SUMMARY Interferon-induced proteins with tetratricopeptide repeats (IFIT) inhibit infection of many viruses by recognizing their RNA, but the mechanisms regulating their remain unclear. We report a crystal structure of cap 0 (m7GpppN)-RNA-bound to human IFIT1 in complex with the C-terminal domain of human IFIT3. Structural, biochemical, and genetic studies suggest that IFIT3 binding to IFIT1 has dual regulatory functions: (a) extending the half-life of IFIT1, which increases its steady-state levels in cells and (b) allosterically regulating the IFIT1 RNA-binding channel, which enhances the specificity of recognition for cap 0 but not cap 1 (m7GpppNm) or 5′-ppp RNA. Mouse Ifit3 lacks this key C-terminal domain and does not bind mouse Ifit1. The IFIT3 interaction with IFIT1 was important for restricting infection of viruses lacking 2′-O methylation in their RNA cap structures. Our experiments establish differences in regulation of IFIT1 orthologs and define targets for modulating the activity of human IFIT proteins.

Although the nucleocytoplasmic large DNA viruses (NCLDVs) are one of the largest group of viruses that infect many eukaryotic hosts, the near-atomic resolution structures of these viruses have remained unknown. Here we describe a 3.5 Å resolution icosahedrally averaged capsid structure of Paramecium bursaria chlorella virus 1 (PBCV-1). This structure consists of 5040 copies of the major capsid protein, 60 copies of the penton protein and 1800 minor capsid proteins of which there are 13 different types. The minor capsid proteins form a hexagonal network below the outer capsid shell, stabilizing the capsid by binding neighboring capsomers together. The size of the viral capsid is determined by a tape-measure, minor capsid protein of which there are 60 copies in the virion. Homologs of the tape-measure protein and some of the other minor capsid proteins exist in other NCLDVs. Thus, a similar capsid assembly pathway might be used by other NCLDVs.

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The detection and quantitation of protein-ligand binding interactions is critical in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g. drugs like tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologies (iTRAQ) with a covalent labeling technique, termed Stability of Proteins from Rates of Oxidation (SPROX). In SPROX, the thermodynamic properties of proteins and protein-ligand complexes are assessed using the hydrogen peroxide-mediated oxidation of methionine residues as a function of the chemical denaturant (e.g. guanidine Hydrochloride or urea) concentration. The proteome-wide SPROX experiments described here enable the ligand binding properties of hundreds of proteins to be simultaneously assayed in the context of complex biological samples. The proteomic capabilities of the protocol render it amenable to detection of both the on- and off-target effects of ligand binding.

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.

Current methods for the large-scale characterization of disease states generally rely on the analysis of gene and/or protein expression levels. These existing methods fail to detect proteins with disease-related functions and unaltered expression levels. Here we describe the large-scale use of thermodynamic measurements of protein folding and stability for the characterization of disease states. Using the Stable Isotope Labeling with Amino Acids in Cell Culture and Stability of Proteins from Rates of Oxidation (SILAC-SPROX) technique, we assayed ~800 proteins for protein folding and stability changes in three different cell culture models of breast cancer including the MCF-10A, MCF-7, and MDA-MB-231 cell lines. The thermodynamic stability profiles generated here created distinct molecular markers to differentiate the three cell lines, and a significant fraction (~45%) of the differentially stabilized proteins did not have altered expression levels. Thus, the differential thermodynamic profiling strategy reported here created novel molecular signatures of breast cancer and provided additional insight into the molecular basis of the disease. Our results establish the utility of protein folding and stability measurements for the study of disease processes, and they suggest that such measurements may be useful for biomarker discovery in disease.

Reported here is the use of stable isotope labeling with amino acids in cell culture (SILAC) and pulse proteolysis (PP) for detection and quantitation of protein-ligand binding interactions on the proteomic scale. The incorporation of SILAC into PP enables the PP technique to be used for the unbiased detection and quantitation of protein-ligand binding interactions in complex biological mixtures (e.g., cell lysates) without the need for prefractionation. The SILAC-PP technique is demonstrated in two proof-of-principle experiments using proteins in a yeast cell lysate and two test ligands including a well-characterized drug, cyclosporine A (CsA), and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). The well-known tight-binding interaction between CsA and cyclophilin A was successfully detected and quantified in replicate analyses, and a total of 33 proteins from a yeast cell lysate were found to have AMP-PNP-induced stability changes. In control experiments, the method's false positive rate of protein target discovery was found to be in the range of 2.1% to 3.6%. SILAC-PP and the previously reported stability of protein from rates of oxidation (SPROX) technique both report on the same thermodynamic properties of proteins and protein-ligand complexes. However, they employ different probes and mass spectrometry-based readouts. This creates the opportunity to cross-validate SPROX results with SILAC-PP results, and vice-versa. As part of this work, the SILAC-PP results obtained here were cross-validated with previously reported SPROX results on the same model systems to help differentiate true positives from false positives in the two experiments.

Japanese encephalitis virus (JEV) remains a leading cause of viral encephalitis worldwide. Although JEV-specific antibodies have been described, an assessment of their ability to neutralize multiple genotypes of JEV has been limited. Here, we describe the development of a panel of mouse and human neutralizing monoclonal antibodies (MAbs) that inhibit infection in cell culture of four different JEV genotypes tested. Mechanism-of-action studies showed that many of these MAbs inhibited infection at a postattachment step, including blockade of virus fusion. Mapping studies using site-directed mutagenesis and hydrogen-deuterium exchange with mass spectrometry revealed that the lateral ridge on domain III of the envelope protein was a primary recognition epitope for our panel of strongly neutralizing MAbs. Therapeutic studies in mice demonstrated protection against lethality caused by genotype I and III strains when MAbs were administered as a single dose even 5 days after infection. This information may inform the development of vaccines and therapeutic antibodies as emerging strains and genotypic shifts become more prevalent.