A potential mechanism for Wolbachia pathogen blocking
Wolbachia was dragged out of the obscurity of phenomenology literature and into the limelight of medical relevance by two high impact publications in 2008 (here and here). Both revealed that Wolbachia can protect insects (in this case, Drosophila melanogaster) from RNA viruses, resulting in a reduction of viral titers during the course of infection or increasing host survival. This trait has obvious vector control applications and indeed, Wolbachia-infected mosquitos are being released in different parts of the world to control the spread of Dengue, among other human pathogens. Although we are currently using Wolbachia to control arboviruses, we do not yet understand the mechanism by which Wolbachia confers pathogen protection. This week, Rainey et al published a potential mechanism in our favorite journal, PLoS Pathogens. I was excited to read it and share the figures with y'all (the actual figures, not the mixed up ones originally published, and thanks for Alain for sending them along).
In Rainey et al., the authors use a Wolbachia-infected Drosophila cell line (Jw18 of Serbus and Sullivan fame), infected with Semliki Forest virus (SFV), a positive stranded, RNA virus - part of the alpha virus family. They first showed that Jw18 cells are competent for SFV infection. To do this they are using a reporter construct, which expresses luciferase. One very nice thing about the SFV system is the suite of genetic tools that you can use to query which part of the virus is being transcribed and translated. Also, because they are using Drosophila, they can introduce a replicase system into the flies and expressed using the Drosophila actin promotor. But I am getting ahead of myself! Let’s look at Figure 1 below.
Fig 1. Virus, replicon and transreplicase systems used in this study.
(A) Schematic representation of genome of SFV4(3H)-RLuc, carrying the RLuc reporter gene flanked by duplicated nsP2-protease cleavage sites at the nsP3/4 junction. Note that the genome is split into two major ORFs, 1 and 2, encoding non-structural and structural proteins respectively. (B) Schematic representation of the genome of viral replicon pSFV1(3F)RLuc-SG-FFLuc, where RLuc is fused to the region encoding for nsP3 and the structural genes have been replaced by the reporter gene firefly luciferase (FFLuc). Expression of FFLuc occurs only from subgenomic RNA produced from the subgenomic promoter; hence detection of this marker is dependent on the active replication of transfected RNA. (C-D) Schematic representation of the SFV-derived transreplicase constructs used in this study. Expression of the replicase proteins is under the control of the Drosophila Actin promoter (C). Expression of SFV template RNA is also under the control of the Actin promoter (D). When the replicase proteins are expressed this leads to active replication of the template RNA. FFLuc expression is therefore under both the control of the Actin promoter and the SFV genomic promoter. Whereas Gluc is exclusively under the control of the subgenomic promoter and therefore requires active replication of the template RNA in order for expression to occur. Two replicase constructs were used in this study: one functional, and one non-functional due to the insertion of a GDD-GAA mutation in nsP4 as indicated in (C).
They then went on to use these constructs in Jw18 cells with and without Wolbachia using the construct in Figure 1A, where luciferase is a proxy for virus replication (or infectivity). In Figure 2 they present their data - let's pass by 2A&B for now (which just show that this arbovirus is not affecting Jw18 growth - as expected) and focus on 2C.
(A) Jw18Free cells were infected with SFV(3H)-RLuc at an MOI of 20. Cells were lysed 4, 8, 12 and 24 hpi and RLuc activity determined. The figure represents data from three independent experiments, where each treatment was carried out in triplicate. (B) The density of Jw18Wol and Jw18Free cells 7 and 24 hpi with SFV(3H)-RLuc at an MOI of 20; 0 (seed) is 24 h prior to infection. Data represents three independent experiments carried out in duplicate. (C) Jw18Wol and Jw18Free cells were infected at an MOI of 20 with SFV4(3H)-RLuc and RLuc activity was measured at 7 and 24 hpi. The graph indicates the mean ratio of RLuc activity in Jw18Wol and Jw18Free cells, where Jw18Wol at 7hpi and 24 hpi is equal to one. The data represents five independent experiments carried out in duplicate. Error bars represent the standard error of mean in all figures. Stars indicate significance P = <0.05 in T-Test analysis.
It looks like the Jw18 with Wolbachia was able to repress RNA virus replication - by 2-3 fold, is what they say in text. In and of itself, that is not surprising as the pathogen blocking effect has been observed in cell lines before. However, but look at the time course. At only 7 hpi, they see an effect of Wolbachia. This means that Wolbachia is blocking early in the infection.
As I said before, one of the cool things about their model system is that they can then use the genetic tools available to examine where the block in replication occurs. To disentangle effects of viral entry or exit from replication within the cell, they use a clever transfection of a SFV replication construct (Figure 1C). Because they will transfect the construct in, defects observed in their readout (in this case, the luciferase reporter) can be interpreted as problems with translation and/or transcription, not entry.