人中性粒细胞多肽HNP1，3体外抗单纯疱疹病毒Ⅰ型的作用

体外观察人中性粒细胞多肽1,3(Humanneutrophilpeptide,HNP1,3)及阿昔洛韦(Acyclovir,ACV)对单纯疱疹病毒-Ⅰ型(Herpessimplexvirus1,HSV-1)的抑制作用。以Vero细胞为靶细胞,用各种浓度HNP1,3与游离病毒颗粒(直接失活组)及感染病毒后的靶细胞(复制抑制组)进行相互作用,镜下观察各药物对HSV-1致细胞病变效应的抑制作用,并采用ELISA法测定感染48h后药物对HSV-1囊膜糖蛋白分泌的抑制作用。MTT法检测各药物对细胞的毒性作用。结果显示直接失活组中,HNP1,3可使HSV-1的致细胞病变效应减轻,对HSV-1直接失活的50%有效浓度(EC50)为8.1μg/mL、10.03μg/mL;复制抑制组中,ACV使HSV-1的致细胞病变效应减轻,EC50为0.68μg/mL。MTT检测结果表明HNP1,3在治疗浓度范围内无明显细胞毒性。以上结果表明HNP1,3除具有较强的抗菌作用和抗人类免疫缺陷病毒Ⅰ型(Humanimmunodeficiencyvirus1,HIV-1)活性外,还能失活HSV-1病毒颗粒,从而逆转病毒及其蛋白的病毒效应(致细胞病变)和抑制病毒蛋白质的合成。     

人中性粒细胞多肽HNP1,3体外抗单纯疱疹病毒Ⅰ型的作用

体外观察人中性粒细胞多肽1,3(Human neutrophil peptide,HNP1,3)及阿昔洛韦(Acyclovir,ACV)对单纯疱疹病毒Ⅰ型(Herpes simplex virus 1,HSV-1)的抑制作用.以Vero细胞为靶细胞,用各种浓度HN1,3与游离病毒颗粒(直接失活组)及感染病毒后的靶细胞(复制抑制组)进行相互作用,镜下观察各药物对HSV-1致细胞病变效应的抑制作用,并采用ELISA法测定感染48h后药物对HSV-1囊膜糖蛋白分泌的抑制作用.MTT法检测各药物对细胞的毒性作用.结果显示直接失活组中,HNP1,3可使HSV-1的致细胞病变效应减轻,对HSV-1直接失活的50%有效浓度(ECs0)为8.1μg/mL、10.03μg/mL;复制抑制组中,ACV使HSV-1的致细胞病变效应减轻,EC5o为0.68μg/mL.MTT检测结果表明HNP1,3在治疗浓度范围内无明显细胞毒性.以上结果表明HNP1,3除具有较强的抗菌作用和抗人类免疫缺陷病毒Ⅰ型(Human immunodeficiency virus 1,HIV-1)活性外,还能失活HSV-1病毒颗粒,从而逆转病毒及其蛋白的病毒效应(致细胞病变)和抑制病毒蛋白质的合成.     

单纯疱疹病毒1型VP16蛋白和VHS蛋白研究进展

单纯疱疹病毒1型(HSV-1)为有包膜的DNA病毒,能引起皮肤性疱疹、角膜炎、脑炎等症状。HSV-1感染细胞后,要么进入裂解性感染阶段,要么进入潜伏感染阶段。受感染的细胞常会启动免疫系统抵抗病毒,而病毒却通过某种机制巧妙地逃避宿主的免疫反应并进入潜伏。进入潜伏感染阶段的病毒又会因机体受某种刺激而被激活进入裂解感染期。在这期间,有两个关键的病毒蛋白一间层蛋白(Viral protein16,VP16)和内膜蛋白(Virion host shutoff protein,VHS)倍受关注,它们既是HSV-1的结构蛋白,在病毒复制晚期参与病毒颗粒的组装,同时又作为重要的功能蛋白…     

