APOBEC-mediated editing of viral RNA

Retroviral DNA can be subjected to cytosine-to-uracil editing through the action of members of the APOBEC family of cytidine deaminases. Here we demonstrate that APOBEC-mediated cytidine deamination of human immunodeficiency virus (HIV) virion RNA can also occur. We speculate that the natural substrates of the APOBEC enzymes may extend to RNA viruses that do not replicate through DNA intermediates. Thus, cytosine-to-uracil editing may contribute to the sequence diversification of many viruses.     

Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells

Integrase is essential for human immunodeficiency virus-type 1 (HIV-1) replication; however, potent inhibition of the isolated enzyme in biochemical assays has not readily translated into antiviral activity in a manner consistent with inhibition of integration. In this report, we describe diketo acid inhibitors of HIV-1 integrase that manifest antiviral activity as a consequence of their effect on integration. The antiviral activity of these compounds is due exclusively to inhibition of one of the two catalytic functions of integrase, strand transfer.     

Suppression of HIV infection in AZT-treated SCID-hu mice

The SCID-hu mouse, engrafted with human hematolymphoid organs, is permissive for infection with the human immunodeficiency virus (HIV). This mouse model was used to test compounds for antiviral efficacy. Two weeks after infection with HIV, 100 percent (40/40) of SCID-hu mice were positive for HIV by the polymerase chain reaction. When first treated with 3'-azido-3'-deoxythymidine (AZT), none (0/17) were HIV-positive by this assay. However, AZT-treated SCID-hu mice did have a few infected cells; after AZT treatment was stopped, viral spread was detected by polymerase chain reaction in such mice. Thus, the SCID-hu mouse provides a means to directly compare new antiviral compounds with AZT and to further improve antiviral efficacy.     

Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1

COP9 signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit was found to underlie CSN's Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria, and eukaryotes, including the Rpn11 subunit of the 26S proteasome. Metal chelators and point mutations within JAMM abolished CSN-dependent cleavage of Nedd8 from Cul1, yet had little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster required an intact Csn5 JAMM domain. We propose that JAMM isopeptidases play important roles in a variety of physiological pathways.     

Protein design of an HIV-1 entry inhibitor

Human immunodeficiency virus type-1 (HIV-1) membrane fusion is promoted by the formation of a trimer-of-hairpins structure that brings the amino- and carboxyl-terminal regions of the gp41 envelope glycoprotein ectodomain into close proximity. Peptides derived from the carboxyl-terminal region (called C-peptides) potently inhibit HIV-1 entry by binding to the gp41 amino-terminal region. To test the converse of this inhibitory strategy, we designed a small protein, denoted 5-Helix, that binds the C-peptide region of gp41. The 5-Helix protein displays potent (nanomolar) inhibitory activity against diverse HIV-1 variants and may serve as the basis for a new class of antiviral agents. The inhibitory activity of 5-Helix also suggests a strategy for generating an HIV-1 neutralizing antibody response that targets the carboxyl-terminal region of the gp41 ectodomain.     

Molecular modeling of the HIV-1 protease and its substrate binding site

The human immunodeficiency virus (HIV-1) encodes a protease that is essential for viral replication and is a member of the aspartic protease family. The recently determined three-dimensional structure of the related protease from Rous sarcoma virus has been used to model the smaller HIV-1 dimer. The active site has been analyzed by comparison to the structure of the aspartic protease, rhizopuspepsin, complexed with a peptide inhibitor. The HIV-1 protease is predicted to interact with seven residues of the protein substrate. This information can be used to design protease inhibitors and possible antiviral drugs.     

Selective elimination of HIV-1-infected cells with an interleukin-2 receptor-specific cytotoxin

Infection by human immunodeficiency virus type 1 (HIV-1) is associated with cellular activation and expression of the interleukin-2 (IL-2) receptor. A genetically engineered fusion toxin, DAB486 IL-2, that contains the enzymatic site and translocation domain of diphtheria toxin and the receptor binding domain of IL-2 specifically kills cells that express high-affinity IL-2 receptors. This toxin selectively eliminated the HIV-1-infected cells from mixed cultures of infected and uninfected cells and inhibited production of viral proteins and infectious virus. Thus, cellular activation antigens present a target for early antiviral intervention.     

The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility

Segmental duplications in the human genome are selectively enriched for genes involved in immunity, although the phenotypic consequences for host defense are unknown. We show that there are significant interindividual and interpopulation differences in the copy number of a segmental duplication encompassing the gene encoding CCL3L1 (MIP-1alphaP), a potent human immunodeficiency virus-1 (HIV-1)-suppressive chemokine and ligand for the HIV coreceptor CCR5. Possession of a CCL3L1 copy number lower than the population average is associated with markedly enhanced HIV/acquired immunodeficiency syndrome (AIDS) susceptibility. This susceptibility is even greater in individuals who also possess disease-accelerating CCR5 genotypes. This relationship between CCL3L1 dose and altered HIV/AIDS susceptibility points to a central role for CCL3L1 in HIV/AIDS pathogenesis and indicates that differences in the dose of immune response genes may constitute a genetic basis for variable responses to infectious diseases.     

水稻GS3蛋白及其与Gβ蛋白复合物的表达及结晶初筛

【目的】阐明GS3蛋白在水稻中的调控机制,为解析植物G蛋白的结构及完善G蛋白的调控网络打下基础。【方法】分别采用表达载体pGEX-6p-1和pET-28b-sumo诱导表达GS3蛋白N端结构域OSR和GS3蛋白C端结构域,再使用凝胶过滤柱Superdex 200/7510/300和阴例子交换层析柱Mono Q 5/50纯化表达蛋白,通过筛选优化晶体和X射线衍射的方法对GS3及GS3与Gβ共表达复合物的结构进行研究。【结果】将构建的重组蛋白OSR-pGEX-6p-1与GβN- pET-28b-sumo转入大肠杆菌BL21(DE3)中共表达,经GST beads亲和层析可获得2条条带,分别为OSR-GST(31 kD)和GβN-sumo(26 kD),再过Ni2+ beads亲和层析同样有2条条带,而SDS-PAGE凝胶电泳结果显示有4条条带,分别为GST(26 kD)、sumo(17 kD)、GβN(9 kD)和OSR(5 kD)。经阴例子交换层析柱Mono Q 5/50分离纯化可获得GβN(9 kD)和OSR(5 kD)二者的复合物。将纯化后的复合物浓缩至12 mg/mL进行晶体初筛,但未获得结晶。【结论】GS3和Gβ互作是通过GS3蛋白N端结构域OSR与Gβ N端结构域的结合而实现,其结果符合经典的G蛋白异源三聚体模型。     

Herpes simplex virus encephalitis in human UNC-93B deficiency

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is the most common form of sporadic viral encephalitis in western countries. Its pathogenesis remains unclear, as it affects otherwise healthy patients and only a small minority of HSV-1-infected individuals. Here, we elucidate a genetic etiology for HSE in two children with autosomal recessive deficiency in the intracellular protein UNC-93B, resulting in impaired cellular interferon-alpha/beta and -lambda antiviral responses. HSE can result from a single-gene immunodeficiency that does not compromise immunity to most pathogens, unlike most known primary immunodeficiencies. Other severe infectious diseases may also reflect monogenic disorders of immunity.     

鸡α-干扰素在毕赤酵母中的分泌表达、纯化及其抗病毒活性测定

本试验将已构建的鸡α-干扰素重组酵母表达质粒pPIC-ChIFNα克隆至Pichia pastorisGS115基因组中,从阳性转化子中筛选出2株能高效表达鸡α-干扰素的重组酵母菌株,并对表达产物进行了纯化和抗病毒活性的测定。结果表明,Pichia pastoris表达的重组ChIFNα具有较高的抗病毒活性,其活性单位为570×104U/ml,结合蛋白定量计算其比活性为2 980×104U/mg。     

Containing pandemic influenza at the source

Highly pathogenic avian influenza A (subtype H5N1) is threatening to cause a human pandemic of potentially devastating proportions. We used a stochastic influenza simulation model for rural Southeast Asia to investigate the effectiveness of targeted antiviral prophylaxis, quarantine, and pre-vaccination in containing an emerging influenza strain at the source. If the basic reproductive number (R0) was below 1.60, our simulations showed that a prepared response with targeted antivirals would have a high probability of containing the disease. In that case, an antiviral agent stockpile on the order of 100,000 to 1 million courses for treatment and prophylaxis would be sufficient. If pre-vaccination occurred, then targeted antiviral prophylaxis could be effective for containing strains with an R0 as high as 2.1. Combinations of targeted antiviral prophylaxis, pre-vaccination, and quarantine could contain strains with an R(0) as high as 2.4.     

mtDNA A3243G 点突变小鼠模型的建立及其致病机制探讨

    利用显微注射线粒体技术建立转人线粒体小鼠模型,研究外源突变mtDNA在不同组织的分布及遗传规律,探讨mtDNA A3243G点突变对线粒体功能的影响.从健康成人及2型糖尿病患者(携带mtDNA 3243A-G突变)血液标本中分离有活性的线粒体,将其显微注射至小鼠受精卵,胚胎移植,产出仔鼠后利用分子生物学方法检测人mtDNA及mtDNA A3243G点突变.获得嵌合体小鼠后,对其空腹血糖和全血乳酸进行测定,并使用荧光法和比色法分析A3243G点突变小鼠重要脏器组织细胞活性氧生成量(ROS)、线粒体复合酶Ⅰ和Ⅳ活力及线粒体ATP合成活力的变化.研究结果显示:在1只雌性(转健康人线粒体)和2只雄性小鼠(转患者线粒体)中检测到人mtDNA,其中2只雄性小鼠携带mtDNA 3243A-G突变;将嵌和体雌鼠与野生型C57BL/6J 雄鼠交配后,在1只后代仔鼠中检测到人mtDNA;人mtDNA仅在嵌合小鼠的部分组织中表达.在含有mtDNA A3243G突变的组织中发现,线粒体复合酶Ⅰ、Ⅳ活力降低,ATP合成速率下降,ROS水平升高,说明A3243G点突变能损伤线粒体正常功能从而导致疾病的发生.综上所述,本研究利用显微注射法成功建立了嵌和小鼠,引入了致病性的点突变,为线粒体疾病的研究提供了良好的思路.     

A novel Arabidopsis miRNA,ath-miR38-3P,is involved in response to Sclerotinia sclerotiorum infection

Plant defense responses against penetration or colonization of pathogens are mediated by activation and repression of a large array of genes. Host endogenous small RNAs are essential in gene expression reprogramming process. We identified a new Arabidopsis micro RNA(mi RNA) ath-mi R38-3P by high-throughput sequencing and further confirmed it by Northern blot assay. Interestingly, ath-miR38-3P was highly induced after infection of the pathogen Sclerotinia sclerotiorum. Further analysis based on the mi RNA target database demonstrated that ath-mi R38-3P might target to five putative genes: AT2G03140, AT5G59430, AT5G66320, AT1G36620 and AT3G03820. To confirm the target, we conducted the quantitative real-time PCR to observe the expression pattern of each candidate gene. The results showed that only AT3G03820 was down-regulated after inoculation of S. sclerotiorum. In addition, overexpression of ath-miR38-3P down-regulates AT3G03820, suggesting AT3G03820 might represent the target for ath-mi R38-3P. Our results may provide the useful information for further studying the biological function of a novel ath-mi R38-3P and its targets in Arabidopsis-Sclerotinia interaction.     

