Principal Investigator: Dr. Pablo Gastaminza, Centro Nacional de Biotecnologia-CSIC, Madrid, Spain
Objective
In hepatitis C virus (HCV) infected cells the administration of antiviral drugs leads to the reversion of virus induced alteration of the host cell structure. Imaging and analyses of the cellular processes that facilitate regaining normal subcellular structure, will progress state-of-the-art understanding of specific effects of the drug and help speed up the development of antiviral compounds against different viruses.
This makes an excellent use case for demonstrating the imaging potential of a lab-based SXM as it will extend a related study previously carried out at the ALBA synchrotron SXM (Perez Berna et al. 2016). Furthermore, there is an extensively characterized disease model readily available which provides excellent antivirals with demonstrated efficacy, known mechanism of action, and devoid of side effects on the host cells.
Concept
Chronic HCV infection induces structural alterations of mitochondria and ER. These changes caused by active HCV replication can be reversed by a prolonged treatment with a clinically approved direct-acting HCV antivirals (DAAs) e.g. combination of sofosbuvir and daclatasvir.
Our goal is to understand how virus-induced changes of cellular structures are reversed after the drug-induced block of infection. Recent studies have shown that various antiviral drugs lead to differential efficiency in the reversion of the structural alterations in HCV infected cells (Perez-Berna et. al. 2016). Our specific aim is to analyze the morphological changes in large ER-derived membranes and mitochondria.
To gain further insight into the mechanisms of cellular processes that govern recovery of transformed cellular components, we will use advanced cellular imaging e.g. SXM and data analysis of infected cells in the presence of cellular proteostasis machinery inhibitors in and well characterized antiviral molecules.
Methodology
Aim 1. Time-resolved structural assessment of the reversion of morphological alterations induced by HCV replication.
HCV-replicating cell lines (replicon cells) will be cultured directly on holey carbon gold Quantifoil (R2/2) grids, as previously described (Perez-Berna et. al. 2016). Replicons will be treated with antiviral doses (2X effective concentration 90; EC90) of compounds that target different components of the viral replicase: sofosbuvir (NS5B polymerase), daclatasvir (NS5A replicase component) or glecaprevir (viral NS3/4A protease) or combinations thereof for various time points and assessed by real-time quantitative PCR and Western-Blot.
In addition, we will monitor reversion of virus-induced host cell transcriptional alterations in order to monitor the kinetics needed for regaining normal cell homeostasis. Grids containing the treated replicon cells or parental Huh-7 cells, will undergo cryofixation as previously described and imaged using the prototype SXM.
Tomograms will be analysed by computing the fraction of damaged mitochondria and the degree of morphological alteration as previously described (Perez-Berna et. al. 2016). We will also compute the areas occupied by the virus-induced vesicular network, which is expected to be reduced as well as the emergence of mitophagosomes, which are readily distinguishable as mitochondria surrounded by an additional membrane.
Using this approach, we expect to be able to determine the sequence of events that lead to elimination of the virus-induced alterations by antiviral drugs used in the clinical setting and to relate them with the presence/absence of viral components and the reversion of virus-induced stress by monitoring virus-induced host gene expression.
Aim 2. Impact of the inhibition of major host cell proteostasis pathways in the reversion of the virus-induced structural alterations.
Once the major sequence of events leading to elimination of virus-induced aberrant subcellular structures has been established in Aim 1, we will manipulate the cultures pharmacologically (inhibitors) and genetically (CRISPR/Cas9), in combination with the aforementioned antiviral drugs. We expect to be able to define major contributions of the cellular pathways involved in the elimination of virus-induced aberrant structures as well as to identify intermediate stages of these processes that may be too dynamic to be observed otherwise.
Figure: Tri-dimensional reconstruction of whole-cell volumes of HCV replicon-bearing cell lines by cryo-SXT.
Control (A-C) and HCV replicon-bearing cell lines (E-G) were vitrified and subjected to cryo-SXT.Tile-scanned projections showing the area selected for SXT (square; panels A, E) as well as volume slices of the tomograms from the boxed areas (B, F) are shown. Color-coded segmentation of the selected areas identifying different organelles is presented in control (C) and HCV-replicating cells (G).
Panels D and H display similar alterations observed using chemically-fixed stained cell sections observed by TEM. Yellow arrows indicate putative lysosomes/lipid droplets. Scale bars: 10 µm (cryo-SXT projections) 0.5 µm (cryo-SXT slices) and 2 µm (TEM). Published in ACS Nano 2016, 10, 7, 6597-6611