Erratum: Proof-of-concept study for liver-directed miQURE technology in a dyslipidemic mouse model.

[This corrects the article DOI: 10.1016/j.omtn.2023.04.004.].

Proof-of-concept study for liver-directed miQURE technology in a dyslipidemic mouse model.

A gene-silencing platform (miQURE) has been developed and successfully used to deliver therapeutic microRNA (miRNA) to the brain, reducing levels of neurodegenerative disease-causing proteins/RNAs via RNA interference and improving the disease phenotype in animal models. This study evaluates the use of miQURE technology to deliver therapeutic miRNA for liver-specific indications. Angiopoietin-like 3 (ANGPTL3) was selected as the target mRNA because it is produced in the liver and because loss-of-function ANGPTL3 mutations and/or pharmacological inhibition of ANGPTL3 protein lowers lipid levels and reduces cardiovascular risk. Overall, 14 candidate miRNA constructs were tested in vitro, the most potent of which (miAngE) was further evaluated in mice. rAAV5-miAngE led to dose-dependent (≤-77%) decreases in Angptl3 mRNA in WT mice with ≤-90% reductions in plasma ANGPTL3 protein. In dyslipidemic APOE∗3-Leiden.CETP mice, AAV5-miAngE significantly reduced cholesterol and triglyceride levels vs. vehicle and scrambled (miSCR) controls when administrated alone, with greater reductions when co-administered with lipid-lowering therapy (atorvastatin). A significant decrease in total atherosclerotic lesion area (-58% vs. miSCR) was observed in AAV5-miAngE-treated dyslipidemic mice, which corresponded with the maintenance of a non-diseased plaque phenotype and reduced lesion severity. These results support the development of this technology for liver-directed indications.

Viral genotype correlates with distinct liver gene transcription signatures in chronic hepatitis C virus infection.

BACKGROUND: Chronic hepatitis C virus (HCV) infection of the liver with either genotype 1 or genotype 3 gives rise to distinct pathologies, and the two viral genotypes respond differently to antiviral therapy. METHODS: To understand these clinical differences, we compared gene transcription profiles in liver biopsies from patients infected with either gt1 or gt3, and uninfected controls. RESULTS: Gt1-infected biopsies displayed elevated levels of transcripts regulated by type I and type III interferons (IFN), including genes that predict response to IFN-α therapy. In contrast, genes controlled by IFN-γ were induced in gt3-infected biopsies. Moreover, IFN-γ levels were higher in gt3-infected biopsies. Analysis of hepatocyte-derived cell lines confirmed that the genes upregulated in gt3 infection were preferentially induced by IFN-γ. The transcriptional profile of gt3 infection was unaffected by IFNL4 polymorphisms, providing a rationale for the reduced predictive power of IFNL genotyping in gt3-infected patients. CONCLUSIONS: The interactions between HCV genotypes 1 and 3 and hepatocytes are distinct. These unique interactions provide avenues to explore the biological mechanisms that drive viral genotype-specific differences in disease progression and treatment response. A greater understanding of the distinct host-pathogen interactions of the different HCV genotypes is required to facilitate optimal management of HCV infection.

Modulation of triglyceride and cholesterol ester synthesis impairs assembly of infectious hepatitis C virus.

In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.

Characterization of hepatitis C virus NS3 modifications in the context of replication.

Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins, hepatitis C virus (HCV) can orchestrate its complete viral life cycle. HCV non-structural protein 3 (NS3) has many functions. It has protease and helicase activities, interacts with several host-cell proteins and plays a role in translation, replication and virus-particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6-NS3 yielded multiple spots. Individual protein spots were digested in gel and analysed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (Q1067R). Further analysis of other NS3 variants revealed phosphorylation of NS3.

Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain.

BACKGROUND: Hepatitis C virus (HCV) induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B) has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD) of NS4B is highly conserved in Hepaciviruses, though its function remains unknown. RESULTS: A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH). The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD) of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system. CONCLUSION: Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

Repair and polyadenylation of a naturally occurring hepatitis C virus 3' nontranslated region-shorter variant in selectable replicon cell lines.

The 3' nontranslated region (NTR) of the hepatitis C virus (HCV) genome is highly conserved and contains specific cis-acting RNA motifs that are essential in directing the viral replication machinery to initiate at the correct 3' end of the viral genome. Since the ends of viral genomes may be damaged by cellular RNases, preventing the initiation of viral RNA replication, stable RNA hairpin structures in the 3' NTR may also be essential in host defense against exoribonucleases. During 3'-terminal sequence analysis of serum samples of a patient with chronic hepatitis related to an HCV1b infection, a number of clones were obtained that were several nucleotides shorter at the extreme 3' end of the genome. These shorter 3' ends were engineered in selectable HCV replicons in order to enable the study of RNA replication in cell culture. When in vitro-transcribed subgenomic RNAs, containing shorter 3' ends, were introduced into Huh-7 cells, a few selectable colonies were obtained, and the 3' terminus of these subgenomic RNAs was sequenced. Interestingly, most genomes recovered from these colonies had regained the wild-type 3' ends, showing that HCV, like several other positive-stranded RNA viruses, has developed a strategy to repair deleted 3' end nucleotides. Furthermore, we found several genomes in these replicon colonies that contained a poly(A) tail and a short linker sequence preceding the poly(A) tail. After recloning and subsequent passage in Huh-7 cells, these poly(A) tails persisted and varied in length. In addition, the connecting linker became highly diverse in sequence and length, suggesting that these tails are actively replicated. The possible terminal repair mechanisms, including roles for the poly(A) tail addition, are discussed.

Exosomes contain ubiquitinated proteins.

Multivesicular bodies (MVB) are endosomal compartments that contain multiple vesicles, which derive from a delimiting membrane by inward budding. Incorporation of membrane proteins into the luminal vesicles requires, at least for some model proteins, monoubiquitination of their cytoplasmic domain. The ubiquitin tags are recognized by a sorting machinery, of which some components are also monoubiquitinated. The ubiquitin tags and the sorting machinery are both removed before the vesicles bud into the MVB lumen. MVB vesicles are therefore not expected to contain monoubiquitinated proteins. The MVB content is degraded upon fusion of MVB with lysosomes. In many cell types, however, MVB can also fuse with the plasma membrane, resulting in secretion of their luminal vesicles into the extracellular milieu. Such secreted vesicles are termed exosomes, and their protein composition should, due to their origin, be identical to that of MVB luminal vesicles. We here demonstrate that exosomes contain polyubiquitinated proteins, many of which are not integrated into the membrane and relatively enriched as compared to total cell lysates. These results suggest that a subset of polyubiquitinated cytoplasmic proteins is incorporated into the MVB pathway. The potential cell biological relevance of this observation is discussed. Furthermore, these data indicate that ubiquitinated proteins can serve as markers for exosomes.