MEK1-ERKs级联激活方式是调控单纯疱疹病毒Ⅱ型复制的重要机制

本研究通过阐明MEK1和MEK2亚型在单纯疱疹病毒Ⅱ型(herpes simplex virus type 2,HSV2)复制中介导的Raf/MEK/ERK(简称ERK)通路活化中的作用,以期进一步阐明该通路调控病毒复制的机制.研究中应用了MEK抑制剂U0126、针对MEK1和MEK2的特异性小干扰RNA(small ...     

一种包装AAV2/5的重组单纯疱疹病毒的构建

为了得到一种可以包装AAV2/5和表达绿色荧光蛋白的重组单纯疱疹病毒,设计并构建了一个由AAV2rep基因和AAV5cap基因嵌合而成的rep2cap5基因,然后,利用一套携带HSV1基因组的粘粒系统(cos6、cos28、cos14、cos56、cos48),将rep2cap5基因插入cos6粘粒上HSV1基因组片段的UL2基因中,而将EGFP的表达单位插入cos56粘粒上HSV1基因组片段的UL44基因中,用这2个重组粘粒与其它3个粘粒(cos14、cos28、cos48)共转染BHK-21细胞获得了重组病毒HSV1-r2c5-EGFP并进行了空斑纯化。HSV1-r2c5-EGFP病毒能够在BHK-21细胞连续传代,并且可以观察到几乎所有的感染细胞都能产生绿色荧光。用PCR方法以及Southern杂交方法表明所获得的HSV1-r2c5-EGFP中携带有rep2cap5基因,用HSV1-r2c5-EGFP感染携带报告基因LacZ的AAV载体细胞株,获得了具有感染性的重组AAV2/5-LacZ。结果表明,所获得的重组单纯疱疹病毒HSV1-r2c5-EGFP可提供AAV2/5载体包装所需的全部辅助功能,是一种能简便、高效制备重组AAV2/5病毒的通用性辅助病毒。     

Specific Deposition of Passively Transferred Monoclonal Antibodies against Herpes Simplex Virus Type 1 in Rat Brain Infected with the Virus

The kinetics of human monoclonal antibody (anti-gB) to herpes simplex virus type 1 (HSV-1) were investigated after intravenous injection of anti-gB into an HSV-1 encephalitis animal model. Immunohistochemical study revealed specific deposition of passively tansferred anti-gB in the hippocampus and thalamus of the infected rat brain, and it bound to the same neurons in which HSV-1 antigen was positively stained. To examine the macroscopic distribution of anti-gB in the infected brain, we undertook an ¹²⁵I-labeled anti-gB injection study, and the same distribution of ¹²⁵I-labeled anti-gB deposition was observed by brain semimicroautoradiography as in the immunohistochemical study. These results suggest that anti-gB easily permeates the capillary wall and is deposited in the inflammatory site where HSV-1-specific antigen is detectable. The use of radioisotope-labeled anti-gB injection and external brain imaging could lead to a noninvasive diagnostic tool for the early detection of HSV-1 antigen in cases of suspected HSV-1 encephalitis.     

Effect of Heparin on Hemagglutinin of Herpes Simplex Virus Type 1

Heparin inhibited the hemagglutinin activity of herpes simplex virus (HSV) type 1. The minimal inhibitory concentration of heparin required to inhibit 8 hemagglutination (HA) U of HSV ranged from 0.005 to 0.01 U/ml. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. On the other hand, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by HSV. Virus-heparin complex formation was observed by sedimenting heparin with the virus particles.     

2型单纯疱疹病毒体外感染周期的研究进展

2型单纯疱疹病毒(Herpes simplex virus type 2,HSV-2)是引起生殖器疱疹的主要病原体。生殖器疱疹主要表现为生殖器和肛周皮肤黏膜疱疹或溃疡,是一种慢性、复发性、难以治愈的性传播疾病,临床治疗多以抑制病毒复制的核苷类药物阿昔洛韦及其衍生物更昔洛韦、喷昔洛韦等为主。这些药物对缓解症状、缩短病程有一定作用,但难以达到根治的目的,长期应用易产生耐药,且对其合并症基本无效。作用于HSV-2其他感染周期的药物成为人类探索的新领域。HSV-2感染宿主细胞是一个复杂的多阶段过程,包括黏附、穿入、核转运、基因组复制、衣壳组装、子代病毒释放等多个步骤,涉及多种细胞和病毒蛋白的活性。本文对HSV-2体外感染周期详细步骤及其分子机制作一综述,以期深入了解其感染机制,并为研制作用于不同感染阶段的抗HSV-2药物提供参考。     

Antiviral Activity of Trichothecene Mycotoxins (Deoxynivalenol,Fusarenon-X,and Nivalenol) against Herpes Simplex Virus Types 1 and 2

The effect of trichothecene mycotoxins, deoxynivalenol (DON), fusarenon-X (FX) and nivalenol (NIV), on plaque formation of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in HEp-2 cells was examined. The 50% effective concentrations (EC₅₀) of DON, FX, and NIV for HSV-1 plaque formation were 160, 56, and 120 ng/ml, respectively. Those for HSV-2 plaque formation were 94, 26, and 50 ng/ml, respectively. These three mycotoxins showed about 2-fold higher selectivity to HSV-2 than to HSV-1. Plaque formation of HSV-1 was not inhibited with trichothecenes at concentrations completely inhibiting plaque formation when cells were treated during virus adsorption period or 15 hr before infection. These results indicate that trichothecenes affect replication of HSV-1 after virus adsorption, but not before or during virus adsorption to the host cells.     

