Budding of many enveloped NS RNA virus require interactions between cellular proteins and the VBP. Interfering with these interactions presents an opportunity to both understand the cellular biology of virus budding and to identify potential novel antiviral targets and drugs. We sought to develop a Z budding assay that would facilitate the identification of small molecule inhibitors of these interactions and therefore arenavirus budding. To this end we tested the use of LASV-Z-luciferase fusion proteins as tools to develop an assay to rapidly and quantitatively measure Z-mediated budding. A chimeric LASV-Z-FLuc protein showed 1000-fold lower activity in TCS than in cells, indicating that only small amounts of LASV-Z-FLuc could bud into TCS. In contrast, about 90% of GLuc activity in LASV-Z(WT)-GLuc expressing cells was in TCS. Moreover, levels of TCS luciferase activity were dramatically reduced (≥8-fold) in cells transfected with LASV-Z(G2A)-GLuc compared to LASV-Z(WT)-GLuc transfected cells. Previously, we showed that LASV Z tagged at its C-terminus with HA (LASV-Z(WT)-HA) has WT Z budding activity as determined by levels of Z present in VLP collected from TCS of transfected cells (Perez, Craven, and de la Torre, 2003
). Using the same biochemical assay, we observed that LASV-Z(WT)-HA and LASV-Z(WT)-GLuc had similar budding activity. These results argue strongly that the LASV-Z(WT)-GLuc chimera is functionally equivalent to WT Z in budding activity and therefore levels of Z-mediated budding can be assessed based on GLuc activity in TCS from Z-GLuc transfected cells.
The reasons why LASV-Z(WT)-FLuc was severely impaired in its budding activity remain to be determined. However, a significant difference is the larger size (three times bigger) of FLuc compared to GLuc. Notably, a TAP-tagged Z (LASV-Z(WT)-TAP) retained WT Z budding activity (MP and JCT, unpublished data), and interestingly the TAP tag and GLuc are similar in size (185aa and 168aa respectively), while FLuc is 550aa. It is therefore plausible that the budding efficiency of chimeric Z proteins may depend on the size of the fusion partner added to Z.
GLuc activity from cells expressing either GLuc-ΔSP or LASV-Z(G2A)-GLuc were 1000-fold lower than GLuc WT. These results showed that consistent with previous results GLuc secretion requires its SP (Tannous et al., 2005
), but also that a Z mutant defective in budding cannot functionally substitute for the SP of GLuc. We also observed that levels of GLuc in TCS from LASV-Z(WT)-GLuc transfected cells were consistently lower (50–60 fold) compared to those in TCS from GLuc WT transfected cells. Differences in the mechanism by which GLuc and LASV-Z(WT)-GLuc are released from cells likely contributed to these findings. The former involves secretion mediated by the native SP in WT GLuc (Knappskog et al., 2007
), whereas the latter occurs via budding mediated by WT Z. Together, these data indicate that GLuc release from cells depends on the efficiency of SP-mediated secretion, whereas levels of released Z-GLuc chimera proteins into TCS reflect the relative efficiency of Z-mediated budding.
When LASV-Z(WT)-GLuc and LASV-Z(G2A)-GLuc were used as positive and negative controls, respectively, our assay had Z′ values significantly >0.5, thus indicating that our assay has excellent features for its use in HTS.
We showed that WT GLuc expression and secretion was somewhat affected, though modestly, by OHM (). These results suggest some effects of OHM that are independent of inhibiting myristoylation. Since GLuc is released from cells via secretion, OHM could have affected one or more steps in secretion by a mechanism that we do not presently understand. To address the possibility that GLuc could be myristoylated and therefore be susceptible to OHM, the amino acid sequence for GLuc was submitted to the prediction program NMT-The Predictor
), which is available through the ExPASy (Ex
stem) proteomics server (http://www.expasy.org/
) of the Swiss Institute of Bioinformatics (SIB). We found no myristoylation sites within GLuc (data not shown), supporting the notion that OHM was somehow affecting secretion. We had also observed a decrease in intracellular GLuc-ΔSP following OHM treatment (). This effect was similar to the effects of OHM on intracellular expression of the Z-GLuc chimeras, suggesting that the effect of OHM on intracellular Z-GLuc was likely due to effects on expression of GLuc-ΔSP. Interestingly, these findings suggest that GLuc intracellular expression could be used to assess pleiotropic effects of small molecules to more general processes such cell viability, PolII-mediated transcription, or both, which provides this assay with an important internal control to normalize values of Z-GLuc budding.
The susceptibility of LASV-Z(WT)-GLuc to RNAi knockdown of Tsg101 indicates that Z-GLuc was also sensitive to disruption of late domain function. While the effect of Tsg101-specific siRNA on Z-GLuc budding was modest compared to previously published findings, the results presented here reflect effects following a single siRNA transfection, whereas stronger effects have been observed with two transfections of siRNA prior to transfection with the VBP of choice (Garrus et al., 2001
; Urata et al., 2006
). These results suggest that with refinement this assay could be adapted for use in siRNA-based HTS approaches to identify additional cellular proteins necessary for Z-mediated budding. The same screens might also identify cellular proteins involved in Z-mediated budding via interactions in regions outside the L-domains of Z. Therefore, cellular proteins identified using this method would need to be evaluated for their L-domain dependent or independent interaction with Z.
We have shown that GLuc fusion to the arenavirus LASV Z matrix protein results in a chimeric protein with similar budding properties to native Z. This would allow the use of BSL2 facilities to implement HTS aimed at identifying inhibitors of budding of LASV and HF arenaviruses, for which use of live virus requires BSL4 facilities. Importantly, because LASV Z protein contains two late domain motifs, the LASV Z-GLuc chimera can be used to identify targets necessary for VBP-mediated budding via either domain. Further, we think that similar GLuc chimeras could be done with VBP from other highly pathogenic riboviruses, making it possible to conduct assays under BSL-2 conditions to rapidly identify inhibitors of virus budding that might be developed into antiviral drugs. Likewise, the use of VBP-GLuc based assays should facilitate the use of siRNA-based HTS to globally identify host cellular proteins involved in VBP-mediated budding in different RNA viruses, including important human pathogens.