单纯疱疹病毒1型VP16蛋白和VHS蛋白研究进展

单纯疱疹病毒1型(HSV-1)为有包膜的DNA病毒,能引起皮肤性疱疹、角膜炎、脑炎等症状.HSV-1感染细胞后,要么进入裂解性感染阶段,要么进入潜伏感染阶段.受感染的细胞常会启动免疫系统抵抗病毒,而病毒却通过某种机制巧妙地逃避宿主的免疫反应并进入潜伏.进入潜伏感染阶段的病毒又会因机体受某种刺激而被激活进入裂解感染期.在这期间,有两个关键的病毒蛋白一间层蛋白(Viral protein 16,VP16)和内膜蛋白(Virion host shutoff protein,VHS)倍受关注,它们既是HSV-1的结构蛋白,在病毒复制晚期参与病毒颗粒的组装,同时又作为重要的功能蛋白,在病毒感染早期发挥重要的转录调节功能.下面就这两个蛋白相关功能的研究进展作一简要综述:     

HSV-1感染对人星形胶质瘤细胞增殖、凋亡和细胞周期的影响

目的:研究单纯疱疹病毒1型(Herpes simplexvires,HSV-1)感染对人星形胶质瘤细胞U251增殖、凋亡和细胞周期的影响.方法:以感染复数(MOI)为5的HSV-1感染体外培养的U251细胞,在感染后24 h、48 h、72 h和96 h用倒置显微镜观察U251细胞的形态改变:用MTT法、流式细胞术观察HSV-1感染对U251细胞增殖、凋亡和细胞周期的影响.结果:①U251细胞在感染24h后开始出现细胞融合,48 h后开始出现典型的细胞病变效应(Cytopathic effect,CPE),72h后超过80%的细胞出现CPE,.96h后细胞大部分死亡.②MTT法显示HSV-1感染U251细胞24 h、48 h、72 h及96 h的U251细胞OD值均低于对照组(P<0.05).③HSV-1感染U251细胞12h后凋亡率与对照组无显著差异(P0.05),感染24h和36h后凋亡率比相应对照组有显著差别(p<0.05).④HSV感染12h、24h和36h后均可引起U251细胞S期细胞增多和G0/G1期细胞减少,24 h后G2/M期细胞比例开始增加.结论:HSV.1能感染体外培养的U251细胞,抑制其增殖,促进其凋亡并影响其细胞周期.     

人类巨细胞病毒、EB病毒和单纯疱疹病毒1型与慢性牙周炎的相关性研究

目的　研究人类巨细胞病毒( HCMV)、Epstein- Barr病毒( EBV)和单纯疱疹病毒1型( HSV- 1)与慢性牙周炎的相关性。方法　收集6 2例慢性牙周炎患者(男性2 7例,女性35例;平均年龄5 3岁)的牙周炎部位,轻度龈炎部位的龈下菌斑,提取DNA后使用巢式PCR检测HCMV、EBV和HSV- 1,比较分析它们在同一患者不同部位的检出率。结果　牙周炎部位的HCMV检出率为38.7% ,EBV的检出率为5 8.0 % ,HSV- 1的检出率为30 .6 % ,2种以上病毒合并感染的检出率为4 0 .3% ;轻度龈炎部位的HCMV检出率为12 .9% ,EBV为19.4 % ,HSV- 1为9.7% ,2种以上病毒合并感染的检出率为8.0 %。这3种病毒及其合并感染在牙周炎部位的检出率均高于轻度龈炎部位,差异有统计学意义( P<0 .0 5 )。结论　提示HCMV、EBV、HSV- 1与慢性牙周炎有相关性。     

靶向ICP4的小干扰RNA对HSV-1病毒复制能力的影响

HSV-1是一种嗜神经病毒,能引起一系列神经系统严重症状,然而目前抗HSV-1药物易反弹、不能完全清除潜伏的病毒。ICP4对HSV复制、转录起主要调节作用,决定着溶细胞型感染或潜伏状态的平衡点。为了探寻新的抗病毒策略,本课题以HSV-1ICP4基因为靶点,设计合成2对siRNA,并构建重组真核慢病毒表达质粒pL-KO-puror-hU6-siRNA,通过脂质体转染和嘌呤霉素筛选建立靶向ICP4的4个siRNA单克隆细胞系,Real-timePCR法检测细胞系中ICP4的mRNA表达水平,TCID50法检测siRNA对HSV-1病毒复制能力的影响。结果显示靶向siRNA能有效抑制单克隆细胞系中的ICP4表达,并且抑制ICP4的表达后HSV-1病毒复制能力明显减弱,表明靶向ICP4的siRNA对HSV-1复制有明显抑制作用,且多位点siRNA联合干扰对病毒复制有协同抑制效果,有望应用于生物抗病毒药物的制备。     

