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.     

Association of a cellular 21-kDa transmembrane protein (VAP21) with enveloped viruses.

We reported previously that the rabies virions contained a 21-kDa cellular transmembrane protein (referred to as VAP21) as a minor component (Sagara, J. et al, Microbiol. Immunol. 41(12): 947-955, 1997). In this study, we further examined the possible interactions of VAP21 with other enveloped viruses, including the vesicular stomatitis virus (VSV; negative-stranded RNA virus), Sindbis virus (positive-stranded RNA virus) and herpes simplex virus type 1 (HSV-1; double-stranded DNA virus). An immunoblot analysis demonstrated that all of these enveloped viruses contained VAP21 in the virion as a minor component. Immunoprecipitation studies suggested that VAP21 was associated with certain viral proteins in the cell, such as the matrix (M) protein of VSV, a capsid protein of Sindbis virus, and at least a capsid protein (VP5) of HSV-1. The association was disrupted by treatment with 0.5% sodium dodecyl sulfate, but resistant to the treatment with 1% NP-40 plus 1% deoxycholate. These results suggest that: 1) VAP21 is not primarily associated with the viral transmembrane glycoprotein but rather with the internal viral protein, and, 2) this association would cause the efficient incorporation of VAP21 into the virion.     

Antiviral Activity of Some 2'-Deoxyuridine 5-Arylethynyl Derivatives

5-(3-Perylenylethynyl)-2'-deoxyuridine was prepared by crosslinking 5-iodo-2'-deoxyuridine derivatives with 3-ethynylperylene followed by deprotection. 5-(1-Perylenylethynyl)-, 5-(3-perylenylethynyl)-, and 5-[4-(2-benzoxazolyl)phenylethynyl]-2'-deoxyuridine were found to inhibit in Vero cells the replication of type 1 herpes simplex virus and its drug-resistant strains.     

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。     

Thymidine kinase activity in mouse embryo fibroblast cells infected with murine cytomegalovirus

Thymidine kinase activity was found in whole cell extracts of growing and stationary mouse embryo fibroblast cells after infection with murine cytomegalovirus. Determination of the kinetic constants and heat stability characteristics indicated that the enzyme activity from infected cells was different to that found in uninfected cells in the growth phase. The expression of thymidine kinase activity during virus replication was reflected by the incorporation of (6-³H) thymidine into acid precipitable fractions of infected cell cultures. Preliminary data from kinetic studies showed a reduction in the phosphorylation of thymidine by this enzyme activity in the presence of Acyclovir, a potent inhibitor of herpes virus replication.     

细胞MEK1/2蛋白及其相关通路在单纯疱疹病毒Ⅱ型（HSV-2）复制中的作用

基于细胞Raf/MEK/ERK信号通路与病毒复制的关系,应用Western印迹检测 p-ERK1/2蛋白的表达、用终点滴定法测定病毒增殖量（TCID_５０）,以及观察感染细胞的细胞病变效应（CPE）等,揭示单纯疱疹病毒Ⅱ型(HSV-2)复制与 ERK通路的关系. 结果表明,HSV-2的复制可引起细胞ERK通路的活化;用U0126预先抑制ERK通路的活化,或用特异性siRNA敲减MEK1/2基因的表达可显著地抑制病毒复制.提示ERK信号通路以及MEK1/2蛋白对HSV-2的复制具有重要的作用.该研究对进一步阐明细胞ERK通路各激酶蛋白在病毒复制中的作用机制、寻找抗病毒作用靶标等奠定了良好的基础.     

Human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells

The antigrowth effects of 5 different cloned human leukocyte IFN subtypes (IFN-alpha A, B, C, D, F) and 2 molecular hybrids between them (IFN-alpha AD(Bg1II) and IFN-alpha DA(Bg1II)) were examined on 6 different human cell lines. The results indicate that the interferons sort into two distinct groups: IFN-alpha B, C and F showed comparable antiproliferative activity which was greater than that of IFN-alpha A, D, AD(Bg1II) and DA(Bg1II). The interferons could also be assigned to one of two groups on the basis of their antiviral activity. IFN-alpha A, D and AD(Bg1II) were observed to be more protective than IFN-alpha B, C and F against HSV-2 and EMCV infections, i.e. the relative antiviral efficacies of the cloned IFN subtypes were the reverse of their antiproliferative activities.     

