Current Research

Viruses and the NFkappaB cascade

Viral infection leads to the activation of an array of signaling cascades. One of the most broadly studied; the NFkappaB signalosome cascade, is known to be strongly activated by the Hepatitis C virus (HCV). The laboratory investigates the effects of structural proteins, in particular capsid, on the NFkappaB cascade, focusing on its inhibitor IkappaBalpha. HCV is used for those studies as proof of principle. We are also investigating the effects of the Chikungunya virus, a member of the Togaviridae, on the nuclear translocation of members of the NFkappaB cascade.

As some of those members are phosphorylated by the COP9 signalosome (see below) we aim at understanding the relationship between viral infection, the NFkappaB signalosome and the COP9 signalosome.

(Above) Effect of HCV infection on well-established signaling cascades. JFH-1-infected cells were analyzed by intracellular staining for PhosphoFlow for p38MAPK, ERK and p65NFkB phosphorylation, one, three and five days post-infection.

(Left) Fluorescence microscopy analysis of cells infected with the JFH1 HCV strain. Infected cells were analyzed for NS3 (left panel) and Core and GRP78 Endoplasmic Reticulum resident protein (right panel).

Viruses and the COP9 Signalosome

The COP9 Signalosome, also referred to as CSN, is a eight/nine subunit complex originally discovered in Arabidopsis Thaliana, where it serves as a center for light-regulated genes. It shares structural homology with the 26S proteosomal lid and the Translation Initiation Factor 3 (eIF3), and seems to be highly conserved through evolution. Its role in mammalian cells is less than clear. Protein degradation via de-neddylation of cullins and direct or indirect roles in phosphorylation events make the CSN an intriguing complex for the study of signaling cascades. The lab focuses on the role of individual subunits and their connection to viral infection.

(Above) Purification of the CSN from T-cells. (A) Coomassie stain of the eluted fractions isolated from SBP-Citrine control and SBP-CSN1 cell line lysates. (B) Confirmation by Western blot of the presence of all the CSN subunits in the eluted fractions from the SBP-CSN1 lysate

(Left) Representation of the SBP-based pull-down. The technique was used for the purification of the COP9 Signalosome (CSN) and its binding partners

Monitoring Proteolytic Cleavage by the Virus

Most viruses rely on proteolytic cleavage for the maturation of their proteome, a crucial step in their life cycle. While for many human viral pathogens protease inhibitors are available, terrible side effects and high mutational rates driving resistance beg for novel inhibitors and assays that facilitate their discovery. With this in mind, the lab has developed an assay that monitors the activity of the HIV-1 protease in cells, in a reliable and robust manner. The assay can easily discriminate between cleavage and lack off based on the appearance of fluorescence. The use of retroviral technology for cell engineering allows us to obtain stable clonal populations. We further miniaturize the cell-based assays for drug discovery.

We are now adapting the assay to other important viral pathogens from the Flaviviridae family (HCV, DenV, WNV and ZIKV) and the Togaviridae family (CHIKV). Flaviviridae members embed their proteome in vesicle membranes, mostly the ER. This requires further engineering of the assay while keeping the biological activity of the viral protease intact. For Togaviridae we focus on both the ‘classical’ protease NSP2 and the Capsid, which has autocatalytic activity.

(Above) Flow cytometry analysis of selected clones. Gal4 and non functional Protease mutant (PRm) are used as controls. Induction (Dox) with/out the FDA-approved inhibitor Indinavir (IDV), proves the efficacy of the assay for monitoring proteolytic activity (Hilton et al. 2010).

Maturation of the viral proteome. HIV-1 proteome (Above) and HCV (Left) are processed by both virally-encoded and host-encoded proteases. Note that the only protein cleaved by host proteases in Retroviridae is Envelope while many are cleaved in Flaviviridae.

Monitoring Proteolytic Cleavage by the Host

While viral proteases are obvious targets for antivirals, the laboratory explores the cleavage of the viral proteome by the host as a possible additional target. Many viruses exploit the vesicles of the classical secretory pathway for the maturation of one (ex: Envelope protein for HIV-1) or many (ex: Flaviviridae) of their proteins. With this in mind, the laboratory has developed an assay that monitors cleavage within the classical secretory pathway. First developed to monitor the cleavage of the gp120/gp41 boundary of HIV-1 Envelope protein, it was then adapted to the premature membrane protein of Dengue virus (prM). The assay can easily discriminate between cleavage and lack off based on classical staining for flow cytometry. As with other assays developed in the lab, the use of retroviral technology for cell engineering allows us to obtain stable clonal populations, enabling miniaturization for drug discovery.

