Hepatitis C Virus Entry Is Impaired by Claudin-1 Downregulation in Diacylglycerol Acyltransferase-1-Deficient Cells

Abstract

Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells by using either the transcription activator-like effector nuclease (TALEN) or lentivirus vector short hairpin RNA (shRNA) and achieved complete long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of the gene coding for hepatocyte nuclear factor 4␣ (HNF4␣) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 expression and HCV entry were also restored by low-dose palmitic acid treatment, indicating that the downregulation of CLDN1 was associated with altered fatty acid homeostasis in the absence of DGAT1. Our findings provide novel insight into the role of DGAT1 in the life cycle of HCV. IMPORTANCEIn this study, we report the novel effect of complete silencing of DGAT1 on the entry of HCV. DGAT1 was recently reported as a host factor of HCV, involved in the assembly of HCV by facilitating the trafficking of the HCV core protein to lipid droplets. We achieved complete and long-term silencing of DGAT1 by either TALEN or repeated transduction of lentivirus shRNA. We found that HCV entry was severely impaired in DGAT1-silenced cell lines. The impairment of HCV entry was caused by CLDN1 downregulation, and the expression of HNF4␣ and other hepatocyte-specific genes was also downregulated in DGAT1-silenced cell lines. Our results suggest new roles of DGAT1 in human liver-derived cells: maintaining intracellular lipid homeostasis and affecting HCV entry by modulating CLDN1 expression. H epatitis C virus (HCV) infection poses a major threat to human health, with a prevalence of more than 160 million people worldwide (1). In addition to a combination regimen of pegylated interferon alpha (IFN-␣) and ribavirin, drugs acting directly on HCV have now been developed, although these direct-acting antivirals may prompt the emergence of resistant strains (2, 3). Our increasing understanding of the HCV-host interactions is allowing novel therapeutic approaches to be developed that modulate host factors that are required for viral entry, replication, and egress; these factors may have a higher genetic barrier to viral resistance (4).Diacylglycerol acyltransferase-1 (DGAT1) is one of two known DGAT enzymes catalyzing the final step in triglyceride biosynthesis (5, 6). The expression of DGAT1 and its physiologic role differ in humans and mice. In mice, DGAT1 is expressed in many organs, including the skeletal muscle, heart, and intestines, but it is bar...

Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry

“…HCV pseudoparticles (HCVpp) with E1 and E2 glycoproteins derived from genotype 1a (H77) or genotype 2a (JFH-1) and vesicular stomatitis virus pseudoparticles (VSVpp) were generated as previously described (20). Briefly, HEK293T cells were transfected with the glycoprotein-expressing plasmid, Gag-Pol (polymerase)-packaging plasmid, and transfer vector encoding the firefly luciferase reporter protein by using polyethyleneimine (Sigma-Aldrich).…”

Activation of human natural killer cells by the soluble form of cellular prion protein

“…RNA extraction, cDNA synthesis, and real-time quantitative PCR NK cells were harvested 8 h after PrP C -Fc treatment. Total RNA isolation, cDNA synthesis and TaqMan real-time quantitative PCR were performed as previously described [18]. Briefly, total RNA was isolated using an RNeasy Mini kit (Qiagen, Valencia, CA, USA), and first-strand cDNA was synthesized using a High Capacity cDNA Synthesis Kit (Applied Biosystems, Foster City, CA, USA).…”

Transcription Activator-Like Effector (TALE) Nucleases and Repressor TALEs for Antiviral Gene Therapy

“…A number of adverse side effects and the emergence of viral escape mutants are major drawbacks of currently available therapies. Recently, two independent studies have demonstrated that TALEN-based disruption of host genes in liver derived cells inhibits HCV replication and cell entry [24,25]. An APOB -/-Huh-7 stable cell line was established to show that the expression of the apoB-100 protein is necessary for active HCV replication [24].…”

“…An APOB -/-Huh-7 stable cell line was established to show that the expression of the apoB-100 protein is necessary for active HCV replication [24]. Similarly, cell entry and replication cycle of HCV may be obstructed by using TALENs to knock out the diacylglycerol acyltransferase-1 (DGAT1) gene [25]. Although these are interesting observations, the feasibility of using TALENs for treating HCV infection remains to be established.…”