HCV (hepatitis C virus) infects nearly 3% of the population worldwide and has emerged as a major causative agent of liver disease, resulting in acute and chronic infections that can lead to fibrosis, ...cirrhosis and hepatocellular carcinoma. Hepatitis C represents the leading cause of liver transplantation in the United States and Europe. A positive-strand RNA virus of the Flaviviridae family, HCV contains a single-stranded RNA genome of approx. 9600 nucleotides. The genome RNA serves as both mRNA for translation of viral proteins and the template for RNA replication. Cis-acting RNA elements within the genome regulate RNA replication by forming secondary structures that interact with each other and trans-acting factors. Although structural proteins are clearly dispensable for RNA replication, recent evidence points to an important role of several non-structural proteins in particle assembly and release, turning their designation on its head. HCV enters host cells through receptor-mediated endocytosis, and the process requires the co-ordination of multiple cellular receptors and co-receptors. RNA replication takes place at specialized intracellular membrane structures called 'membranous webs' or 'membrane-associated foci', whereas viral assembly probably occurs on lipid droplets and endoplasmic reticulum. Liver inflammation plays a central role in the liver damage seen in hepatitis C, but many HCV proteins also directly contribute to HCV pathogenesis. In the present review, the molecular and cellular aspects of the HCV life cycle and the role of viral proteins in pathological liver conditions caused by HCV infection are described.
Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the ...mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.
Envelope glycoprotein (Env) spikes on AIDS retroviruses initiate infection of host cells and are therefore targets for vaccine development. Though crystal structures for partial Env subunits are ...known, the structure and distribution of native Env spikes on virions is obscure. We applied cryoelectron microscopy tomography to define ultrastructural details of spikes. Virions of wild-type human immunodeficiency virus 1 (HIV-1) and a mutant simian immunodeficiency virus (SIV) had approximately 14 and approximately 73 spikes per particle, respectively, with some clustering of HIV-1 spikes. Three-dimensional averaging showed that the surface glycoprotein (gp120) 'head' of each subunit of the trimeric SIV spike contains a primary mass, with two secondary lobes. The transmembrane glycoprotein 'stalk' of each trimer is composed of three independent legs that project obliquely from the trimer head, tripod-like. Reconciling available atomic structures with the three-dimensional whole spike density map yields insights into the orientation of Env spike structural elements and possible structural bases of their functions.
We describe the design and synthesis of two compact multicoordinating (lipoic acid-appended) zwitterion ligands for the capping of luminescent quantum dots, QDs. This design is combined with a novel ...and easy to implement photoligation strategy to promote the in situ ligand exchange and transfer of the QDs to buffer media. This method involves the irradiation of the native hydrophobic nanocrystals in the presence of the ligands, which promotes in situ cap exchange and phase transfer of the QDs, eliminating the need for a chemical reduction of the dithiolane groups. Applied to the present LA-zwitterion ligands, this route has provided QDs with high photoluminescence yields and excellent colloidal stability over a broad range of conditions, including acidic and basic pH, in the presence of growth media and excess salt conditions. The small lateral extension of the capping layer allowed easy conjugation of the QDs to globular proteins expressing a terminal polyhistidine tag, where binding is promoted by metal-affinity interactions between the accessible Zn-rich surface and imidazoles in the terminal tag of the proteins. The ability to carry out conjugation in acidic as well as basic conditions opens up the possibility to use such self-assembled QD-protein conjugates in various biological applications.
