BACKGROUND The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mono-nuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibro-blasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients’ lymphocytes was reduced by JAK inhibitors. CONCLUSIONS STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173.

277 transactivator proteins of human retroviruses may mediate both viral and cellular effects. However, the events associated with the release, the uptake, and the biological effects of extracellular Tat are not yet well understood. In this study, we investigated whether during acute infection of T cells by HIV-1, Tat is released at concentrations sufficient to stimulate HIV-1 gene expression in a paracrine fashion and the events associated with the release of biologically active protein by cells transfected with the tat gene. As the Tat-containing supematants from both HIV-1-infected and Tat-transfected cells stimulate maximal AIDS-KS cell growth but only low levels of HIV-1 gene expression, we analyzed the kinetics of dose-response for these activities. This analysis was done by using recombinant purified Tat protein in assays of AIDS-KS cell growth, LTR-directed gene expression, and viral replication. Since the nuclear uptake of Tat is a prerequisite for transactivation, we also determined the concentration of exogenous protein necessary to detect nuclear-localized Tat with AIDS-KS cells. The results from these studies suggest that different pathways mediate the cellular and viral effects of extracellular Tat protein. MATERIALS AND METHODS Cell cultures. The T-cell lines H9 and Jurkat, the epithelial cell line COS-1, and HeLa-CD4+ cells containing a Tatdefective HIV-1 provirus (HLM1 cell line) have all been previously described (14, 44). Primary cell cultures derived from KS lesions of patients with AIDS were established and cultured as previously described (14, 16, 37, 38, 46). HIV-1 infection. H9 or Jurkat cells (106/ml) were infected by a cell-free method (14) with HIV-1 (IIIB isolate), as

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Measurements of proton transverse relaxation rates, R 2 and R 1 ρ, have not been commonly performed for proteins because cross correlations among the numerous 1H−1H dipolar interactions complicate analysis of the data. In addition, these interactions make large contributions to the relaxation of the amide protons, making it difficult to detect if an exchange of chemical shifts also makes a contribution, R ex, to relaxation. To overcome these problems, we have investigated proton relaxation of a perdeuterated protein, HIV-1 protease, bound to a small protonated inhibitor DMP323. Perdeuteration significantly reduces the contributions of 1H−1H dipolar interactions to the relaxation of the amide protons. The ROESY R 1 ρ experiment further reduces the overall relaxation rate as compared with the usual R 1 ρ experiment because the protons relax as unlike spins, with rate R 1 ρ ,unlike, in the former experiment but as like spins, with rate R 1 ρ, in the latter. These reductions of the proton transverse-relaxation rate facilitated the detection of R ex contributions at several sites in the protein (1) from the B 1-field dependence of R 1 ρ ,unlike and (2) by comparing R 1 ρ ,unlike values with relaxation rates, R 2, obtained from Carr−Purcell−Meiboom−Gill (CPMG) and Hahn-echo experiments. The significant reduction of the proton spin-flip rate in the perdeuterated protein enabled measurement of 15N R 2 values using the CPMG method and the same large duration between 180° pulses as used in the 1H CPMG experiments. Hence, relaxation data of both nuclei were utilized to obtain complementary information about sites experiencing exchange of chemical shifts in the protein.

Preface vi Supplement 41 Current Protocols in Protein Science would like to thank two people who played important roles in initiating this project. Sarah Greene initially conceived this project and had a critical role in developing it. John A. Smith made important contributions during the early planning phases of the project.

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Human immunodeficiency virus type 1 Rev protein modulates the distribution of viral mRNAs from the nucleus to the cytoplasm by interaction with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To identify the minimal functional elements of RRE, we evaluated mutant RREs for Rev binding in vitro and Rev response in vivo in the context of a Gag expression plasmid. The critical functional elements fold into a structure composed of a stem-loop A, formed by the ends of the RRE, joined to a branched stem-loop B/B1/B2, between bases 49 and 113. The 5' 132 nucleotides of RRE, RREDDE, which possessed a similar structure, bound Rev efficiently but were nonfunctional in vivo, implying separate binding and functional domains within the RRE. Excision of stem-loop A reduced Rev binding significantly and abolished the in vivo Rev response. The B2 branch could be removed without severe impairment of binding, but deletions in the Bl branch significantly reduced binding and function. However, deletion of 12 nucleotides, including the 5' strand of stem B, abolished both binding and function, while excision of the 3' strand of stem B only reduced them. Maintenance of the native RRE secondary structure alone was not sufficient for Rev recognition. Many mutations that altered the primary structure of the critical region while preserving the original RNA conformation were Rev responsive. However, mutations that changed a 5'. . CACUAUGGG. . 3' sequence in the B stem, without affecting the overall structure abolished both the in vitro Rev binding and the in vivo Rev response.