Tewis Bouwmeester

Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

ABSTRACT Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.

Biochemical characterization of USP7 reveals post‐translational modification sites and structural requirements for substrate processing and subcellular localization

Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130‐kDa protein with a cysteine peptidase core, N‐ and C‐terminal domains required for protein–protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post‐translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N‐ and C‐terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N‐terminal domain are over 100‐fold less active than the full length enzyme, whereas a construct including the C‐terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C‐terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N‐terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzymes first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor‐like region (amino acids 70–205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.

Structure, expression and in vitro functional characterization of a novel RNA binding zinc finger protein from Xenopus.

Large multigene families of zinc finger proteins are expressed in vertebrates. One way of approaching their function is to characterize their structure, expression and biochemical properties. XFG 5‐1 is a Xenopus zinc finger protein which is widely transcribed in oocytes, embryos and adult tissues. It carries a novel, non‐finger repeat structure, which is common to a subfamily of Xenopus zinc finger proteins. The bacterially expressed protein exhibits specific RNA homopolymer binding activities with the zinc finger domain being sufficient for this ability. These findings suggest that XFG 5‐1 serves a general biological function involving its RNA binding capacity.

The FAR domain defines a new Xenopus laevis zinc finger protein subfamily with specific RNA homopolymer binding activity

The zinc finger motif defines a large superfamily of nucleic acid binding proteins. Conserved amino acid sequence elements associated with structurally variant zinc finger clusters define subfamilies of zinc finger proteins (ZFPs). The FAR domain (Finger Associated Repeats) is a novel type of repeat element found at the amino-terminus in a subfamily of Xenopus laevis ZFPs. Northern blot analyses of three different members of the FAR subfamily (XFO 6, XFO 9-3 and XFG 68) revealed that each of these genes is transcribed during oogenesis, embryogenesis and in all investigated tissues of adult animals thereby indicating a ubiquitous distribution of transcripts. All FAR-ZFPs tested so far have specific RNA homopolymer binding activity; they associate preferentially with poly(U). The FAR repeats possess limited primary sequence homology with a sequence in the nucleolar shuttling protein NO38, within a region that contains a casein kinase II phosphorylation site.

The maternal store of zinc finger protein encoding mRNAs in fully grown Xenopus oocytes is not required for early embryogenesis.

A large family of C2H2 (Krüppel‐like) zinc finger protein genes is maternally transcribed in Xenopus oocytes; many of the corresponding mRNAs are actively translated post‐fertilization, before the onset of zygotic activation of transcription. With the aim of asking if any of these stored mRNAs have a function in Xenopus development, we made use of antisense oligonucleotide mediated, targeted RNA destruction. Injected oocytes lose the entire pool of C2H2 zinc finger protein encoding mRNAs. They are indistinguishable from control oocytes in their abilities to mature in vitro and to be fertilized in vitro. Embryos generated from such oocytes develop normally until tadpole stage. These findings do not rule out the possibility that C2H2 zinc finger protein genes are involved in developmental control in Xenopus. However, they do suggest that the biological function for at least some of the early expressed zinc finger proteins in Xenopus differs in important aspects from the way Krüppel or other DNA binding factors act as developmental regulators in Drosophila.

Screening of Intestinal Crypt Organoids: A Simple Readout for Complex Biology

Oral and intestinal mucositis is a debilitating side effect of radiation treatment. A mouse model of radiation-induced mucositis leads to weight loss and tissue damage, reflecting the human ailment as it responds to keratinocyte growth factor (KGF), the standard-of-care treatment. Cultured intestinal crypt organoids allowed the development of an assay monitoring the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay resembles the mouse model as KGF and roof plate-specific spondin-1 (RSPO1) enhanced crypt organoid recovery following radiation. Screening identified compounds that increased the survival of organoids postradiation. Testing of these compounds revealed that the organoids changed their responses over time. Unbiased transcriptome analysis was performed on crypt organoid cultures at various time points in culture to investigate this adaptive behavior. A number of genes and pathways were found to be modulated over time, providing a rationale for the altered sensitivity of the organoid cultures. This report describes an in vitro assay that reflects aspects of human disease. The assay was used to identify bioactive compounds, which served as probes to interrogate the biology of crypt organoids over prolonged culture. The pathways that are changing over time may offer potential targets for treatment of mucositis.