Hepatitis C virus (HCV) is an enveloped RNA virus that often manifests into chronic infections of the liver, which represents a threat to human health due to the morbidity it presents within infected ...patients. The virus is especially persistent as current antiviral treatments have difficulty in restricting the infections, allowing most new cases of viral hepatitis to develop into persistent infections. It has emerged as a major causative agent of liver diseases, resulting in cirrhosis, liver failure, and hepatocellular carcinoma as the disease progresses with time. There is no prophylactic vaccine currently available to protect against HCV, nor is there an effective therapy capable of generating a sustained virologic response. HCV has shown the capability of developing resistance to antiviral compounds that target specific viral enzymes necessary for replication. HCV is found as quasispecies within an individual due to its error prone RNA-dependent RNA polymerase. The genetic heterogeneity of HCV allows it to be capable of escaping the effect of compounds that specifically target viral proteins. Several cellular cofactors have been identified recently that permit HCV to be infectious. The interactions of these cofactors with viral proteins shed light on the life cycle of HCV and warrant further study as potential cellular targets to restrict viral infectivity. One such cofactor is cyclophilin A (CyPA) which has peptidylproline isomerase (PPIase) activity. HCV strains with a reduced dependency on CyPA for replication were selected in a CyPA-knockdown cell line. Sequencing of these isolates revealed mutations within a dipeptide motif of domain 2 of NS5A for all clones (D316E and/or Y317N). Analysis of these phenotypes upon insertion into the full-length viral genome revealed the double mutant, termed DEYN, to most efficiently restore the infectivity of the virus in CyPA-knockdown cells to that of control cells. We have hypothesized several possible mechanisms for the reduced dependency on CyPA of DEYN virus. These mutations could potentially affect viral replication via the PPIase activity of CyPA which catalyzes the cis-trans isomerization of proline residues. Presumably, HCV requires CyPA as a host cofactor to isomerize one or more proline residues within NS5A, converting it from a non-functional, unfolded protein into a functional, folded conformer. The work described here identifies structural differences between wt and mutant NS5A sequences, identifies CyPA binding sites within domain II and LCS II, and describes critical determinants within CyPA and NS5A that permit their interactions. Furthermore, this work identified A PXW and APLPP as CyPA binding motifs among the 7 genotypes tested.
Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the ...mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.
We describe the design and synthesis of a series of compact ligands made of lipoic acid (LA)-based coordinating anchors and hydrophilic zwitterion groups. This ligand design is combined with a novel ...photoligation strategy to promote the transfer of QDs to polar and buffer media. This approach has provided hydrophilic QDs that exhibit great colloidal stability over a broad range of pHs and in the presence of cell culture media. Our photoligation strategy drastically improves previous phase transfer methods by eliminating the need for chemical reduction of the dithiolane ring using NaBH4 prior to the cap exchange, and it is adapted to several LA-based ligands. We also found that QDs stabilized with these compact zwitterionic ligands are fully compatible with metal-histidine-driven self-assembly where the protein activity is maintained after forming conjugation with the QDs.
Introduction
The 0.19 mg fluocinolone acetonide (FAc) intravitreal implant delivers a continuous intravitreal corticosteroid dose for the treatment of refractory diabetic macular oedema (DMO). The ...aim of this study was to assess the impact of an FAc intravitreal implant on intraocular pressure (IOP).
Methods
We retrospectively collected anonymised data on the patients’ characteristics, DMO treatment, and IOP and IOP-lowering treatments before and after the FAc intravitreal implant between September 2013 and March 2020 in several European centres.
Results
A total of 221 eyes from 179 patients were included. The mean follow-up duration was 13.4 (± 12.5, range 2.4–33.5) months. Overall, 194 eyes (88.2%) had received an intravitreal dexamethasone injection before the FAc intravitreal implant. For 25 eyes (11.3%) there was a history of glaucoma, and 52 eyes (23.5%) had previous IOP-lowering treatment. Mean IOP before injection was 14.7 (3.4) mmHg and increased to 16.9 (3.7) mmHg 12 months after injection (
P
< 0.0001). During follow-up, 55 eyes (24.9%) required the addition or initiation of topical IOP-lowering medication, only one patient (0.5%) had laser trabeculoplasty and one patient (0.5%) a minimally invasive glaucoma surgery, and no patient required incisional IOP-lowering surgery.
Conclusion
The FAc intravitreal implant led to substantial IOP elevation. This elevation was monitored most of the time with addition or initiation of topical IOP-lowering medication.
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