Outlook

So far, a direct relation between FADS2 and ApoE has neither been described. In the future, the source of enriched ApoE should be elucidated to discriminate between newly synthesized ApoE and recycled ApoE obtained from lipoprotein uptake (Heeren et al., 2006).

The increased surface expression of SR-BI and the increased amount of ApoE, both involved in lipid transport through lipoproteins, suggest that the cell tries to compensate the alterations in the lipid composition caused by the FADS2 inhibition and thereby affects the HCV life cycle.

3. Discussion 83

should be determined. This would help to understand whether HCV induces precise changes of compartments that are known to be important for its replication, such as lipid droplets and the ER, or whether HCV-infection induces global cellular changes affecting all compartments in a similar manner.

Lipidomics will also help to further understand the decline in sphingomyelins observed in this study and previously by others (Diamond et al., 2010). Sphingomyelins are synthesized from ceramides and can be converted back (Ridgway ed., 2015). Therefore, the amount of ceramides in correlation to sphingomyelins will provide information whether the sphingo-myelin metabolism is manipulated in HCV-infected cells. Diamond et al. have additionally suggested that the decline in sphingomyelins is due to their incorporation into the viral particles. This could be studied by additionally analyzing the supernatants from infected and uninfected cells by lipidomics.

One hypothesis for the increase in free fatty acids is an upregulated fatty acid uptake.

Different tools are available to study fatty acid uptake. First of all, the expression of the fatty acid transporter CD36 on the cells surface should be tested on HCV-infected and uninfected Huh7.5 cells by antibody staining, following the analysis by flow cytometry. Furthermore, the fatty acid uptake can be measured by supplementation of fluorescently labeled fatty acids, for example, a Bodipy conjugate. The evaluation can be performed by flow cytometry or in a plate reader (Dubikovskaya et al., 2014). Alternatively, click chemistry can be used to determine the uptake of fatty acids. For this protocol alkyne-fatty acids are supplemented to the cell. Alkyne-fatty acids carry an alkyne group at their methyl terminus, which has the advantage that these fatty acids are not fused to a large fluorophore which may impair their physiology. Upon lipid extraction, the alkyne-group is conjugated with a fluorophore by a click reaction, followed by thin layer chromatography. This method additionally allows the study of the incorporation of fatty acids into other lipid classes, for example, phosphatidylcholines and triglycerides, to elucidate potential substrate preferences (Thiele et al., 2012). The click-chemistry approach would additionally allow time course experiments to study the kinetics and selectivity of the fatty acid incorporation. The kinetics may help to elucidate whether lipid synthesis is decelerated upon infection. Here, the fatty acids identified by mass spectrometry would be of foremost interest. Instead of analyzing the incorporation the lipolysis can be measured with this approach in a pulse-chase setup. A prolonged treatment with a mixture of different alkyne-fatty acids should result in an incorporation of the alkyne-fatty acids into lipids. The loss of alkyne-fatty acid chains upon removal of the alkyne-fatty acids can give hints about the kinetics of lipolysis.

As mentioned previously, mitochondria and peroxisomes have different substrate preferences for β-oxidation. This leads to the question whether the contribution to β-oxidation of one organelle is altered in HCV-infected cells. The peroxisomal β-oxidation can be

measured by the conversion of the fatty acid C24:0 to C16:0, which exclusively occurs in the peroxisomes. Deuterium labeled C24:0 enables the identification by mass spectrometry

(Kemp et al., 2004).

In this study, various knockdown experiments of elongases and desaturases had an impact on viral replication. Therefore, further experiments focusing on the distinct role of these enzymes in the viral life cycle should be performed. One desaturase, FADS2, was already taken under further investigation but there are still several open questions. So far, it was not investigated whether the surface receptor SR-BI is also upregulated upon a 3 day pretreatment with the FADS2 inhibitor SC-26196. Next, it should be elucidated whether the increased surface expression of SR-BI on 1 day and potentially 3 days post FADS2 inhibition indeed results in an increased virus uptake. Therefore, entry assays should be performed with commercially available branched DNA assays to stain viral RNA at early time points post infection following the analysis by fluorescence microscopy (Neufeldt et al., 2016). SR-BI is known to function in lipoprotein uptake, mainly the cholesterol rich HDL (Ridgway ed., 2015).

Therefore, the question arises if FADS2 inhibition increases the intracellular cholesterol or triglyceride level by upregulation of lipoprotein import. As shown in this study, alterations in the intracellular cholesterol level can affect the virus production. An easy way to test the intracellular cholesterol and triglyceride level upon FADS2 inhibition in uninfected and HCV-infected cells would be the analysis by commercially available colorimetric quantification kits.

A second connection of FADS2 inhibition and lipoproteins was the intracellular increase of ApoE. It should further be tested by RT-qPCR whether the ApoE mRNA expression is increased, the protein stabilized, or if ApoE is derived from lipoprotein uptake. Moreover, it would be interesting to determine the localization of ApoE by immunofluorescence co-staining with PDI (ER-marker), G130 (Golgi marker), and recycling endosomes (Rab11). It is of great importance to determine the intracellular and extracellular ApoE levels upon FADS2 inhibition in HCV-infected cells. Additionally, the intracellular localization of the viral protein core to lipid droplets should be determined since changes in the lipid composition might also influence the binding properties of membrane anchored proteins. Overall, solving the mechanism of how FADS2 inhibition impairs virus production will provide further insights to the so far not well understood late steps of the viral life cycle.

Understanding the way in which HCV interferes with the lipid metabolism will raise further options to intervene in the viral replication and to identify potential cellular drug targets.

Although highly effective direct acting antivirals are available, the problem of drug resistance due to the error-prone RNA synthesis remains. Cellular drug targets have a lower potential to give rise to drug resistant viruses, therefore lipid modulating enzymes can be considered as potential targets to indirectly inhibit viral replication (Li et al., 2015).