Development of an iVLP assay with na¨ıve target cells

To date all iVLP systems rely on RNP components in p1, which are provided by either transfection or helper virus infection. Since, in theory, delivery of one minigenome is enough to initiate replication and transcription, these systems produce high signals. How-ever, they do not model the initial transcription of negative-sense RNA in target cells by the RNP components packaged in the virion, a step indispensable in a real infection. We, therefore, decided to develop an iVLP system that is independent of the presence of RNP components in p1.

Figure 25:VP40-R134A in an iVLP assay with pretransfected target cells. iVLPs were produced with either VP40-WT or VP40-R134A. 3 days p.t. the iVLP-containing supernatant of p0 was transferred to 293T target cells (p1) pretransfected with all RNP components. After 3 days reporter activity, reflecting delivery of the minigenome and, therefore, packaging, budding and entry, was determined in these cells.

3.1.5.1 Timecourse of reporter activity in p1 In a first pilot experiment the protocol for an iVLP assay with pretransfected target cells was adopted; however, na¨ıve 293T and VeroE6 cells were used as target cells. In this first experiment na¨ıve VeroE6 cells were much more susceptible to transfection than na¨ıve 293T cells; however, in both cases absolute signals were very low when compared to those obtained using an iVLP assay with pretransfected target cells (data not shown). We, therefore, decided to perform timecourse experiments to determine the optimal timepoint for analyzing p1 cells for reporter activity.

Figure 26: iVLPs assay with na¨ıve target cells: timecourse.iVLPs were produced and used to infect na¨ıve VeroE6 cells. p1 cells were harvested at days 1, 2 and 3 p.i. and reporter activity was determined.

Interestingly, reporter activity increased from day 1 to day 2, but then dropped down again (Figure 26). This can be explained by the fact that after entry and uncoating initial transcription takes place, which leads to the accumulation of reporter protein. However, since RNP components as well as minigenome are only available in very small amounts, and most likely no replication of the minigenome takes place (see section 1.5), this process

comes to a stop after some time, and the previously produced reporter protein is turned over via the degradation pathways in the target cells.

3.1.5.2 Infection of different target cell types To further optimize the system, different target cell types were tested. In particular, we compared 293T cells, which have been used in previous iVLP assays as target cells, VeroE6 cells, which are usually used in our laboratory to propagate EBOV, and human macrophages, which are known to be natural target cells for EBOV.

Figure 27: iVLPs assay with na¨ıve target cells: cell lines. iVLPs were produced and used to infect VeroE6 and 293T cells as well as human macrophages (M0). After 2 days reporter activity was determined.

As already observed in our pilot experiment, infection of 293T cells with iVLPs resulted in an about 6 × lower signal than infection of Vero E6 cells (17% ± 6%; n=3). Also, infection of human primary macrophages produced lower reporter levels than infection of VeroE6 cells; however, only macrophages from one donor were tested.

3.1.5.3 Further characterization of the iVLP assay with na¨ıve target cells To ensure that reporter activity in p1 was not due to unspecific transfer of reporter protein inside iVLPs, the iVLP producing p0 cells were cotransfected with a plasmid encoding Firefly luciferase under the control of an eukaryotic promoter. If reporter activity in p1 was due to unspecific transfer of reporter protein from p0 to p1, there should be no drop in the ratio of Firefly to Renilla from p0 to p1, since unspecific packaging of Renilla and Firefly luciferase would be expected to occur equally.

We observed a drop in the ratio of Firefly luciferase to Renilla luciferase of about 12 × (4.6‰± 1.5‰vs. 0.4‰± 0.1‰; n=4). This indicates that reporter activity in p1 is not

Figure 28: Specific transfer of minigenome in an iVLP assay with na¨ıve target cells. To ensure that reporter activity in p1 is not due to unspecific transfer of reporter protein, the ratio of minigenome encoded Renilla luciferase and plasmid encoded Firefly control luciferase was determined in p0 and p1.

due to unspecific transfer of reporter protein, but rather caused by transfer of minigenome and subsequent transcription of minigenome and translation of the reporter mRNA in p1.

We also compared the absolute signal strength between iVLP assays with pretransfected and na¨ıve target cells (Table 3). It is interesting to note that a positive signal in an iVLP assay with pretransfected 293T target cells is about 100 × higher than a positive signal in an iVLP assay with na¨ıve VeroE6 target cells. Also, a negative signal in an iVLP assay with pretransfected target cells is of similar strength than a positive signal in an iVLP assay with na¨ıve target cells.

cell type sample pretransfected p1 na¨ıve p1 293T -40 1.8×10⁵±5.7×10⁴ 7.4×10²±3.6×10² 293T WT 9.3×10⁶±1.8×10⁵ 1.8×10⁴±8.3×10³

Vero -40 nd 7.1×10²±4.9×10²

Vero WT 2.5×10⁶± nd 1.3×10⁵±8.4×10⁴

Table 3: Absolute signals in iVLP assays. Signals are given in RLU/s/well. nd = not determined.

Finally, we wanted to demonstrate that filamentous particles and not spherical particles, which are usually found in high amounts in VLP preparations, are responsible for transfer of minigenomes. We, therefore produced iVLPs and purified them over a sucrose cushion and a Nycodenz gradient (see sections 2.4.4 and 2.4.5). We then pooled fractions 1-3 and 4-6, which contain the spherical and filamentous particles, respectively (see section 2.4.5), pelleted the iVLPs by ultracentrifugation for 1 hour at 21000 × g in an SW41 rotor, resuspended the pellet in 4 ml DMEM_5%FBS and used this suspension to infect na¨ıve VeroE6 cells.

Figure 29:iVLP assay with Nycodenz gradient separated iVLPs. iVLPs were produced and purified over a sucrose cushion and a Nycodenz gradient. Fractions 1-3 (spherical particles) or 4-6 (filamentous particles) were used to infect na¨ıve VeroE6 cells.

The reporter signal in cells infected with particles from fractions 4-6 was about 10×higher than the signal in cells infected with particles from fractions 1-3, showing that filamentous particles are responsible for the transfer of reporter activity (Figure 29).

3.1.6 Role of VP40 octamerization in an iVLP assay with na¨ıve target cells