Visualizing and quantifying the differential cleavages of the eukaryotic translation initiation factors eIF4GI and eIF4GII in the enterovirus‐infected cell

Abstract

Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A(pro)). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET-based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP(2)) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real-time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A(pro) catalytic activity, irrespective of other viral-encoded protease, the activated caspases or general inhibition of protein synthesis in the EV-infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease-substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus-induced host translation inhibition.

Modulation of Glucagon-like Peptide-1 (GLP-1) Potency by Endocannabinoid-like Lipids Represents a Novel Mode of Regulating GLP-1 Receptor Signaling

“…We probed the potential conformation of GLP-1(7-36) amide based on its susceptibility to trypsin digestion. The two trypsin cleavage products of GLP-1 amide are an inactive GLP-1 (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and a partially active GLP-1(7-34) (Fig. 8A) (32).…”

A Survey of Visualization for Live Cell Imaging

“…Spatial embedding . At a basic level, users are interested in cell growth, cell movement (motility), cell shape (morphology), and cell reproduction (proliferation) as captured by phenotypes visible in the acquired images [GME03, GBBS09, HLLK09, PM07]. Further objectives include understanding the influence of cell positions and migration, that is, their impact on cell fate, tissue formation, organs, and organisms [CBI*08, FHWL12].…”

“…Spatial embedding . Imaging is performed at cellular or sub‐cellular resolution with sub‐cellular structures, such as nuclei [GBBS09], or whole cells [HLLK09, PM07], marked by fluorescent proteins. Cells may be imaged at different light excitation wavelengths to record different fluorescent labels [HLLK09].…”

“…Imaging is performed at cellular or sub‐cellular resolution with sub‐cellular structures, such as nuclei [GBBS09], or whole cells [HLLK09, PM07], marked by fluorescent proteins. Cells may be imaged at different light excitation wavelengths to record different fluorescent labels [HLLK09]. Such sequences of ‘raw’ time‐lapse images are transformed to obtain the following data: temporal sequences of 2D fluorescent intensity maps, each containing measured fluorescence intensities for every pixel in the corresponding image [GBBS09]; temporal sequences of 2D images or 3D volumes [CBI*08, FHWL12]; or animated movies constructed by sequentially animating through the time‐lapse images [PM07].…”

Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells

“…The polyprotein synthesized subsequently to virus entry and uncoating is processed during and following translation by viral proteinases (2A pro , 3C pro and 3CD pro ) into precursor polypeptides: P1 that will constitute capsid proteins (VP1-4); P2 that will be cleaved to produce 2A pro as well as 2BC and 2C, proteins involved in cytoplasmic membrane reorganization and genomic replication; and P3, which is cleaved into proteins involved in protein processing and viral RNA replication (e.g., 3C pro , 3CD pro , VPg, and 3D pol ). In enterovirus-infected cells, translation of 5 capped cellular mRNAs is shut-off due to 2A pro cleavage of the eIF4G component of the eIF4F cap-binding protein complex (Hsu et al, 2009). CV-B translation is, however, initiated using an internal ribosome entry site (IRES) in the 5 NCR in a cap-independent manner (Fernández-Miragall et al, 2009).…”