Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles. © 2012 Meng et al

\u3csup\u3e1\u3c/sup\u3eH, \u3csup\u3e15\u3c/sup\u3eN, \u3csup\u3e13\u3c/sup\u3eC, and \u3csup\u3e13\u3c/sup\u3eCO Assignments of Human Interleukin-4 Using Three-Dimensional Double- and Triple-Resonance Heteronuclear Magnetic Resonance Spectroscopy

The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of \u3e95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)-N(i+l),NH(i+l) correlations are observed in the HNCO experiment, the CαH(i),Cα(i)-N(i+l) correlations in the HCA(CO)N experiment, the Cα(i)-N(i+l),NH(i+ 1) correlations in the HNCA and HN(C0)CA experiments, the CαH(i)-N(i+ l),NH(i+l) correlations in the H(CA)NH and HN(CO)HB experiments, and the Cβ(i)-N(i+ l),NH(i+ 1) correlations in the HN(CO)HB experiments. The backbone intraresidue CαH(i)-15N(i)-NH(i) correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the CβH(i)-15N(i)-NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the l3Cα(i)-l5N(i)-NH(i) correlations by the HNCA experiment, and the CαH(1)-13Cα(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution

Silylation of titanium-containing amorphous silica catalyst: effect on the alkenes epoxidation with H2O2

The surface of a Ti/SiO2 catalyst was silylated using hexamethyldisilazane (HMDS) and tetramethyldisilazane (TMDS) as silylating reagents in vapor phase. The silylation of silanol (Si–OH) on the catalysts was confirmed by diffuse reflectance UV-Vis, DRIFT spectroscopy and solid-state 29Si MAS NMR techniques. Silylation with TMDS improves the catalytic performance of Ti/SiO2 catalyst more significantly than with HMDS and this is mainly due to the steric bulk effects of trimethylsilyl groups from HMDS as compared to dimethylsilyl groups from TMDS. The silylation degree with TMDS was found to be higher than with HMDS and no changes in the silylation degree with TMDS was observed for silylation times longer than 2 h. Despite the silylation route employed, the silylated surface is hydrophobic which enhances not only in hydrogen peroxide efficiency but also and in selectivity to epoxide in the epoxidation of alkenes with H2O2. These effects are more evident when the hydrogen peroxide concentration is higher and particularly for the catalyst modified with TMDS.Peer reviewe

Genomic and dietary discontinuities during the Mesolithic and Neolithic in Sicily

Summary Sicily is a key region for understanding the agricultural transition in the Mediterranean, due to its central position. Here, we present genomic and stable isotopic data for 19 prehistoric Sicilians covering the Mesolithic to Bronze Age periods (10,700-4,100 yBP). We find that Early Mesolithic hunter-gatherers (HGs) from Sicily are a highly drifted lineage of the Early Holocene western European HGs, while Late Mesolithic HGs carry ∼20% ancestry related to northern and (south)eastern European HGs, indicating substantial gene flow. Early Neolithic farmers are genetically most similar to farmers from the Balkans and Greece, with only ∼7% ancestry from local Mesolithic HGs. The genetic discontinuities during the Mesolithic and Early Neolithic match changes in material culture and diet. Three outlying individuals dated to ∼8,000 yBP, however, suggest that hunter-gatherers interacted with incoming farmers at Grotta dell’Uzzo, resulting in a mixed economy and diet for a brief interlude at the Mesolithic-Neolithic transition.- Introduction - Results -- Genetically-distinct groups of prehistoric Sicilians -- Genomic and dietary transitions in Sicily during the Mesolithic and Early Neolithic -- Did Sicilian Late Mesolithic foragers adopt some aspects of early farming? - Discussion -- Limitations of the stud

Rhesus TRIM5α disrupts the HIV-1 capsid at the inter-hexamer interfaces

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5αrh) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5αrh containing coiled-coil and SPRY domains (CC-SPRYrh). Concentration-dependent binding of CC-SPRYrh to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRYrh, but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5αrh on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction. © 2011 Zhao et al