Cell culture and in vivo analyses of cytopathic hepatitis C virus mutants

AbstractHCV-JFH1 yields subclones that develop cytopathic plaques (Sekine-Osajima Y, et al., Virology 2008; 371:71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection

Lnk regulates integrin αIIbβ3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk-/- mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin αIIbβ3-mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk-/- mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk-/- platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the β3 integrin subunit, and reduced binding of Fyn to integrin αIIbβ3. These results provide new insight into the mechanism of αIIbβ3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events

Inhibition of Hepatitis C Virus Replication by a Specific Inhibitor of Serine-Arginine-Rich Protein Kinase ▿

Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection

Regulation of Hepatitis C Virus Replication by Interferon Regulatory Factor 1

Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the interferon-mediated antiviral system in cells expressing an HCV replicon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor 1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replicon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replicon. Moreover, in cured Huh7 cells from which the HCV replicon had been eliminated, the expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of interferon-stimulated-gene-mediated antiviral responses