Project description:The influenza A virus polymerase, consisting of a heterotrimer of three viral proteins, carries out both transcription and replication of the viral RNA genome. These distinct activities are regulated by viral proteins that vary in abundance during infection, and by various co-opted host cell proteins, which serve as targets for the development of novel antiviral interventions. However, little is known about which host proteins direct transcription and which replication. In this report, we performed a differential interactome screen to identify host proteins co-opted as either transcription- or replication-specific factors. We found that distinct sets of host proteins interact with the influenza polymerase as it carries out the different activities. We functionally characterised HMGB2 and RUVBL2 as replication-specific cofactors and RPAP2 as a transcription-specific cofactor. Our data demonstrate that comparative proteomics can be used as a targeted approach to uncover virus-host interactions that regulate specific stages of the viral lifecycle.
Project description:Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)- containing domain translocates ~90 A ̊ to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs.</br></br>Extra contact information:</br><a href="mailto:cusack@embl.fr">Stephen Cusack</a>, EMBL Grenoble Outstation, Unit of Virus Host-Cell Interactions, France ( corresponding author and lab head )
Project description:Analyses of AP-MS experiments performed in HEK 293T cells infected with the influenza A/WSN/33 virus. In half of the experiments the virus was modified to contain a C-terminal Strep tag on the polymerase subunit PB2. Full details in York et al. 'Interactome analysis of the influenza A virus transcription/replication machinery identifies protein phosphatase 6 as a cellular factor required for efficient virus replication.'
Project description:During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitin landscape of the IAV polymerase during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-ε-GG- (di-glycyl)-remnant-bearing peptides. We identified 59 modified lysines across all three subunits of the viral polymerase of which 17 distinctively affected mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, our results demonstrate that the ubiquitinated residue K578 in the PB1 thumb domain is crucial for the dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R impeded the steps of cRNA stabilization and vRNA transcription, respectively, and affected NP binding as well as polymerase dimerization. Collectively, our results indicate that ubiquitin-mediated disruption of the charge-dependent interaction between PB1-K578 and PB2-E72 is required to coordinate polymerase dimerization and facilitate vRNA replication, which demonstrates that IAV exploit the cellular ubiquitin system to modulate the activity of the viral polymerase for the regulation of viral replication.
Project description:The PB2-E627K mutation is a key determinant for mammalian adaptation of avian influenza A viruses, yet its system-wide impact on the host microenvironment of the viral polymerase remains unknown. Here, we employed TurboID proximity labeling coupled with quantitative mass spectrometry to map the proximal interactomes of the reconstituted H5N1 polymerase trimer (PA-PB1-PB2) in its wild-type (WT) and PB2-E627K forms in human cells. We identified 204 high-confidence interactions and revealed that E627K extensively rewires the host interaction landscape, gaining associations with cytoskeletal regulators while losing connections to mRNA splicing machinery. AlphaFold3 structural modeling provided high-confidence predictions for key interactions, including PB2-KPNA1 and PA-CPSF6, which were validated biochemically. This study provides the first spatial interactome map of a pivotal influenza host-adaptation mutation, demonstrating how PB2-E627K reprogrammes the host-virus interface to facilitate cross-species infection, and offers a resource for novel antiviral strategies.