The Herpes Simplex Virus Type 1 Infected Cell Protein 22

As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction wit...     

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.     

格尔德霉素抗病毒作用的相关基因研究

格尔德霉素(Geldanamycin,GA)作为一种苯醌安莎霉素类抗生素,能与热休克蛋白90特异性结合,具有广谱的抗病毒作用.为了从转录水平上研究GA抗病毒的分子机制,本研究以单纯疱疹病毒Ⅰ型(Herpes simplex vi-rus type 1,HSV-1)为对象,在确定药物有效抗病毒作用的基础上,采用基因芯片技术分析了在HeLa细胞中病毒感染和药物处理对细胞表达谱的影响,并筛选出GA抗病毒作用的可能相关基因.同时用半定量RT-PCR方法对GA诱导上调、HSV-1诱导下调的基因(ACTG1、RAN、SODl)以及GA诱导下调、HSV-1诱导上调的基因(HYALl)进行了验证.研究GA抗病毒作用对细胞表达谱的影响,有利于深入理解药物的抗病毒机制.     

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。     

疱疹病毒Ⅱ型感染SH-SY5Y细胞的生物学效应

目的：探讨疱疹病毒Ⅱ型（HSV-2）感染人神经母细胞瘤细胞株SH-SY5Y的生物学效应。方法：病毒液接种SH-SY5Y细胞后,用相差和电子显微镜观察感染细胞的形态变化,RT-PCR检测病毒在细胞中的增殖,MTT法检测病毒感染对细胞增殖的影响,流式细胞仪测定感染后的细胞凋亡状况。结果：相差显微镜显示细胞病变,从24～72h,细胞变性、坏死的程度和数量随感染时间延长而增加;电镜结果显示感染24h后,细胞核染色质固缩,出现多核巨细胞,线粒体内嵴紊乱、断裂,出现不同程度的自噬化、溶酶体化、空泡化,并可见大量鹰眼样已包装成熟的病毒颗粒及正在包装的病毒粒子;HSV-2LAT基因RT-PCR扩增表明,病毒能在SH-SY5Y细胞中增殖;凋亡检测显示HSV-2在体外细胞感染中并未使细胞出现凋亡现象;感染后24、48及72h,SH-SY5Y细胞的抑制率分别为11.3%、31.2%和63.1%,与对照组相比均存在显著性差异（P〈0.05）;分别用0.1、1、10MOI的病毒感染SH-SY5Y细胞,上述不同组在24、48、72h时细胞形态变化基本一致,感染结果相似,各组之间病毒毒力无明显差异（P〉0.05）。结论：初步在人神经母细胞瘤细胞株SH—SY5Y中建立了HSV-2感染的细胞模型,并研究了感染对细胞生物性状的影响,为探讨HSV-2的潜伏与激发机制、了解HSV-2的致病机制打下基础。     

Analysis of the Relationship between Cellular Thymidine Kinase Activity and Virulence of Thymidine Kinase-Negative Herpes Simplex Virus Types 1 and 2

The virulence of thymidine kinase-negative herpes simplex virus type 1 (HSV-1; VRTK^? strain) and type 2 (HSV-2; UWTK^? strain) was studied in comparison with that of their parental strains (VR-3 and UW-268, respectively) in an encephalitis model of adult (4-week-old) and newborn (3-day-old) mice. Viral thymidine kinase (TK) activity was essential for the maximum expression of virulence of HSV-1, because the 50% lethal dose (LD₅₀) of VRTK^? was 60 times higher than that of VR-3 in the brains of newborn mice expressing high levels of cellular TK activity. However, the UWTK^? strain showed the same virulence as the parental strain in newborn mice, despite the lack virulence in adults, suggesting that replication of the UWTK^? strain was completely supported by cellular TK activity. This difference in the role of viral and cellular TKs for virus growth between HSV-1 and HSV-2 was confirmed with the one-step growth of virus strains in L-M and L-M(TK^?) cells.     

