Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy.

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.

Large-Scale Arrayed Analysis of Protein Degradation Reveals Cellular Targets for HIV-1 Vpu.

Accessory proteins of lentiviruses, such as HIV-1, target cellular restriction factors to enhance viral replication. Systematic analyses of proteins that are targeted for degradation by HIV-1 accessory proteins may provide a better understanding of viral immune evasion strategies. Here, we describe a high-throughput platform developed to study cellular protein stability in a highly parallelized matrix format. We used this approach to identify cellular targets of the HIV-1 accessory protein Vpu through arrayed coexpression with 433 interferon-stimulated genes, followed by differential fluorescent labeling and automated image analysis. Among the previously unreported Vpu targets identified by this approach, we find that the E2 ligase mediating ISG15 conjugation, UBE2L6, and the transmembrane protein PLP2 are targeted by Vpu during HIV-1 infection to facilitate late-stage replication. This study provides a framework for the systematic and high-throughput evaluation of protein stability and establishes a more comprehensive portrait of cellular Vpu targets.

Identification of Polo-like kinases as potential novel drug targets for influenza A virus.

In recent years genome-wide RNAi screens have revealed hundreds of cellular factors required for influenza virus infections in human cells. The long-term goal is to establish some of them as drug targets for the development of the next generation of antivirals against influenza. We found that several members of the polo-like kinases (PLK), a family of serine/threonine kinases with well-known roles in cell cycle regulation, were identified as hits in four different RNAi screens and we therefore studied their potential as drug target for influenza. We show that knockdown of PLK1, PLK3, and PLK4, as well as inhibition of PLK kinase activity by four different compounds, leads to reduced influenza virus replication, and we map the requirement of PLK activity to early stages of the viral replication cycle. We also tested the impact of the PLK inhibitor BI2536 on influenza virus replication in a human lung tissue culture model and observed strong inhibition of virus replication with no measurable toxicity. This study establishes the PLKs as potential drug targets for influenza and contributes to a more detailed understanding of the intricate interactions between influenza viruses and their host cells.

Systems-based analysis of RIG-I-dependent signalling identifies KHSRP as an inhibitor of RIG-I receptor activation.

Retinoic acid-inducible gene I (RIG-I) receptor recognizes 5'-triphosphorylated RNA and triggers a signalling cascade that results in the induction of type-I interferon (IFN)-dependent responses. Its precise regulation represents a pivotal balance between antiviral defences and autoimmunity. To elucidate the cellular cofactors that regulate RIG-I signalling, we performed two global RNA interference analyses to identify both positive and negative regulatory nodes operating on the signalling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, Wnt signalling and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate its sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signalling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge.

PQBP1 Is a Proximal Sensor of the cGAS-Dependent Innate Response to HIV-1.

Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell-intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection.

Identification of small molecules that interfere with H1N1 influenza A viral replication.

Successful replication of the influenza A virus requires both viral proteins and host cellular factors. In this study we used a cellular assay to screen for small molecules capable of interfering with any of such necessary viral or cellular components. We used an established reporter assay to assess influenza viral replication by monitoring the activity of co-expressed luciferase. We screened a diverse chemical compound library, resulting in the identification of compound 7, which inhibits a novel yet elusive target. Quantitative real-time PCR studies confirmed the dose-dependent inhibitory activity of compound 7 in a viral replication assay. Furthermore, we showed that compound 7 is effective in rescuing high-dose influenza infection in an in vivo mouse model. As oseltamivir-resistant influenza strains emerge, compound 7 could be further investigated as a new and potentially suitable scaffold for the development of anti-influenza agents that act on novel targets.

Positive regulation of Itk PH domain function by soluble IP4.

Pleckstrin homology (PH) domain-mediated protein recruitment to cellular membranes is of paramount importance for signal transduction. The recruitment of many PH domains is controlled through production and turnover of their membrane ligand, phosphatidylinositol 3,4,5-trisphosphate (PIP3). We show that phosphorylation of the second messenger inositol 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) establishes another mode of PH domain regulation through a soluble ligand. At physiological concentrations, IP4 promoted PH domain binding to PIP3. In primary mouse CD4+CD8+ thymocytes, this was required for full activation of the protein tyrosine kinase Itk after T cell receptor engagement. Our data suggest that IP4 establishes a feedback loop of phospholipase C-gamma1 activation through Itk that is essential for T cell development.