DENV type 4 Envelope protein (45 kDa)

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.

The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins fromDENVand ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 degrees C). At 23 degrees C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 degrees C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from < 1 mu M at 25 degrees C to 50 mu M at 41 degrees C, due to a large exothermic enthalpy of binding of similar to 79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 degrees C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine.