Ribosome inactivating proteins from plants inhibiting viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.     

核糖体失活蛋白抗HIV-1作用的研究进展

核糖体失活蛋白(RIPs)抗HIV-1活性研究已有十几年的历史。RIPs类化合物代表了抗HIV/AIDS天然产物或先导化合物发展的一个重要方向。本文从介绍RIPs的酶活性及其抗HIV-1活性入手,对RIPs抗HIV-1的可能机制,从与RIPs酶活性的关系、诱导HIV-1感染细胞的凋亡及相应的信号转导、诱发活性氧的产生,以及对HIV-1整合酶的抑制作用等几个方面做了较详尽的阐述,并对RIPs的结构修饰和抗HIV-1构效关系进行了综述。对RIPs类化合物在抗病毒领域进行深入而系统地研究,能拓宽其在抗HIV/AIDS临床上的进一步应用。     

17种植物中蛋白质提取物的抗HIV—1活性

以化合物对ＨＩＶ－１诱导Ｃ８１６６细胞形成合胞体的抑制实验和化合物对ＨＩＶ－１感染细胞的保护实验作为初筛方法,筛选了来源７科１７种植物的４７个样品,其中８个样品经测定是核糖体失活蛋白,其余为粗提蛋白。     

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCS(C2), TCS(C4), and TCS(C14)) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCS(C19aa) and TCS(KDEL) having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS.     

重组蓖麻毒素A链与几种天然单链核糖体失活蛋白的活性研究

采用ｐＥｘＳｅｃⅠ载体系统进行了蓖麻毒素Ａ链的原核表达,经ＣＭ－Ｓｅｐｈａｒｏｓｅ一步纯化后,获得了纯度约８０％的重组蓖麻毒素Ａ链．将其与几种天然单链核糖体失活蛋白进行了超螺旋ＤＮＡ裂解研究和无细胞体系中蛋白合成抑制试验,结果表明,重组蓖麻毒素Ａ链具有类似于天然单链核糖体失活蛋白的活性,但两种测活方法之间没有明显的相关性     

Purification and Characterization of a New Ribosome Inactivating Protein from Cinchonaglycoside C-treated Tobacco Leaves

A new ribosome-inactivating protein(RIP)with a molecular weight of 31 kDa induced by Cinchonaglycoside C(1)designatedCLP31,was isolated from tobacco leaves.Analysis of this protein sequence indicated that it belongs to the RIP family and itwas distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence.CIP31 can directly impairsynthesis of coat protein(CP)of tobacco mosaic virus(TMV),which resulted in inhibition of TMV long distance movementand multiplication in tobacco plants at concentrations of ng/mL.Furthermore,no toxicity was shown to the growth andfertility of the plants.CIP31 was synthesized only in the presence of Cinchonaglycoside C(1)and was independent of thesalicylic acid(SA)signal pathway.We provided evidence for the SA-independent biological induction of resistance.     

Purification and Characterization of a New Ribosome Inactivating Protein from Cinchonaglycoside C-treated Tobacco Leaves

A new ribosome-inactivating protein （RIP） with a molecular weight of 31 kDa induced by Cinchonaglycoside C （1） designated CIP31, was isolated from tobacco leaves. Analysis of this protein sequence indicated that it belongs to the RIP family and it was distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence. CIP31 can directly impair synthesis of coat protein （CP） of tobacco mosaic virus （TMV）, which resulted in inhibition of TMV long distance movement and multiplication in tobacco plants at concentrations of ng/mL. Furthermore, no toxicity was shown to the growth and fertility of the plants. CIP31 was synthesized only in the presence of Cinchonaglycoside C （1） and was independent of the salicylic acid （SA） signal pathway. We provided evidence for the SA-independent biological induction of resistance.     

快速检测核糖体失活蛋白活性质粒的构建及应用

核糖体失活蛋白专一地断裂28S rRNA第4 324位的腺嘌呤与核糖之间的N-糖苷键,具有特异破坏核糖体的结构,抑制蛋白质生物合成的功能。核糖体失活蛋白在医疗方面有极大的应用价值。为了能简单快速筛选出核糖体失活蛋白,本实验构建了一种包含核糖体失活蛋白识别位点的双荧光素酶质粒psiCHECKTM-2-F28RNA。用具有N 糖苷酶活性的苦荞凝集素(tartary buckwheat lectin,TBL)作用于psiCHECKTM-2-F28RNA质粒,电泳检测发现,TBL可以将质粒DNA由超螺旋型切割为缺刻型。将psiCHECKTM-2-F28RNA转染HCT116细胞,发现海肾/萤火虫荧光比值也明显降低,表明构建的质粒可以用于检测核糖体失活蛋白对细胞的毒性作用。当将psiCHECKTM-2-F28RNA中的GAGA序列中腺嘌呤分别突变后进行同样实验,确定该质粒中的GAGA为核糖体失活蛋白的识别位点。进一步构建包含GAGA特征序列的Wnt1-3′UTR区的质粒psiCHECKTM-2-Wnt1-3′UTR,实验也发现,在胞外和胞内TBL与psiCHECKTM-2-Wnt1-3′UTR都具有相互作用,表明细胞内具有GAGA序列的mRNA也可能成为核糖体失活蛋白的靶点。选用几种食源性作物中提取的蛋白质,分别与psiCHECKTM-2-F28RNA作用,进行体外检测,结果显示,该质粒能快速地筛选来源于不同生物的核糖体失活蛋白。这些结果表明,本实验构建的psiCHECKTM-2-F28RNA质粒,可用于核糖体失活蛋白的快速筛选和酶活性鉴定。     