以重组人巨细胞病毒pp65制备的多克隆抗体建立检测感染性病毒颗粒方法

人巨细胞病毒(HCMV)活动性感染是造成器官移植失败的重要原因。建立灵敏、特异的检测方法可为及早发现感染、及时给予抗病毒治疗提供依据。以离子交换和分子筛方法纯化重组表达的HCMVpp65(rp65hp)抗原,经两步纯化后,rp65hp纯度达到95%。免疫家兔制备的抗血清,ELISA抗体滴度高达1∶2560000;免疫印迹证明能与HCMVpp65抗原特异反应。将HCMV病毒感染细胞,以纯化的多克隆抗体建立了间接免疫荧光法,成功地检测到细胞中的病毒。因此,该方法为临床检测HCMV病毒活动性感染奠定了良好的基础。     

HSV-1 HF株扩增子载体的构建及其在不同HSV血清型间的通用性研究

旨在构建HSV-1HF株的扩增子载体,研究其在不同血清型HSV辅助下的包装通用性。经酶切HF株的BAC-HSV-1,获得oriS和pac元件并测序。以pSilencer2.0-U6为骨架,以DsRed为报告基因构建HSV-1HF株的扩增子载体,利用脂质体2000转染扩增子载体至Vero细胞,分别应用HSV-1HF株和HSV-2HG52辅助HSV-1扩增子载体进行包装,待产生细胞病变效应后取上清,再次感染Vero细胞,观察Vero细胞内红色荧光蛋白表达情况。本研究首次构建了HSV-1HF株的扩增子载体,鉴定了HSV-1HF株oriS和pac元件,HSV-1HF株扩增子载体可以被HSV-1HF株和HSV-2HG52株包装并扩增。     

Polyamine metabolism in MRC5 cells infected with different herpesviruses.

Both methyglyoxal bis(guanylhydrazone), an inhibitor of S-adenosyl-L-methionine decarboxylase (EC.4.1.1.50) and DL-α-methylornithine, an inhibitor of ornithine decarboxylase (EC.4.1.1.17), are shown to be potent inhibitors of the replication of human cytomegalovirus (HCMV) in MRC-5 cells. These compounds, both inhibitors of polyamine biosynthesis, do not affect the replication of either herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). This difference in antiviral effect is shown to be related to the stimulation of spermidine and spermine synthesis in host cells following HCMV infection and the inhibition of polyamine metabolism in HSV-1 or HSV-2-infected cells. Inhibition of HCMV replication by the inhibitors of polyamine biosynthesis is accompanied by a marked decrease in the formation of intranuclear, DNA-containing inclusions characteristic of HCMV infection. These results suggest significant differences in the mechanisms of replication of different herpesviruses.     

单纯疱疹病毒Ⅰ型新开放读码框UL57的鉴定

单纯疱疹病毒1型(Herpes simplex virus type 1,HSV-1)潜伏感染期间LATs的活跃转录可能与其启动子与增强子两侧的CTCF结合序列有关。本研究对位于UL56下游与LAT启动子上游之间并与CTCF结合序列重叠存在的一个新开放读码框(本研究中命名为UL57)进行了鉴定。首先利用HSV-1(F)细菌人工染色体(HSV-BAC)系统构建重组病毒HSV-EGFP-UL57,将EGFP序列插入UL57 5’端;然后分别通过Northern Blot和Western Blot检测EGFP标记的UL57的转录和表达;同时构建敲除UL57的重组病毒HSV-ΔUL57,观察UL57对病毒增殖的影响。结果显示,重组病毒HSV-EGFP-UL57感染HEp-2细胞17h后,EGFP探针检测到两条转录产物,其中1.8kb转录产物与预测大小相符;使用放线菌酮(Cycloheximide,CHX)阻断病毒即刻早期蛋白/早期蛋白合成后,UL57转录受到明显抑制。重组病毒HSV-EGFP-UL57感染Vero细胞后,9h可见融合蛋白表达,24h表达明显;融合蛋白分子量与预测大小(58kD)一致。病毒生长曲线显示,重组病毒HSV-EGFP-UL57及HSV-ΔUL57在Vero细胞中的增殖水平与HSV-1(F)基本一致。本研究表明,在HSV-1基因组(GenBank:GU734771.1)UL56下游与LAT启动子上游之间存在一个新开放读码框UL57(116 921bp～117 799bp),UL57可以进行转录,且其转录受病毒即刻早期蛋白/早期蛋白调控;转录产物可以翻译出融合蛋白,但表达水平较低。删除UL57对病毒增殖无明显影响。     