大蒜茎粉和牛至草粉对镜鲤生长性能、消化酶活性以及血清生化指标的影响

试验研究了大蒜茎粉和牛至草粉对镜鲤(Cyprinus carpio L.minor)生长性能、消化酶活性及血清生化指标的影响,为进一步开发利用天然植物添加剂提供理论参考。试验共设7个处理组:G1空白对照组,G2添加10 mg/kg黄霉素,G3、G4分别添加0.5%和2.5%大蒜茎粉,G5、G6分别添加0.1%和0.5%牛至草粉,G7添加0.5%大蒜茎粉和0.5%牛至草粉。每个处理设3个重复,每个重复10尾鱼,初始体重为(201.45±16.25) g,试验共进行8周。结果表明:与G1和G2相比,G3显著提高了特定生长率和增重率,显著降低了饵料系数(P<0.05),G3、G4和G5、G6显著提高了蛋白质效率(P<0.05)。与G1对比,G3显著提高了肝胰脏、前肠和中肠蛋白酶活性(P<0.05),肝胰脏和中肠脂肪酶活性(P<0.05)。G4显著提高了肝胰脏蛋白酶活性(P<0.05)。G5、G6、G7比G1和G2显著提高了前肠淀粉酶和脂肪酶活性(P<0.05)。与G1相比,G3、G6和G7血总蛋白、白蛋白、球蛋白浓度显著提高(P<0.05),G2血清总胆固醇、谷草转氨酶和谷丙转氨酶显著降低(P<0.05),各试验组肌酐均无显著差异(P>0.05)。结论:饲料中添加0.5%大蒜茎粉可以有效提高镜鲤生长性能,但是添加牛至草粉促生长作用不明显。     

长期施用袋控缓释肥对南方红壤中磷素形态及有效性的影响

磷供应不足是南方红壤区农、林业生产所面临的主要问题,为探究施用袋控缓释肥是否对提高磷素有效性及有效磷组分含量有显著正向效应,以广西国有高峰林场桉树人工林红壤为研究对象,设计袋控缓释肥常规施肥(G1)、袋控缓释肥减量施肥(G2)、传统复混肥(G3)、施用基肥(G4)、不施肥(G5)等不同肥料种类与施肥量对比试验,采用土壤磷分级方法测定土壤中磷素各组分含量,以冗余分析方法研究各组分对林木生长量的影响。结果表明：（1）相比传统施肥,袋控缓释肥处理显著提升了土壤中AP、Pi、Fe-P等组分含量;（2）G2、G1的磷活化系数显著高于其余3个处理,PAC排序顺序为G2>G1>G4>G5>G3;（3）冗余分析结果表明,PAC、Fe-P提升对植物生长量均有显著正效应。在南方红壤区桉树人工林经营过程中施用袋控缓释肥可以促进土壤中磷素的累积并提高磷活化率。袋控缓释肥具有较高磷素利用效率的机制可能是通过磷元素的持续输入以及阻隔磷元素与酸性土壤的接触进而减少磷素在土壤中的固定,达到保证林木生长过程中的磷素供给。     

建鲤IGFⅠa基因的SNPs位点筛选及其与增重的相关性分析

机体生长主要受到促生长激素释放激素—生长激素—胰岛素样生长因子(GHRH-GH-IGFs)生长轴调控,类胰岛素生长因子IGFⅠ是生长激素（GH）发挥生物学功能的重要传导因子。实验特异性扩增了建鲤(Cyprinus carpio var. jian) IGFⅠa基因的5个外显子和3个内含子（内含子1、内含子3和内含子4）。通过比对10尾建鲤的序列,共找到SNPs位点8个。使用PCRRFLP方法检测了5个家系共372尾建鲤的内含子1_C175G,12个家系共987尾的内含子1_A993G和内含子4_A511C 3个位点。内含子1_C175G雌雄个体均以GG型频率为最高,分别是0.44和0.43;此位点在幼鱼和成鱼时期与雌、雄建鲤增重均无相关性;内含子1_A993G雌雄个体均以AG型频率最高,分别是0.76和0.72;此位点在成鱼阶段与雄性建鲤增重呈显著相关(P<0.05);内含子4_A511C在雌雄个体中均以CC型频率最高,分别是0.48和0.47;其在幼鱼和成鱼阶段均与增重存在极显著的相关性(P<0.01)。本次试验表明在建鲤体内,IGFⅠ基因在不同生长阶段的表达量不同,且处于同一生长阶段的雌雄个体间的表达也存在差异。内含子1_A993G、内含子4_A511C均与建鲤增重存在相关性,可以考虑作为建鲤分子育种的相关依据。     

N-terminal acetylation acts as an avidity enhancer within an interconnected multiprotein complex

Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.