黑木耳菌丝体核糖体失活蛋白的研究

目的：由悬浮培养的黑木耳菌丝体中分离纯化黑木耳的核糖体失活蛋白,对其生化性质及生物学活性进行研究。方法：实验中采用了DEAE-离子交换纤维素,Affi-Gel Blue Gel亲和与Bio-Gel 100柱层析方法。结果：从100g悬浮培养黑木耳菌丝体中得到4.14mg的核糖体失活蛋白,命名为Auriculin。同时证明它在家兔网织红细胞裂解系统中具有抑制蛋白质的翻译活性。结论：经试验证明和文献检索,Auriculin为黑木耳菌分离纯化获得的核糖体失活蛋白,一种新蛋白质。     

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCS(M(120-123)), TCS(E160A/E189A), with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCS(R122G), which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property.     

繁缕核糖体失活蛋白基因克隆及序列分析

采用同源克隆结合RACE法,克隆了繁缕核糖体失活蛋白的全长cDNA,命名为q3(GenBank accession GQ870262)。序列分析结果表明,q3的开放阅读框(ORF)长780 bp,编码259个氨基酸。序列G+C含量为41.5%,与大部分Ⅰ型RIP基因相近。q3编码的蛋白质命名为Q3,理论分子量为28.16 kD,pI为9.44,均与Ⅰ型核糖体失活蛋白相近;包含由23个氨基酸组成的信号肽。功能结构域分析发现,该蛋白含有3个蛋白激酶磷酸化位点、4个络氨酸蛋白激酶磷酸化位点和7个N-肉豆蔻酰化位点。三级结构预测发现,有35.52%的氨基酸残基参与了α螺旋,24.32%的氨基酸残基组成延伸链,40.15%的氨基酸残基随机缠绕其中。基于繁缕及其近缘种核糖体失活蛋白的氨基酸序列构建的系统发育树显示,其结构与经典分类结果基本一致。     

苦瓜核糖体失活蛋白的分离纯化及其抑制烟草花叶病毒活性

目的:探讨分离纯化苦瓜种子核糖体失活蛋白(RIP)的方法,并研究利用纯化后的RIP抑制烟草花叶病毒(TMV)的活性。方法:用盐溶液抽提总蛋白后,经30%～90%的(NH4)2SO4分级沉淀,制成粗提液。用CMSepharoseFastFlow离子交换层析结合SephadexG-75凝胶柱分离纯化RIP。结果与结论:经SDS-PAGE检测及RNAN-糖苷酶活性鉴定,确定最终得到的蛋白为苦瓜RIP;所纯化的RIP对感染黄瓜和番茄的TMV有明显的抑制作用,但RIP的浓度并不与其抑制TMV的活性呈正相关。     

转望江南核糖体失活蛋白基因cassin烟草及其对烟草花叶病毒的抗性

通过构建植物表达载体,由农杆菌介导,将望江南核糖体失活蛋白基因cassin转入烟草。PCR和Southern blot杂交结果证明：外源基因已经以单拷贝整合到烟草基因组内,并且在后代发生遗传分离。RT—PCR和Northern blot杂交结果显示：外源基因可以正常转录。用不同浓度的TMV机械摩擦接种转基因T1、T2代各3个自交株系,以非转基因烟草为阴性对照,实验结果表明转基因烟草对TMV表现出不同程度的抗性。     

Isolation and Characterization of cDNAs Encoding Ribosome Inactivating Protein from Dianthus sinensis L.

To isolate a ribosome inactivating protein (RIP) gene, six plant species were surveyed for antiviral activity. Crude proteins extracted from these plants were tested for the antiviral activity against tobacco mosaic virus (TMV) in Nicotiana glutinosa. All the plants, Spinacia oleracea, Amaranthus lividus, Dianthus superbus, Dianthus sinensis and Celosia cristata, with an exception of Oenanthe stolonifera, presented 70–90% inhibition of viral infectivity. In an attempt to search for the RIP gene from D. sinensis, partial cDNA was obtained by polymerase chain reaction (PCR) of the poly(A)⁺ RNA from D. sinensis leaves. DNA gel blot analysis showed that D. sinensis has multi-copy RIP genes. The expression of RIP gene was investigated in the flower, leaf, root and stem of D. sinensis, and was found to be most abundant in the leaf. Using the partial cDNA as a probe, seven full-length cDNAs were isolated from a library prepared from D. sinensis leaves. They were divided into three groups on the basis of their nucleotide sequence homology. The three representative clones, cDsRIP1, cDsRIP2 and cDsRIP3 were completely sequenced. They all had an open reading frame of 882 bp. The cDsRIP2 showed 79% homology with dianthin 30 and saporin genes; 59% with PAP and Mirabilis antiviral protein MAP genes. From the analysis of deduced amino acid sequences, it was predicted that D. sinensis RIP cDNAs might have a putative signal peptide of 23 amino acid residues at their N-terminus. When the cDNA was expressed in E. coli, the bacteria was unable to grow upon IPTG induction, suggesting that expression of the gene renders toxicity to E. coli cells.     

