Proteomic Analysis of Virus-Host Interactions in an Infectious Context Using Recombinant Viruses

Abstract

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells.

The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing

“…Moreover, the generation of recombinant viruses able to replicate expressing tagged viral proteins allowed identification of protein complexes in the context of measles, influenza and HIV infections [9–11]. A technical limitation for this kind of proteomic approach is the feasibility to design recombinant viruses that tolerate the addition of tags.…”

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

“…Compounds are tested first on human cells infected with a recombinant measles virus (MV) expressing firefly luciferase 25 (rMV2/Luc, Figure 1, primary screen). MV belongs to Mononegavirales order, and is often considered as a prototypical member of negative-strand RNA viruses.…”

Protein-protein Interactions: Network Analysis and Applications in Drug Discovery

“…By defining the interactions between viral and host proteins we will obtain a better knowledge on how viruses reproduce and cause disease, which may ultimately reveal novel targets for therapeutic intervention. Systematic maps capturing viral-host physical PPIs have been obtained using Y2H for Epstein-Barr virus [142], hepatitis C virus [143], several herpesviruses [144,145], vaccinia virus [146], influenza virus [147], dengue virus [148] and Tcell leukemia virus [149], and by AP-MS methodologies for HIV1 [150] and measles virus [151].…”