Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

Site-Specific Structural Variations Accompanying Tubular Assembly of the HIV-1 Capsid Protein

The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent ¹⁵N and ¹³C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide ¹H nuclei, and quantitative measurements of site-specific ¹⁵N–¹⁵N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 3₁₀-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant.     

中药联合微生态制剂防治呼吸、消化系统病毒病

本文阐述了中药防治病毒病的历史及其应用概况,中药联合微生态制剂防治呼吸、消化系统病毒病的理论依据、临床应用和发展前景。     

Salivary apyrase in New World blackflies (Diptera: Simuliidae) and its relationship to onchocerciasis vector status

Abstract. Salivary gland apyrase is believed to be critical to blood-feeding in arthropod vectors. This enzyme was measured in six New World blackflies representing three taxonomic pairs of non-vectors and vectors of Onchocerca volvulus. In Simulium (Psilopelmia) ochraceum , a highly anthropophilic vector in Mexico and Guatemala, apyrase exhibited maximum activity between pH 8.0 and 9.0, mean 39.8 pM 4.7 milliUnits/pair of gland equivalents (mU), and was enhanced when ATP was used as a substrate. In the zoophilic non-vector Simulium (Psilopelmia) bivittatum maximum activity was significantly less (5.1 pM 0.7 mU) under all conditions examined. Preference for ADP or ATP as substrate was a function of the pH of the reaction for this species. Apyrase activity in Simulium (Simulium) metallicum Bellardi (29.5 pM 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S.(Ps.)ochraceum (24.8 pM 13.7 mU at pH 8.5) with ADP as substrate, but showed reduced activity with ATP. Both these Central American vectors had higher apyrase activity than found in Simulium (Notolepria) exiguum , a vector of O. volvulus in Ecuador and Colombia. However, maximum apyrase activity, measured at pH 8.0 with ADP as substrate, was greater in S.(N.) exiguum (10.9 pM 0.6mU) than in Simulium (Notolepria) gonzalezi (5.9pM1.9mU), a non-vector species widespread in Central America. Therefore, for the consubgeneric species pairs examined, a positive association was detected between higher concentrations of apyrase activity and their vector status for O. volvulus.     

一个RNAi载体上反向重复片段的测序策略

相邻的反向重复DNA片段有形成单链内二级结构的倾向,属于一种测序困难的DNA模板。解决RNAi载体插入的反向重复片段的测序问题,为该类载体正确性的测序验证奠定基础。采用常规分子克隆方法构建表达小麦TaATG2串联反向重复片段的RNAi载体,设计2种策略对经菌落PCR初步鉴定的载体进行测序验证:一种是以完整的载体质粒为模板进行测序;另一种是先对载体进行酶切处理,切除反向重复片段中的一个后对保留另一个片段的线性载体进行测序。结果表明,第一种测序策略受到串联反向重复片段形成的单链内部二级结构的影响,测序信号在反向重复片段处出现衰减或乱峰,无法读取序列。第二种测序策略排除了2个反向重复片段之间的干扰,保留在载体上的片段测序信号清晰,序列准确。采用酶切切除一个片段后进行测序的方法,经过2次酶切和2次测序可以有效地对载体上的2个反向重复片段分别进行序列测定,进而确认构建载体的正确性。     

Detecting early-warning signals for influenza A pandemic based on protein dynamical network biomarkers

The outbreak of influenza A comes from a relatively stable state is a critical phenomenon on epidemic. In this paper, influenza A varying from different states is studied in the method of dynamical network biomarkers (DNB). Through studying DNB of influenza A virus protein, we can detect the warning signals of outbreak for influenza A and obtain a composite index. The composite index varies along with the state of pandemic influenza, which gives a clue showing the turn point of outbreak. The low value (<1) steady state of the composite index means influenza A is normally in the relatively steady stage. Meanwhile, if the composite index of a certain year increases by more than 0.8 relative to the previous year and it is less than 1 and it increases sharply and reaches a peak being larger than 1 in next year, it means the year is normal in the critical state before outbreak and the next year is normally in the outbreak state. Therefore, we can predict the outbreak of influenza A and identify the critical state before influenza A outbreak or outbreak state by observing the variation of index value.     

Increase of cyclin B by overexpression of cystatin α

Degradation of cyclin B was effectively suppressed when cells were treated with ALLN (N-acetylleucylleucylnorleucinal) which inhibits proteasome, calpain and cysteine proteinase cathepsins. In order to examine which protease degrades cyclin B, the effect of a cathepsin inhibitor, cystatin α, was investigated. The cystatin α gene was inserted into an inducible expression vector, pMSG, and transfected into NIH3T3 mouse fibroblasts. The expression of cystatin α was induced effectively in the transfected cells after treatment with dexamethasone. Overexpression of cystatin α resulted in an increase of the amount of cyclin B, suggesting that cysteine proteinase cathepsins might be involved in the degradation of cyclin B.     

Hepatitis C virus represses E-cadherin expression via DNA methylation to induce epithelial to mesenchymal transition in human hepatocytes

Hepatitis C virus (HCV) core protein is known to induce promoter hypermethylation of tumor suppressor genes including E-cadherin to repress their expression when overexpressed in human hepatocytes; however, its actual role during HCV infection is still unknown. Here, we report that infection with HCV derived from pJFH-1 replicon system that mimics natural infection elevates protein levels of DNA methyltransferase 1 and 3b to enhance DNMT activity in human hepatocytes. As a consequence, HCV induced promoter hypermethylation of E-cadherin, resulting in repression of its expression. In addition down-regulation of E-cadherin by HCV led to epithelial–mesenchymal transition that is known to be a critical event during the late stage of tumorigenesis.