STD门诊生殖器溃疡性疾病的病原学研究

目的 了解生殖器溃疡性疾病（GUD）的发病率、病因及其与HIV感染的关系。方法 用暗视野显微镜检查（D-F）、梅毒血清学试验（STS）、酶免疫法（EIA）检测HSV抗原和HD培养等方法检测322例生殖器溃疡标本中TP、HSV和HD,并进行血清HIV抗体检测。结果 在8 999例STD门诊患者中,GUD患者322例（3.58%,322/8 999）;在322例GUD患者中,梅毒85例（26.4%,85/322）,生殖器疱疹74例（26.09%,84/322）,软下疳0例（0,0/322）,病因不明的GUD为153例（47.52%,153/322）;其中GUD患者的HIV感染率为1.86%（6/322）,梅毒患者的HIV感染率为4.70%（4/85）,生殖器疱疹患者的HIV感染率为1.19%（1/84）,其他GUD的HIV感染率为0.65%（1/153）。比较三者的HIV感染率发现,梅毒的HIV感染率高于生殖器疱疹和其他GUD患者（4.70%vs1.19%,χ^2=0.24,P＞0.05,OR=3.04,95%CI=0.31-29.93;4.70% vs 0.65%,χ^2=1.29,P＞0.05,OR=5.63,95%CI=0.58-55.06）,但两者差异无显著性;GH患者的HIV感染率与其他GUD患者的HIV感染率相比,差异无显著性（1.19% vs 0.65%,χ^2=0.00,P＞0.05,OR=1.85,95% CI=0.11-30.00）。结论 在性病门诊中,GUD的主要病因为梅毒和生殖器疱疹,且存在混合性感染;梅毒与HIV的感染有一定关系.     

Chemical modifications of pokeweed antiviral protein: effects upon ribosome inactivation, antiviral activity and cytotoxicity

Pokeweed antiviral protein (PAP) is a protein known to inactivate eukaryotic ribosomes by an unknown enzymatic action and inhibit the production of mammalian viruses in tissue culture. This protein was subjected to a variety of chemical modifications to determine their effects upon ribosomal inactivation, antiviral action, and cytotoxicity. It was found that modifications of a number of different amino acid residues had similar effects upon all 3 activities. Also the inactivation of PAP with diethylpyrocarbonate was not due to its reaction with a histidine residue but to a modification of an unidentified amino acid residue.     

子宫颈糜烂病毒病因的探讨

491份宫颈拭子病毒分离结果表明:糜烂宫颈单纯疱疹病毒(HSV)分离阳性率(30.8％)是正常宫颈(2.6％)的11.8倍,用人干扰素治疗一个疗程后,病毒分离率下降至疗前的1/4.36例糜烂宫颈活体组织DNA分子杂交表明,乳头瘤病毒16型(HPV-16)阳性者占52.8和,HPV-18占17.9％,HPV-6B占28.1％,HPV-11占7.7％,251例宫颈糜烂患者经人(?)D型基因工程干扰素双盲对比治疗后,总有效率达93.8％,显效率达60％,分析临床疗效与HSV分离率的变化表明,临床有效病例中有35％(49/140)在治疗后病毒阴转,有57％在疗前疗后均未分离出HSV,有5％在疗前疗后保持阳性不变,有2.9％疗前阴性,疗后阳性,上述结果表明,HSV和HPV与慢性宫颈炎有一定关系。     

PCR在猴B病毒与猴病毒8型鉴别中的应用

目的为了使BV147与SA8相区别,并进一步分析BV147基因结构特点.方法用PCR方法扩增BV147DNA,并对扩增片段进行克隆测序.结果该序列与美国BVE2490株部分基因(UL27)相对应位置的核苷酸同源性为99.54%,与SA8相对应位置的核苷酸同源性为89.91%.结论进一步证明了新分离物为B病毒.     

速成单层法半微量病毒空斑技术的研究

本文报道一种不需预先制备细胞单层的速成单层法半微量病毒空斑技术(简称速成法)及其在空斑中和试验与空斑纯化试验中的应用。作者以脊髓灰质炎病毒为代表,对速成法的实验条件、可靠性、敏感性、重复性等作了系统研究,并以此法对单纯疱疹病毒、呼吸道合胞病毒和水泡性口炎病毒进行了空斑滴定。结果提示速成法简单快速、经济方便、敏感稳定、可靠易行,可能具有较大的实用和推广价值。