We are now adapting the assay to ZIKV,  another member of the Flaviviridae family. In addition, we are working on MMP-14, an important molecule involved in the extracellular matrix remodeling, with deleterious effects on tumor invasion and metastasis and other disease conditions. MMP-14 was chosen as a proof of principle to further adapt the assay to monitor cleavage at the cell surface rather than within the vesicles of the classical secretory pathway.

(Above) Fluorescence microscopy analysis of cells expressing MMP-14 substrate. Cells expressing GFP as control or an MMP-14 substrate engineered as a fusion with mCitrineshow clear localization of the substrate at the cell surface.

(Above) Flow cytometry analysis of clones harboring the assay. Naïve cells (top), cells expressing the wild-type Envboundary (middle) or the mutant version (bottom) were stained with anti-HA and anti-FLAG antibodies, allowing to clearly distinguish between cleaved and un-cleaved boundary.

(Above) A depiction of the assay that monitors cleavage during transport to the cell surface. Cells can be discriminated by 1. no transport, 2. transport, and cleavage or 3. transport but no cleavage, based on staining with no, anti-HA or anti-HA and anti-FLAG antibodies, respectively (Stolp, Z. et al. PLoSOne. 2013).

Multiplexing for Enhanced Applications

The use of retroviral technology for the stable expression of ectopic information in mammalian cells has been exploited in the laboratory for multiplexing applications. We use multiplexing through genetic bar-coding rather than classical multi-staining conditions. This has allowed us to analyze distinct HIV-1 clinically prevalent proteases engineered in the context of our assay, in the same sample. In addition, we have exploited multiplexing to analyze individual mixed samples carrying distinct viral targets or targets from diverse viruses.

To further demonstrate the power of multiplexing we have recently engineered a cell line that expresses both assays: the assay that monitor the activity of the viral protease and the assay that monitors cleavage by the host within the classical secretory pathway.

(Below) Genetically bar-coded cells for multiplexing – HIV-1 protease. Three clinically prevalent HIV-1 mutant proteases and wild-type protease are expressed in genetically bar-coded cells (non-fluorescent, E2-Crimson, td-Tomato and both). Flow cytometry analysis shows the four distinct populations in one sample. The histograms show the results in the presence of Dox and inhibitor (Darunavir), proving the utility of the multiplexed assay for drug screening.

(Left) Genetically bar-coded cells for multiplexing – Cleavage within the classical secretory pathway. Three individual clones expressing the wild-type or mutant HIV-1 envelope gp120/gp41 boundary, or the Dengue virus prMboundary where genetically bar-coded with no fluorescence or td-Tomato at two different intensities. Flow cytometry analysis of the mixed populations reveals the population positive for FLAG (HIV-1 Env-mut) (Smurthwaite C.  et al. J VisExp2015).

Cell-based Assays for Drug Discovery

The assays developed in the laboratory are aimed at helping shed light into the requirements for full proteolytic activity by the viral proteases or on the viral proteome. In addition, the laboratory exploits the assays in an attempt to speed up the early stages of drug discovery. The cellular nature of the assays and the pinpointed biological process they investigate facilitates targeted drug screening for the search of antivirals.

The laboratory thus engineers the cell-based assays as a platform for drug discovery. For that purpose we ensure the assays are miniaturized, adapted to 96 or 484-well plate format, and with a high Z score. In addition to pilot screens with chemical libraries such as the Prestwick Chemical Library (PCL), the laboratory is embarking in screening endeavors with collaborators.

(Above) Results of a screen with the PCL. A screen performed with the cell-based assay carrying the HIV-1 Env boundary or the DenV prM boundary revealed two compounds; A and B, that inhibited both HIV-1 and DenV cleavage, but only one compound; C, that was specific to DenV.

(Above) Miniaturization of the HIV-1 Env assay in a 96-well plate. The assay carrying the wild-type Envelope boundary was plated without (bottom half of plate) or with the pan-pro-convertase inhibitor DCK (top half of plate) in order to obtain the Z score of the assay.

(Above) Parameters set for a pilot screen aimed at finding inhibitors of prM cleavage. A stringent set of parameters was set to identify hits that block DenV prM cleavage but not HIV-1 Env (Stolp, Z. et al. JBS 2015).