From structural regulation to cellular function : a multiscale view on the human TRPML2 ion channel and its role in Zika virus infection

Abstract

Zika virus (ZIKV) gained public attention during a major outbreak in the Americas in 2015/2016 when the infection was first associated with severe neurological complications, such as neonatal microcephaly. At that time, the epidemic was even declared as a public health emergency of international concern (PHEIC) by the WHO (World Health Organisation). Until now, no specific treatment or prophylactic vaccine is available. Therefore, a more in-depth knowledge about the Zika virus life cycle is required to identify host factors, which can be targeted for the development of novel drug candidates. In recent years, ion channels have emerged as important modulators of viral infections. The human cation channel TRPML2 (transient receptor potential mucolipin 2) is located in the membranes of the endolysosomal system, which plays an important role in the Zika virus life cycle. In this thesis, the potential to address TRPML2 as an antiviral target against ZIKV was investigated. Treatment of human A549 cells with the synthetic TRPML2 agonist ML2-SA1 reduced intracellular ZIKV mRNA (messenger ribonucleic acid), protein levels and amount of released viral particles of two ZIKV strains (ZIKV French Polynesia and ZIKV Uganda) in vitro. Confocal laser scanning microscopy (cLSM) showed increased acidification of vesicular structures and super-resolution microscopy revealed enlarged size of multivesicular bodies (MVBs). Inhibition of lysosomal degradation did not lead to intracellular ZIKV accumulation, indicating that the antiviral effect of ML2-SA1 is not caused by increased ZIKV degradation in lysosomal compartments. Moreover, time-of-drug-addition experiments revealed that ML2-SA1 does not affect early stages of the ZIKV life cycle (such as entry), but later stages, for example viral replication. The effect on ZIKV replication was confirmed via experiments with a ZIKV Renilla luciferase reporter virus. A potential cause of the antiviral effect ML2-SA1 might be changes in the intracellular cholesterol distribution, which is an important factor of ZIKV replication. In fact, cLSM analyses revealed intracellular cholesterol accumulations in ML2-SA1 treated cells. Investigations on proteins involved in complex biological processes, such as viral infections, require a profound understanding of the (structural) regulation of these proteins. TRPML channels are tetramers, one protomer consists of six transmembrane-spanning helices, the cytosolic N- and C-termini and the extracytosolic/lumenal domain (ELD), which connects the first two transmembrane helices and is the structural hallmark of the TRPML family. In this thesis, a potential function of the ELD for the structural regulation of human TRPML2 was analyzed. The ELD faces the endolysosomal lumen where, depending on whether the channel resides in early or late endosomes or lysosomes, it encounters variations in pH values and ion concentrations. Isothermal titration calorimetry (ITC) revealed that calcium interacts with an acidic amino acid motif in the central pore loop of the ELD in a pH-dependent manner. At neutral pH, calcium binds to the ELD potentially forming a block for other cations, while at acidic pH the decreased calcium affinity might allow unhindered cation flux. This indicates that the ELD serves an important function in the endolysosomal compartment-specific regulation of TRPML2. However, interactions with Ca2+ did not induce significant structural changes of the isolated TRPML2 ELD as assessed via NMR (nuclear magnetic resonance) and EPR (electron paramagnetic resonance) spectroscopy.