Moonlighting glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is required for efficient hepatitis C virus and dengue virus infections in human Huh-7.5.1 cells.

Hijacking of cellular biosynthetic pathways by human enveloped viruses is a shared molecular event essential for the viral lifecycle. In this study, the accumulating evidence of the importance of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the host secretory pathway led us to hypothesize that this moonlighting enzyme could play a key role in the lifecycle steps of two important Flaviviridae members, hepatitis C virus (HCV) and dengue virus (DENV). We used short interfering RNA (siRNA)-mediated knockdown of human GAPDH in Huh-7.5.1 cells- both pre- and post-HCV infection- to demonstrate that GAPDH is a host factor for HCV infection. siRNA-induced GAPDH knockdown performed pre-HCV infection inhibits HCV core production in infected cells and leads to a decrease in infectivity of the HCV-infected cell supernatants. siRNA-induced GAPDH knockdown performed post-HCV infection does not have an effect on HCV core abundance in infected cells, but does lead to a decrease in infectivity of the HCV-infected cell supernatants. Exogenous expression of V5-tagged human GAPDH, pre- and post-infection, increases the infectivity of HCV-infected cell supernatants, suggesting a role for GAPDH during HCV post-replication steps. Interestingly, siRNA-induced GAPDH knockdown in HCV replicon-harbouring cells had no effect on viral RNA replication. Importantly, we confirmed the important role of GAPDH in the HCV lifecycle using Huh-7-derived stable GAPDH-knockdown clones. Finally, siRNA-induced GAPDH knockdown inhibits intracellular DENV-2 E glycoprotein production in infected cells. Collectively, our findings suggest that the moonlighting enzyme, GAPDH, is an important host factor for HCV infection, and they support its potential role in the DENV lifecycle.

A proteolytic fragment of Mcl-1 exhibits nuclear localization and regulates cell growth by interaction with Cdk1.

Mcl-1 (myeloid cell leukaemia-1) is a Bcl-2 family member with short-term pro-survival functions but whose other functions, demonstrated by embryonic lethality of knockout mice, do not involve apoptosis. In the present study, we show a cell-cycle-regulatory role of Mcl-1 involving a shortened form of the Mcl-1 polypeptide, primarily localized to the nucleus, which we call snMcl-1. snMcl-1 interacts with the cell-cycle-regulatory protein Cdk1 (cyclin-dependent kinase 1; also known as cdc2) in the nucleus, and Cdk1 bound to snMcl-1 was found to have a lower kinase activity. The interaction with Cdk1 occurs in the absence of its cyclin partners and is enhanced on treatment of cells with G2/M blocking agents, but not by G1/S blocking. The snMcl-1 polypeptide is present during S and G2 phases and is negligible in G1. Overexpression of human Mcl-1 in a murine myeloid progenitor cell line resulted in a lower rate of proliferation. Furthermore, Mcl-1-overexpressing cells had lower total Cdk1 kinase activity compared with parental cells, in both anti-Cdk1 and anti-cyclin B1 immunoprecipitates. The latter results suggest that binding to snMcl-1 alters the ability of Cdk1 to bind its conventional partner, cyclin B1. Given the important role of Cdk1 in progression through G2 and M phases, it is probable that the inhibition of Cdk1 activity accounts for the inhibitory effect of Mcl-1 on cell growth.

Development of a red-shifted fluorescence-based assay for SARS-coronavirus 3CL protease: identification of a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata.

SARS-coronavirus (SARS-CoV) encodes a main protease, 3CLpro, which plays an essential role in the viral life cycle and is currently the prime target for discovering new anti-coronavirus agents. In this article, we report our success in developing a novel red-shifted (RS) fluorescence-based assay for 3CLpro and its application for identifying small-molecule anti-SARS agents from marine organisms. We have synthesised and characterised the first generation of a red-shifted internally quenched fluorogenic substrate (RS-IQFS) for 3CLpro based on resonance energy transfer between the donor and acceptor pair CAL Fluor Red 610 and Black Hole Quencher-1 (Km and kcat values of 14 microM and 0.65 min-1). The RS-IQFS primary sequence was selected based on the results of our screening analysis of 3CLpro performed using a series of blue-shifted (BS)-IQFSs corresponding to the 3CLpro-mediated cleavage junctions of the SARS-CoV polyproteins. In contrast to BS-IQFSs, the RS-IQFS was not susceptible to fluorescence interference from coloured samples and allowed for successful screening of marine natural products and identification of a coumarin derivative, esculetin-4-carboxylic acid ethyl ester, a novel 3CLpro inhibitor (IC50=46 microM) and anti-SARS agent (EC50=112 microM; median toxic concentration>800 microM) from the tropical marine sponge Axinella corrugata.