Search

The Ebola virus (EBOV) has emerged as a significant global health concern, notably during the 2013–2016 outbreak in West Africa. Despite the clinical approval of two EBOV antibody drugs, there is an urgent need for more diverse and effective antiviral drugs, along with comprehensive understanding of viral-host interactions. In this study, we harnessed a biologically contained EBOVΔVP30-EGFP cell culture model which could recapitulate the entire viral life cycle, to conduct a genome-wide CRISPR/Cas9 screen. Through this, we identified PIK3C3 (phosphatidylinositide 3-kinase) and SLC39A9 (zinc transporter) as crucial host factors for EBOV infection. Genetic depletion of SLC39A9 and PIK3C3 lead to reduction of EBOV entry, but not impact viral genome replication, suggesting that SLC39A9 and PIK3C3 act as entry factors, facilitating viral entry into host cells. Moreover, PIK3C3 kinase activity is indispensable for the internalization of EBOV virions, presumably through the regulation of endocytic and autophagic membrane traffic, which has been previously recognized as essential for EBOV internalization. Notably, our study demonstrated that PIK3C3 kinase inhibitor could effectively block EBOV infection, underscoring PIK3C3 as a promising drug target. Furthermore, biochemical analysis showed that recombinant SLC39A9 protein could directly bind viral GP protein, which further promotes the interaction of viral GP protein with cellular receptor NPC1. These findings suggests that SLC39A9 plays dual roles in EBOV entry. Initially, it serves as an attachment factor during the early entry phase by engaging with the viral GP protein. Subsequently, SLC39A9 functions an adaptor protein, facilitating the interaction between virions and the NPC1 receptor during the late entry phase, prior to cathepsin cleavage on the viral GP. In summary, this study offers novel insights into virus-host interactions, contributing valuable information for the development of new therapies against EBOV infection. Author summary: Ebola virus (EBOV) remains a significant global health threat. The limited availability of high-containment (BSL-4) facilities hinders our understanding of the interaction of virus with host cells and the development of effective treatments. To address this, we utilized a biologically contained EBOVΔVP30-EGFP model to identify critical host factors necessary for EBOV infection through a genome-wide CRISPR/Cas9 screen conducted at BSL-2 lab. Our study revealed that two proteins, PIK3C3 and SLC39A9, are essential for the virus to enter host cells. We found that PIK3C3 is required for EBOV endosomal trafficking, and inhibiting its kinase activity can significantly reduce infection, making it a promising target for antiviral therapy. Additionally, we discovered that SLC39A9 facilitates viral entry by interacting with the viral glycoprotein (GP) and enhancing its binding to the cell's NPC1 receptor, rather than through its usual role in zinc transport. These findings offer new insights into how EBOV enters cells and highlight potential targets for developing therapies to combat Ebola virus infection. [ABSTRACT FROM AUTHOR]

As members of the membrane-bound O-acyltransferase (MBOAT) enzyme family, acyl-coenzyme A:cholesterol acyltransferases (ACATs) catalyse the transfer of an acyl group from acyl-coenzyme A to cholesterol to generate cholesteryl ester, the primary form in which cholesterol is stored in cells and transported in plasma.sup.1. ACATs have gained attention as potential drug targets for the treatment of diseases such as atherosclerosis, Alzheimer's disease and cancer.sup.2-7. Here we present the cryo-electron microscopy structure of human ACAT1 as a dimer of dimers. Each protomer consists of nine transmembrane segments, which enclose a cytosolic tunnel and a transmembrane tunnel that converge at the predicted catalytic site. Evidence from structure-guided mutational analyses suggests that acyl-coenzyme A enters the active site through the cytosolic tunnel, whereas cholesterol may enter from the side through the transmembrane tunnel. This structural and biochemical characterization helps to rationalize the preference of ACAT1 for unsaturated acyl chains, and provides insight into the catalytic mechanism of enzymes within the MBOAT family.sup.8. The structure of human ACAT1, which catalyses the transfer of an acyl group from acyl-coenzyme A to cholesterol to form cholesteryl ester, is resolved by cryo-electron microscopy., Author(s): Hongwu Qian [sup.1] , Xin Zhao [sup.2] , Renhong Yan [sup.3] [sup.4] , Xia Yao [sup.1] , Shuai Gao [sup.1] , Xue Sun [sup.5] , Ximing Du [sup.6] , [...]

Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans.sup.1. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins.sup.2,3. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity. The structure of human diacylglycerol O-acyltransferase 1, a membrane protein that synthesizes triacylglycerides, is solved with cryo-electron microscopy, providing insight into its function and mechanism of enzymatic activity., Author(s): Lie Wang [sup.1] , Hongwu Qian [sup.2] , Yin Nian [sup.1] [sup.4] , Yimo Han [sup.2] [sup.5] , Zhenning Ren [sup.1] , Hanzhi Zhang [sup.1] , Liya Hu [sup.1] [...]

Niemann-Pick C1 (NPC1) is a membrane protein required for the transport of low-density lipoprotein (LDL)-derived cholesterol from endosomes and lysosomes to the other organelles. Here, we describe the recombinant protein expression, purification, and characterization of the human NPC1. The protein is transiently expressed in human embryonic kidney (HEK) cells. Our purification protocol describes the steps to obtain a pure and homogeneous NPC1 protein. Niemann-Pick C2 (NPC2) is a small soluble protein, which mediates cholesterol transport in tandem with NPC1. Finally, we also describe two biochemical approaches to characterize NPC1 function in vitro-a cholesterol transfer assay from purified NPC2 to NPC1 and a binding assay between NPC1 and NPC2.