7 research outputs found
Isolation and Characterization of Highly Replicable Hepatitis C Virus Genotype 1a Strain HCV-RMT
<div><p>Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed <i>in vitro</i> and <i>in vivo</i>, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient’s serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between <i>in vitro</i> and <i>in vivo</i> replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication <i>in vitro</i> and <i>in vivo</i>.</p></div
<i>In vitro</i> replication ability of HCV-RMT derivative genomes.
<p>(A) Schematic representation of construction of the HCV genome and the sites of adaptive mutations (red bars). (B) Electroporation of the generated HCV-RNA genomes of wild-type HCV-RMT (closed triangles), HCV-RMT with triple mutations (HCV-RMTtri; closed circles), and the JFH-1 strain (open circles) into Huh-7.5.1 or HuH7-K4 cells. The experiments were carried out in duplicate. (C) Comparison of the <i>in vitro</i> replication ability of each HCV-RMT derivative in HuH7-K4 cells. The experiments were carried out in duplicate. Wild type: open squares, E1202G: open circles, E1056V: open triangles, A2199T: closed squares, E1056V and A2199T: closed triangles, triple mutations: closed circles. (D) Immunostaining for the HCV core protein in HCV-RNA-electroporated cells. Scale bar  =  100 µm. The percent of HCV core protein-positive cells (%) was calculated as an average of ten observed areas.</p
<i>In vivo</i> replication ability of HCV-RMT derivatives.
<p>(A) Change in HCV copy numbers in the serum of chimeric mice in which the HCV genome was directly injected into the livers. HCV-RMT (wild type): closed circles, HCV-RMT (E1056V and A2199T): open circles, HCV-RMT (E1202G): closed triangles, HCV-RMTtri: closed squares, JFH-1: open triangles. Data are indicated as the mean ± S.D. (B) HCV copy number in the livers. N.D.: not detected. (C) Serum/liver ratio of HCV copy number.</p
Establishment of HCV-RMTtri highly replicating #11 cell and infectivity of its supernatant on naïve HuH7-K4 cells.
<p>(A) Immunostaining for the HCV core protein in HCV-RMTtri-electroporated parental cells and the cell clone (#11) obtained by limiting dilution cloning. Scale bar  =  100 µm. (B) Immunostaining for the HCV core protein in naïve HuH7-K4 cells infected with supernatants of HCV-RMTtri- or JFH-1-replicating cells. Scale bar  =  50 µm. (C) Infection with the HCV-RMTtri supernatant was inhibited with anti-CD81 antibody in a similar manner as JFH-1. Control IgG (normal mouse IgG<sub>1</sub>): open circles, anti-CD81 mAb (JS-81): closed circles. Data are indicated as the mean ± S.D. (D) Replication of HCV-RMTtri in HuH7-K4 cells was inhibited by HCV replication inhibitors such as cyclosporin A and interferon-α. Drugs were added to #11 cells in 96-well plates 1 day after passaging, and cells were harvested after 72 h of treatment. NT: no treatment.</p
Relationship between the <i>in vitro</i> and <i>in vivo</i> replication ability of HCV-RMT derivatives.
<p><i>in vitro</i> column, +++: maximum replication ability, ++: approximately 1 log lower than the maximum, +: approximately 2 logs lower than the maximum, –: no difference compared to the wild-type strain. For the <i>in vivo</i> column, +++: maximum replication ability, ++: approximately 1 log lower than the maximum, +: approximately 2 logs lower than the maximum, -: no replication.<sup></sup> For the </p
Basic characteristics of the HCV-RMT clone.
<p>Change in HCV copy number in chimeric mice. (A) Two mice were intravenously infected with 10 µl patient serum HCG9. (B) Three mice per group were directly injected with 30 µg HCV RNAs of the HCV-RMT strain or the JFH-1 strain into the liver. Data are indicated as the mean ± S.D. (C) Schematic representation of construction of the replicon and the sites of adaptive mutations. (D) Colony formation assay of replicon clones with adaptive mutations. Each RNA (1 µg) was electroporated into HuH7-K4 cells. (E) Western blot analysis of replicon cells. Each culture of replicon RNA-electroporated cells was maintained and passaged with G418 selection for 2 weeks. Cell lysates (10 µg) were loaded onto an SDS-PAGE gel.</p
Efficient siRNA Delivery by Lipid Nanoparticles Modified with a Nonstandard Macrocyclic Peptide for EpCAM-Targeting
The
development of a specific, effective method for the delivery
of therapeutics including small molecules and nucleic acids to tumor
tissue remains to be solved. Numerous types of lipid nanoparticles
(LNPs) have been developed in attempts to achieve this goal. However,
LNPs are probably not taken up by target cells because cancer-targeting
LNPs are typically modified with polyÂ(ethylene glycol) (PEG), which
inhibits the cellular uptake of LNPs, to passively accumulate in tumor
tissue via the enhanced permeability and retention (EPR) effect. It
would clearly be important to develop a LNP with both a prolonged
circulation and cancer-specific efficient uptake for use in an innovative
nanodrug delivery system. Herein, we assessed the effect of nonstandard
macrocyclic peptides against the epithelial cell adhesion molecule
(EpCAM) Epi-1, which was discovered by a random nonstandard peptides
integrated discovery (RaPID) system, on the cellular uptake and therapeutics
delivery of LNPs. A liposomal siRNA delivery system (MEND) was modified
with an Epi-1 lipid-derivative (EpCAM-targeting MEND; ET-MEND). The
resulting ET-MEND showed a more than 27-fold increase in cellular
uptake in EpCAM-positive cell lines. In the case of negative cells,
cellular uptake and the efficiency of the ET-MEND for delivering therapeutics
were comparable with those of nonmodified MEND. In addition, when
systemically injected, the ET-MEND successfully inhibited gene expression
in the tumor tissue at a dose of 0.5 mg siRNA/kg without any obvious
toxicity. These results suggest that a combination of a specific peptide
ligand can be used to identify a RaPID system and that the use of
such a MEND for liposomal drug delivery has the potential for use
in developing a system for the efficacious delivery of pharmaceuticals
to various cancer cells