SKAP2 Modular Organization Differently Recognizes SRC Kinases Depending on Their Activation Status and Localization.

Dimerization of SRC kinase adaptor phosphoprotein 2 (SKAP2) induces an increase of binding for most SRC kinases suggesting a fine-tuning with transphosphorylation for kinase activation. This work addresses the molecular basis of SKAP2-mediated SRC kinase regulation through the lens of their interaction capacities. By combining a luciferase complementation assay and extensive site-directed mutagenesis, we demonstrated that SKAP2 interacts with SRC kinases through a modular organization depending both on their phosphorylation-dependent activation and subcellular localization. SKAP2 contains three interacting modules consisting in the dimerization domain, the SRC homology 3 (SH3) domain, and the second interdomain located between the Pleckstrin homology and the SH3 domains. Functionally, the dimerization domain is necessary and sufficient to bind to most activated and myristyl SRC kinases. In contrast, the three modules are necessary to bind SRC kinases at their steady state. The Pleckstrin homology and SH3 domains of SKAP2 as well as tyrosines located in the interdomains modulate these interactions. Analysis of mutants of the SRC kinase family member hematopoietic cell kinase supports this model and shows the role of two residues, Y390 and K7, on its degradation following activation. In this article, we show that a modular architecture of SKAP2 drives its interaction with SRC kinases, with the binding capacity of each module depending on both their localization and phosphorylation state activation. This work opens new perspectives on the molecular mechanisms of SRC kinases activation, which could have significant therapeutic impact.

Influenza A virus co-opts ERI1 exonuclease bound to histone mRNA to promote viral transcription.

Cellular exonucleases involved in the processes that regulate RNA stability and quality control have been shown to restrict or to promote the multiplication cycle of numerous RNA viruses. Influenza A viruses are major human pathogens that are responsible for seasonal epidemics, but the interplay between viral proteins and cellular exonucleases has never been specifically studied. Here, using a stringent interactomics screening strategy and an siRNA-silencing approach, we identified eight cellular factors among a set of 75 cellular proteins carrying exo(ribo)nuclease activities or involved in RNA decay processes that support influenza A virus multiplication. We show that the exoribonuclease ERI1 interacts with the PB2, PB1 and NP components of the viral ribonucleoproteins and is required for viral mRNA transcription. More specifically, we demonstrate that the protein-protein interaction is RNA dependent and that both the RNA binding and exonuclease activities of ERI1 are required to promote influenza A virus transcription. Finally, we provide evidence that during infection, the SLBP protein and histone mRNAs co-purify with vRNPs alongside ERI1, indicating that ERI1 is most probably recruited when it is present in the histone pre-mRNA processing complex in the nucleus.

Nonproteolytic K29-Linked Ubiquitination of the PB2 Replication Protein of Influenza A Viruses by Proviral Cullin 4-Based E3 Ligases.

The multifunctional nature of viral proteins is essentially driven by posttranslational modifications (PTMs) and is key for the successful outcome of infection. For influenza A viruses (IAVs), a composite pattern of PTMs regulates the activity of viral proteins. However, almost none are known that target the PB2 replication protein, except for inducing its degradation. We show here that PB2 undergoes a nonproteolytic ubiquitination during infection. We identified E3 ubiquitin ligases catalyzing this ubiquitination as two multicomponent RING-E3 ligases based on cullin 4 (CRL4s), which are both contributing to the levels of ubiquitinated forms of PB2 in infected cells. The CRL4 E3 ligase activity is required for the normal progression of the viral cycle and for maximal virion production, indicating that the CRL4s mediate a ubiquitin signaling that promotes infection. The CRL4s are recruiting PB2 through an unconventional bimodal interaction with both the DDB1 adaptor and DCAF substrate receptors. While able to bind to PB2 when engaged in the viral polymerase complex, the CRL4 factors do not alter transcription and replication of the viral segments during infection. CRL4 ligases catalyze different patterns of lysine ubiquitination on PB2. Recombinant viruses mutated in the targeted lysines showed attenuated viral production, suggesting that CRL4-mediated ubiquitination of PB2 contributes to IAV infection. We identified K29-linked ubiquitin chains as main components of the nonproteolytic PB2 ubiquitination mediated by the CRL4s, providing the first example of the role of this atypical ubiquitin linkage in the regulation of a viral infection.IMPORTANCE Successful infection by influenza A virus, a pathogen of major public health importance, involves fine regulation of the multiple functions of the viral proteins, which often relies on post-translational modifications (PTMs). The PB2 protein of influenza A viruses is essential for viral replication and a key determinant of host range. While PTMs of PB2 inducing its degradation have been identified, here we show that PB2 undergoes a regulating PTM signaling detected during infection, based on an atypical K29-linked ubiquitination and mediated by two multicomponent E3 ubiquitin ligases. Recombinant viruses impaired for CRL4-mediated ubiquitination are attenuated, indicating that ubiquitination of PB2 is necessary for an optimal influenza A virus infection. The CRL4 E3 ligases are required for normal viral cycle progression and for maximal virion production. Consequently, they represent potential candidate host factors for antiviral targets.

Comparative Profiling of Ubiquitin Proteasome System Interplay with Influenza A Virus PB2 Polymerase Protein Recapitulating Virus Evolution in Humans.

The optimized exploitation of cell resources is one cornerstone of a successful infection. Differential mapping of host-pathogen protein-protein interactions (PPIs) on the basis of comparative interactomics of multiple strains is an effective strategy to highlight correlations between host proteome hijacking and biological or pathogenic traits. Here, we developed an interactomic pipeline to deliver high-confidence comparative maps of PPIs between a given pathogen and the human ubiquitin proteasome system (UPS). This subarray of the human proteome represents a range of essential cellular functions and promiscuous targets for many viruses. The screening pipeline was applied to the influenza A virus (IAV) PB2 polymerase proteins of five strains representing different levels of virulence in humans. An extensive PB2-UPS interplay has been detected that recapitulates the evolution of IAVs in humans. Functional validation with several IAV strains, including the seasonal H1N1pdm09 and H3N2 viruses, confirmed the biological relevance of most identified UPS factors and revealed strain-independent and strain-specific effects of UPS factor invalidation on IAV infection. This strategy is applicable to proteins from any other virus or pathogen, providing a valuable resource with which to explore the UPS-pathogen interplay and its relationship with pathogenicity. IMPORTANCE Influenza A viruses (IAVs) are responsible for mild-to-severe seasonal respiratory illness of public health concern worldwide, and the risk of avian strain outbreaks in humans is a constant threat. Elucidating the requisites of IAV adaptation to humans is thus of prime importance. In this study, we explored how PB2 replication proteins of IAV strains with different levels of virulence in humans hijack a major protein modification pathway of the human host cell, the ubiquitin proteasome system (UPS). We found that the PB2 protein engages in an extended interplay with the UPS that evolved along with the virus's adaptation to humans. This suggests that UPS hijacking underlies the efficient infection of humans and can be used as an indicator for evaluation of the potential of avian IAVs to infect humans. Several UPS factors were found to be necessary for infection with circulating IAV strains, pointing to potential targets for therapeutic approaches.