Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.
Engineering the BirAR118G biotin ligase into the MHV replicase transcriptase complex To insert the promiscuous biotin ligase BirAR118G as an integral subunit of the MHV RTC, we used a vaccinia virus-based reverse genetic system (Coley et al., 2005; Eriksson et al., 2008) to generate a recombinant MHV harboring an in-frame fusion of myc-tagged BirAR118G to nsp2. MHV-BirAR118Gnsp2 retained the cleavage site between nsp1 and BirAR118G, while a deleted cleavage site between BirAR118G and nsp2 ensured the expression of a BirAR118G-nsp2 fusion protein (Figure 1a). This strategy was chosen because it was recently employed by Freeman et al. for a fusion of green fluorescent protein (GFP) with nsp2 and represents the only known site tolerating large insertions within the MHV replicase polyprotein (Freeman et al., 2014). MHV-BirAR118G-nsp2 replicated to comparable peak titers and replication kinetics as the parental wild-type MHV-A59 (Figure 1b). MHV-GFP-nsp2, which was constructed in parallel and contained the coding sequence of EGFP (Freeman et al., 2014) instead of BirAR118G, was used as a control and also reached wild-type virus peak titers, with slightly reduced viral titers at 9 hr post- infection (h.p.i.) compared to MHV-A59 and MHV-BirAR118Gnsp2 (Figure 1b). Western blot analysis confirmed that the BirAR118G-nsp2 fusion protein is specifically detected in MHV-BirAR118G-nsp2-infected cells and that the BirAR118G biotin ligase remains fused to nsp2 during MHV-BirAR118G-nsp2 infection (Figure 1—figure supplement 1). To further confirm the accommodation of BirAR118G within the viral RTC, MHV-A59-, MHV-BirAR118G-nsp2-, and mock-infected L929 fibroblasts were visualized using indirect immunofluorescence microscopy. BirAR118G-nsp2 remained strongly associated with the MHV RTC throughout the entire replication cycle, as indicated by the co-localization of BirAR118G-nsp2 with established markers of the MHV replicase, such as nsp2/3 and nsp8 (Figure 1c, Figure 1—figure supplement 2, Figure 1—figure supplement 3). This observation corroborates previous studies demonstrating that nsp2, although not required for viral RNA synthesis, co-localizes with other nsps of the coronaviral RTC (Schiller et al., 1998; Hagemeijer et al., 2010; Graham et al., 2005). Importantly, by supplementing the culture medium with biotin, we could readily detect biotinylated proteins with fluorophore-coupled streptavidin that appeared close to the MHV RTC throughout the entire replication cycle in MHV-BirAR118G-nsp2-infected cells,represent the mean and SEM of three independent experiments, each performed in quadruplicate. (c) Immunofluorescence analysis of MHV-BirAR118Gnsp2-mediated biotinylation of RTC-proximal factors. L929 cells were infected with MHV-BirAR118G-nsp2 (MOI = 1) in medium supplemented with 67 mM biotin. Cells were fixed 15 hr post infection (h.p.i.) and processed for immunofluorescence analysis with antibodies directed against the BirAR118G (antimyc), the viral replicase (anti-nsp2/3) and biotinylated factors (streptavidin). Nuclei are counterstained with DAPI. Z-projection of deconvolved z-stacks acquired with a DeltaVision Elite High-Resolution imaging system are shown. Scale bars: 20 mm; insets 5 mm. (d) Ultrastructural analysis of MHV-APEX2- nsp2 infection. L929 cells were infected with MHV-APEX2-nsp2 and MHV-A59 (MOI = 2), or mock infected. At 10 h.p.i., cells were fixed, stained with DAB and processed for electron microscopy investigations. Representative low (scale bar: 10 mm) and high magnifications (scale bar: 2 mm) are displayed
Reference & Source information: https://elifesciences.org/
Read More on :