

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

Red-Mediated Transposition and Final Release of the Mini-F Vector of a Cloned Infectious Herpesvirus Genome

Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via synchronous Red-recombination in E. coli in an en passant mutagenesis-based procedure. Using this technique, the mini-F elements of a cloned infectious varicella zoster virus (VZV) genome were specifically transposed into novel positions distributed over the viral DNA to generate six different BAC variants. In comparison to the other constructs, a BAC variant with mini-F sequences directly inserted into the junction of the genomic termini resulted in highly efficient viral DNA replication-mediated spontaneous vector excision upon virus reconstitution in transfected VZV-permissive eukaryotic cells. Moreover, the derived vector-free recombinant progeny exhibited virtually indistinguishable genome properties and replication kinetics to the wild-type virus. Thus, a sequence-independent, efficient, and easy-to-apply mini-F vector transposition procedure eliminates the last hurdle to perform virtually any kind of imaginable targeted BAC modifications in E. coli. The herpesviral terminal genomic junction was identified as an optimal mini-F vector integration site for the construction of an infectious BAC, which allows the rapid generation of mutant virus without any unwanted secondary genome alterations. The novel mini-F transposition technique can be a valuable tool to optimize, repair or restructure other established BACs as well and may facilitate the development of gene therapy or vaccine vectors

Structure‐guided insights on the role of NS1 in flavivirus infection

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111115/1/bies201400182.pd

The Hepatitis C Virus Nonstructural Protein 2 (NS2): An Up-and-Coming Antiviral Drug Target

Infection with Hepatitis C Virus (HCV) continues to be a major global health problem. To overcome the limitations of current therapies using interferon-α in combination with ribavirin, there is a need to develop drugs that specifically block viral proteins. Highly efficient protease and polymerase inhibitors are currently undergoing clinical testing and will become available in the next few years. However, with resistance mutations emerging quickly, additional enzymatic activities or functions of HCV have to be targeted by novel compounds. One candidate molecule is the nonstructural protein 2 (NS2), which contains a proteolytic activity that is essential for viral RNA replication. In addition, NS2 is crucial for the assembly of progeny virions and modulates various cellular processes that interfere with viral replication. This review describes the functions of NS2 in the life cycle of HCV and highlights potential antiviral strategies involving NS2

Older adult mistreatment, dementia, and the family caregiver in the Northeastern Ontario home: the influence of context on professional agency

This integrated-article thesis presents the findings of a qualitative critical inquiry with three related aims: to understand the experience of formal care practitioners who encounter mistreatment of an older adult with dementia by their family caregiver in the home; to explain the influences of the domestic, health care, geographical, and socio-political contexts upon that experience; and to facilitate empowerment and collective action to improve policy, practice and care outcomes. Viewing this experience through a Critical Social Theory lens, data collection methods consisted of interviews, reflective journals, and inquiry and action focus groups. Practitioners representing 23 organizations providing care to older adults in their homes in urban and rural Northeastern Ontario participated over the course of two phases of Understanding and Empowerment. In this thesis, Chapter 1 will review the literature, identifying gaps in knowledge and describing the critical theoretical underpinnings, methodology and theoretical thematic analysis which were instrumental to facilitate self-reflection of past cases, critique of socially dominant ideologies and structures, dialogue with other practitioners also encountering these cases, and dialectic reasoning, a process of examining contradictions of what is, versus what should be in cases of older adult mistreatment and dementia. Chapter 2 presents the findings on the experience of practitioners with these cases. More specifically, practitioners described a lack of professional agency defined as the ability to control outcomes and act in a meaningful way in their cases. Next, Chapter 3 examines the home, familial, health care, geographical and socio- political contexts and their influences on professional agency. Those findings describe the oppression of these contexts on practitioners who could not control the outcomes of older adult mistreatment [OAM] within them. Chapter 4 then presents the process of empowerment during which practitioners shared their concerns related to these oppressive contexts and collaborated towards collective action projects to improve policy, practice and outcomes. Chapter 5 situates the significance of the findings within the field of OAM and dementia and discusses cross- cutting themes linking the papers. Limitations of the study will be reviewed as well as recommendations for policy, practice and research.Doctor of Philosophy (PhD) in Interdisciplinary Rural and Northern Healt

Primary hepatocytes as targets for hepatitis C virus replication

Much of our current understanding of hepatitis C virus (HCV) replication has hailed from the use of a small number of cloned viral genomes and transformed hepatoma cell lines. Recent evidence suggests that lipoproteins play a key role in the HCV life cycle and virus particles derived from the sera of infected patients exist in association with host lipoproteins. This report will review the literature on HCV replication in primary hepatocytes and transformed cell lines, focusing largely on host factors defining particle entry

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

Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice

International audienceBACKGROUND: With the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination. METHODOLOGY/PRINCIPAL FINDINGS: A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5' non-coding region (5'NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%-29.5% of cases, and HCV subtype 1b in 9.5%-8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5'NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5'NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases. CONCLUSIONS/SIGNIFICANCE: In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5'NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice