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.

The integrase protein of human immunodefi- (1)(2)(3)(4)(5)(6). Integrase thenjoins the recessed 3' ends to protruding 5' ends of breaks made by integrase in the target DNA (the "strand transfer" reaction) (3,4,(7)(8)(9)(10). According to our present understanding, host DNA repair enzymes process and connect the remaining unjoined ends, thereby completing the formation of a provirus (for reviews see refs. 11 and 12). The polynucleotidyl transfer reactions carried out by the integrase proteins of retroviruses are similar to the reactions carried out by the integrases of retrotransposons and also the reactions carried out by the transposase proteins of several prokaryotic transposons (13). The integrase protein of the yeast retrotransposon Tyl is known to join 3' ends of the element DNA to 5' ends in the host DNA (14). For the case of the transposons, the transposase proteins of phage Mu, TnlO, and Tn7 each direct the hydrolysis of phosphodiester bonds that connect element DNA to host DNA and then join the free 3' ends generated by this cleavage to target DNA (15-17).Two conserved amino acid sequence motifs have been found in the integrase proteins of retroviruses and retrotransposons. One motif, found also in the transposase proteins of some bacterial transposons, consists of three invariant acidic residues in the arrangement DX39-58DX35E with various interdigitated conserved residues (the D,D-35-E motif) (18)(19)(20). Another amino acid sequence motif, HX3_7HX23-32CX2C (the HHCC motif), is present near the amino terminus (21-23). This sequence is reminiscent of the "zinc finger" motif that is known to mediate metal binding in many nucleic acid-binding proteins (for review see ref. 24).To explore the function ofthe various parts ofthe integrase protein of human immunodeficiency virus type 1 (HIV-1), we generated and characterized a series of deletion mutants. We find that a polypeptide of only 137 amino acid residues containing the D,D-35-E motif can carry out a subset of the reactions carried out by the complete protein. Our assays of Zn2+ binding by a series ofintegrase derivatives bearing point mutations indicate that the HHCC motif does in fact bind Zn2+. The activities of the integrase mutants put constraints on the possible roles of the various parts ofthe HIV integrase protein. MATERIALS AND METHODSConstruction of Deletion Mutants. Deletion derivatives of the HIV-1 integrase protein were prepared by constructing plasmids encoding the deletion proteins. Procedures for manipulating DNA molecules were essentially as described (25). DNA fragments encoding each deletion derivative were prepared by PCR using pINSD (26) as template. PCR primers were designed to provide an Nde I site in the DNA encoding the amino terminus, and a stop codon followed by a BamHI site in the DNA encoding the carboxyl terminus. Amplification was carried out for 15 cycles using Vent DNA polymerase (New England Biolabs) under the manufacturer's suggested conditions. PCR products were cleaved with BamHI and Nde I and ligated to pA...

The solution structure of HIV-1 Nef has been solved by multidimensional heteronuclear NMR spectroscopy. The construct employed to circumvent problems associated with aggregation was a double-deletion mutant (delta2-39, delta159-173) in which conformationally disordered regions of the protein at the N terminus and in a long solvent-exposed flexible loop were removed, without affecting the properties or structural integrity of the remainder of the protein. Despite the absence of any sequence similarity, the overall fold of Nef is reminiscent of that of the family of winged helix-turn-helix DNA binding proteins. The binding surface of Nef for the SH3 domain of Hck tyrosine protein kinase has been mapped and reveals a non-contiguous (in terms of amino-acid sequence) interaction surface. This unique feature may suggest possible avenues for drug design aimed at inhibiting the interaction between Nef and SH3 domains.