Human cytomegalovirus TRS1 and IRS1 gene products block the double-stranded-RNA-activated host protein shutoff response induced by herpes simplex virus type 1 infection

Human cytomegalovirus (HCMV) attachment and entry stimulates the expression of cellular interferon-inducible genes, many of which target important cellular functions necessary for viral replication. Double-stranded RNA-dependent host protein kinase (PKR) is an interferon-inducible gene product that limits viral replication by inhibiting protein translation in the infected cell. It was anticipated that HCMV encodes gene products that facilitate the evasion of this PKR-mediated antiviral response. Using a deltagamma1 34.5 herpes simplex virus type 1 (HSV-1) recombinant that triggers PKR-mediated protein synthesis shutoff, experiments identified an HCMV gene product expressed in the initial hours of infection that allows continued protein synthesis in the infected cell. Recombinant HSV-1 viruses expressing either the HCMV TRS1 or IRS1 protein demonstrate that either of these HCMV gene products allows the deltagamma1 34.5 recombinant viruses to evade PKR-mediated protein shutoff and maintain late viral protein synthesis.     

Inhibition of Herpes Simplex Virus-1 Replication by Natural Compound Honokiol

Honokiol is a pleiotropic natural compound isolated from Magnolia and has multiple biological and clinically relevant effects, including anticancer and antimicrobial function. However, the antiviral activity of honokiol has not yet been well studied. Here we showed that honokiol had no effect on herpes simplex virus-1(HSV-1) entry, but inhibited HSV-1 viral DNA replication, gene expression and the production of new progeny viruses. The combination of honokiol and clinical drug acyclovir augmented inhibition of HSV-1 infection. Our results illustrate that honokiol could be a potential new candidate for clinical consideration in the treatment of HSV-1 infection alone or combination with other therapeutics.     

Herpes Simplex Virus 1 Infection Alters the mRNA Translation Processing in L-02 Cells

HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry （MALDI-TOF-MS）. The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.     

Herpes simplex virus 1 infection alters the mRNA translation processing in L-02 cells

HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off. Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.     

Trichosanthin suppresses the elevation of p38 MAPK, and Bcl-2 induced by HSV-1 infection in Vero cells

Trichosanthin (TCS) is a type 1 ribosome-inactivating protein (RIP) effective against HIV-1 and HSV-1 replication. The mechanism of its antiviral activity is not clear. Many believe that it is related to ribosome inactivation. Some RIPs and viral infection affect the phosphorylation of MAPK and Bcl-2 and these proteins may be the common element linking RIP and viral infection. This study investigated the effect of HSV-1 infection on p38 MAPK and Bcl-2 as well as possible interference by TCS. Results showed that HSV-1 infection induced an elevation of phosphorylated p38 and Bcl-2 in Vero cells, which could be partially blocked by TCS. At the same time, both viral replication and host cells viability were lowered. Viral replication, Vero cell viability, p38 MAPK and Bcl-2 were further reduced with the addition of a p38 MAPK inhibitor (SB203580). This suggested that TCS may interfere with MAPK and Bcl-2 signals generated by infection leading to inhibition of viral replication. In summary, our results demonstrated that HSV-1 infection in Vero cells induced an elevation of p38 MAPK and Bcl-2. TCS suppressed this rise and reduced viral replication. The MAPK family may play a role in the antiviral mechanism of TCS.