In Vitro Selection of Variants of Herpes Simplex Virus Type 1 Which Differ in Cytopathic Changes

To analyze the mechanisms for in vitro emergence of the syncytial variants of herpes simplex virus type 1 (HSV-1), several cell lines were infected with a mixture of equal amounts of two HSV-1 variants, one syncytial and the other non-syncytial, and changes in their relative abundance were monitored during passage. With a combination of two variants of the Miyama strain of HSV-1, the syncytial variant became dominant during passage in Vero, RK-13 and FL cells. On the other hand, the ratios of the two variants remained around 1:1 during the passage in HEp-2, MGC and HEL cells. In another set of variants of the SKO strain of HSV-1, the outcomes were different from those of the Miyama strain in the FL, MGC and HEp-2 cells. The ratios of the two variants remained around 1:1 during passage in FL cells, while the non-syncytial variant became dominant during passage in MGC and HEp-2 cells. In addition, we examined the effects of a complement and interferon-β (IFN-β) on the outcome of the selection. As a result, the complement slowed the selection of a syncytial variant, whereas IFN-β facilitated it.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

Effect of Loss-of-function of the Herpes Simplex Virus-1 microRNA H6-5p on Virus Replication

To date, 29 distinct microRNAs(miRNAs) have been reported to be expressed during herpes simplex virus infections.Sequence analysis of mature herpes simplex virus-1(HSV-1) miRNAs revealed five sets of miRNAs that are complementary to each other: miR-H6-5p/H1-3p, miR-H6-3p/H1-5p, H2-5p/H14-3p, miR-H2-3p/H14-5p, and miR-H7/H27.However, the roles of individual miRNAs and consequences of this complementarity remain unclear. Here, we focus on two of these complementary miRNAs, miR-H6-5p and miR-H1-3p, using loss-of-function experiments in vitro and in a mouse model of infection using an miRNA sponge approach, including tandem multiplex artificial miRNA-binding sequences that do not match perfectly to the target miRNA inserted downstream of a green fluorescent protein reporter gene. Infection with recombinant virus expressing the miR-H6-5p sponge reduced viral protein levels and virus yield.Decreased accumulation of viral proteins was also observed at early stages of infection in the presence of both an miR-H6-5p inhibitor and plasmid-expressed miR-H1-3p. Moreover, establishment of latency and reactivation did not differ between the recombinant virus expressing the miR-H6-5p sponge and wild-type HSV-1. Taken together, these data suggest that miR-H6-5p has an as-yet-unidentified role in the early stages of viral infection, and its complement miR-H1-3p suppresses this role in later stages of infection. This report extends understanding of the roles of miRNAs in infection by herpes simplex viruses, supporting a model of infection in which the production of virus and its virulent effects are tightly controlled to maximize persistence in the host and population.     

一种1型单纯庖疹病毒载体系统的建立

1型单纯疱疹病毒（HSV-1）作为溶瘤病毒和病毒载体的研究已有很长的历史. 本研究利用细菌人工染色体技术建立了一种HSV-1载体系统. 首先,将HSV-1内部反向重复序列（internal inverted repeat sequences, IR）两侧的片段克隆入pKO5获得穿梭质粒pKO5/BN,其电转含pHSV-BAC的大肠杆菌后筛选获得删除IR区重组DNA的 pHSVΔIR-BAC. pHSVΔIR-BAC转染Vero细胞获得删除IR区的重组病毒HSVΔIR（MH1001）.上述pKO5/BN和含pHSVΔIR BAC的大肠杆菌构成了HSV-1载体系统. 利用该系统获得了表达绿色荧光蛋白EGFP的重组病毒HSVΔIR/EGFP（MH1002）.MH1001和MH1002在感染的Vero细胞中增殖水平略低于野生型HSV-1,但无显著差异|Western印迹检测表明,重组病毒早期蛋白质ICP0、ICP4、ICP8、ICP22、ICP27在感染细胞中的表达水平下降|免疫荧光及激光共聚焦检测表明,重组病毒与野生型病毒均存在于细胞质中.以上结果表明,删除IR区的重组HSV-1保留了复制能力,能够携载并表达外源基因,建立的HSV-1载体系统可用于构建携载外源基因的复制型重组HSV-1.     

栀子提取物ZG对单纯疱疹病毒1型细胞吸附的影响

采用负染技术,借助高倍电子显微镜观察栀子提取物ZG作用后,病毒颗粒及其病毒吸附蛋白(virus attach-ment protein,VAP)的变化,考察药物是否直接改变或破坏病毒包膜蛋白的结构,使其失去感染性;采用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记病毒,以肝素钠为参照,借助冷却慢扫描电荷耦合器件荧光成象技术,用Aquacomos软件进行图象分析,以探讨栀子提取物ZG不同加药方式对HSV-1吸附量的影响。结果表明栀子提取物ZG对HSV-1包膜表面的VAP无直接破坏作用,不影响病毒对Hep-2细胞的感染性;先加入肝素钠再进行病毒吸附及肝素钠病毒同时加入培养细胞这两种用药方式可明显减少细胞表面病毒的吸附量;栀子提取物ZG各种不同加药方式均能阻止HSV-1对Hep-2细胞表面的吸附,使病毒吸附量减少。     

Herpes Simplex Virus-Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post-infection. HSP70 synthesis and accumulation increased in HSV-infected cells. Irradiation of HSV with UV-light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.