SummaryChemoreceptors play a central role in chemotaxis, allowing bacteria to detect chemical gradients and bias their swimming behavior in order to navigate toward favorable environments. The genome of the kiwifruit pathogen, Pseudomonas syringae pv. actinidiae (Psa) strain NZ-V13 encodes 43 predicted chemoreceptors, none of which has been characterized. We developed a high-throughput fluorescence-based thermal shift assay for identifying the signal molecules that are recognized by a given chemoreceptor ligand binding domain (LBD). Using this assay, we characterized the ligand binding profiles of three Psa homologs of the P. aeruginosa PAO1 amino acid chemoreceptors PctA, PctB and PctC. Each recombinant LBD was screened against 95 potential ligands. The three Psa homologs, named pscA, pscB and pscC (Psa chemoreceptors A, B and C) bound 3, 10 and 3 amino acids respectively. In each case, their binding profiles were distinct from their P. aeruginosa PAO1 homologs. Notably, Psa PscA-LBD only bound the acidic amino acids L-aspartate, D-aspartate and L-glutamate, whereas P. aeruginosa PctA-LBD binds all of the L-proteinogenic amino acids except for L-aspartate and L-glutamate. A combination of homology modeling, site-directed mutagenesis and functional screening identified a single amino acid residue in the Psa PscA-LBD (Ala146) that is critically important for determining its narrow specificity.

More than 10 million people worldwide are infected with the retrovirus human T-cell lymphotropic virus type 1 (HTLV-1). Infection phenotypes can range from asymptomatic to severe adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy. HTLV-1, like human immunodeficiency virus type 1 (HIV-1), is a blood-borne pathogen and viral infection happens in a similar fashion, with the major mode of transmission through breastfeeding. There is a strong correlation between time of infection and disease development, with a higher incidence of ATLL in patients infected during childhood. There is no successful therapeutic or preventative regimen for HTLV-1. It is therefore essential to develop therapies to inhibit transmission or block the onset/development of HTLV-1 associated diseases. Recently, we have seen the overwhelming success of integrase strand transfer inhibitors (INSTIs) in the treatment of HIV-1. Previously, raltegravir was shown to inhibit HTLV-1 infection. Here, we tested FDA-approved and two Phase II HIV-1 INSTIs in vitro and in a cell-to-cell infection model and show that they are highly active in blocking HTLV-1 infection, with bictegravir (EC 50 = 0.30 ± 0.17 nM) performing best overall. INSTIs, in particular bictegravir, are more potent in blocking HTLV-1 transmission than tenofovir disproxil fumarate (TDF), an RT inhibitor. Our data suggest that HIV-1 INSTIs could present a good clinical strategy in HTLV-1 management and justifies the inclusion of INSTIs in clinical trials.

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.

Full-spectrum flow cytometry is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With capacity to use larger and more complex staining panels, standardized protocols are required for optimal panel design and analysis. Importantly, for ex vivo analysis, tissue preparation methods also need to be optimized to ensure samples are truly representative of tissues in situ. This is particularly relevant given the recent interest in adaptive immune cells that form residency in specific organs. Here we provide optimized protocols for tissue processing and phenotyping of memory T cells and natural killer T (NKT) cell subsets from liver, lung, spleen, and lymph node using full-spectrum flow cytometry. We provide a 21-color antibody panel for identification of different memory subsets, including tissue-resident memory T (T RM ) cells, which are increasingly regarded as important effectors in adaptive immunity. We show that processing procedures can affect outcomes, with liver T RM cells particularly sensitive to heat, such that accurate evaluation requires fast processing at defined temperatures.

Protein phosphatase 2A (PP2A) is one of the most ubiquitous cellular proteins and is responsible for the vast majority of Ser/Thr phosphatase activity in eukaryotes. PP2A is a heterotrimer, and its assembly, intracellular localization, enzymatic activity, and substrate specificity are subject to dynamic regulation. Each of its subunits can be targeted by viral proteins to hijack and modulate its activity and downstream signaling to the advantage of the virus. Binding to PP2A is known to be essential to the life cycle of many viruses and seems to play a particularly crucial role for oncogenic viruses, which utilize PP2A to transform infected cells through controlling the cell cycle and apoptosis. Here we summarise the latest developments in the field of PP2A viral targeting; in particular recent discoveries of PP2A hijacking through molecular mimicry of a B56-specific motif by several different viruses. We also discuss the potential as well as shortcomings for therapeutic intervention in the face of our current understanding of viral PP2A targeting.

2The Retroviridae delta-retrovirus genus includes the most oncogenic pathogen -human T-cell lymphotropic virus type 1 (HTLV-1)(1). Many of the ~20 million people infected with HTLV-1 will develop severe leukaemia (2) or an ALS-like motor disease (3) unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the viral integrase (IN) enzyme. Here we used X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of functional delta-retroviral IN assembled on viral DNA ends and bound the B56g subunit of its human host factor, the protein phosphatase 2A (4). The structure reveals a tetrameric IN assembly bound to the phosphatase via a conserved short linear motif found within the extended linker connecting the catalytic core (CCD) and C-terminal (CTD) IN domains. Unexpectedly, all four IN subunits are involved in B56g binding, taking advantage of the flexibility of the CCD-CTD linkers. Our results fill the current gap in the structural understanding of the delta-retroviral integration machinery. Insight into the interactions between the delta-retroviral intasome and the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.

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