Ornithine decarboxylase protein diversity and activity modulation in HTC cells

Ornithine decarboxylase of HTC cells was chromatographically separated into three ionically distinct but kinetically similar forms of this protein. The sequential appearance of these ornithine decarboxylase species during enzyme induction, and the accumulation of normally minor species under conditions that stabilize this enzyme, suggest that these represent modifications that are associated with the extremely rapid turnover of this protein $\text{in vivo}$. These forms may also be differentially active or unequally distributed $\text{in vivo}$ as indicated by the selective inactivation of one of the forms by short exposure to α-difluoromethylornithine.     

Antiviral activity of Basidiomycete mycelia against influenza type A (serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.     

Expression and immunogenicity of recombinant glycoprotein D of herpes simplex virus 1 in Drosophila S2 cells

Herpes simplex virus type 1 (HSV-1) is responsible for cold sores in the general population, but also contributes to the development of other more serious diseases in some circumstances. The viral glycoprotein D (gD) is essential for virus entry into host cells. In the present study, the Drosophila melanogaster Schneider 2 (S2) expression system (DES) was evaluated for the expression of recombinant gD1. The DNA sequences encoding the full-length gD1 (369aa, FLgD1) and a truncated gD1 form corresponding to the ectodomain (314aa, EgD1) were cloned into S2 expression vector pMT/BiP/V5-HisA to generate pMT-EgD1 and pMT-FLgD1, respectively. Two forms of gD1 gene were fitted with a hexahistidine tag to facilitate their purification. Cell populations expressing the highest gD1 levels were selected by using a limiting dilution assay. Western blot, flow cytometry (FACS), and confocal immunofluoresence assay demonstrated that the full-length form is restrained in the lipid membranes of the cell and the ectodomain form is secreted into the medium. Recombinant ectodomain gD1 was scaled up and purified from the culture medium using nickel nitrilotriacetic acid affinity chromatography, and a maximum production level of 56.8 mg/L of recombinant gD1 was obtained in a shake-flask culture of S2 cells after induction with 5 µM CdCl2 for 4 days. Mice were then immunized with recombinant purified gD1 and produced high titers of antibody measured by enzyme-linked immunosorbent assay (ELISA; 1:5,120,000) as well as high plaque neutralization titer (1:320). Overall, the data indicated that stable expression in S2 cells is a practical way of synthesizing gD1 for use in structural and functional studies in the further study.     

IFIT1负反馈上调干扰素β表达促进抗HSV-1病毒保护

Ⅰ型单纯疱疹病毒（HSV-1）是危害人类健康的常见病原体之一,能够通过受损皮肤或黏膜感染宿主细胞并引起多种疾病。HSV-1的侵入激活先天免疫模式识别受体,诱导干扰素β（IFN-β）的产生,通过表达干扰素刺激基因（ISG）发挥抗病毒功能。近年来,干扰素诱导的四肽重复蛋白1（IFIT1）在病毒感染过程中的作用引起了广大研究者的关注。然而,其具体机制尚未完全清楚。本研究利用CRISPR/Cas9技术构建了小鼠成纤维细胞（L929）IFIT1敲除细胞株,免疫印迹方法检测敲除细胞株IFIT1在蛋白质水平的表达。转染HT-DNA和Poly［I:C］刺激L929 WT和IFIT1敲除细胞株,实时定量PCR技术检测发现,HT-DNA刺激敲除细胞时,IFN-β及下游ISGs的表达量显著升高。IFN-β的表达量比 L929-WT组平均高出13.4倍,IFIT1和趋化因子10（CXC chemokine ligand-10,CXCL10）的表达量比L929 WT组分别平均高出 6.7倍和21倍(P<0.001）,而Poly［I:C］刺激无明显变化（P>0.05）,表明IFIT1是通过DNA信号通路来行使其负反馈调节作用。为研究IFIT1基因的抗病毒作用,利用CRISPR/Cas9技术改造的HSV-1-VP26 mCherry 病毒感染该敲除细胞株,通过测定病毒荧光数及病毒拷贝数,发现IFIT1敲除细胞株与L929 WT细胞相比,存活率提高了60%（P<0.001）,病毒增殖能力在48 h后降低28.6倍（P<0.001）。该结果表明,IFIT1基因的缺失有利于抵抗HSV-1的感染。综上所述,IFIT1通过DNA信号通路负反馈上调IFN-β及ISG的表达,IFIT1的缺失对病毒入侵发挥了保护作用。该结果为后续研究开发治疗HSV-1感染相关的治疗药物提供了一个新思路。     

Inhibition of polyamine biosynthesis by alpha-difluoromethyl ornithine potentiates the cytotoxic effects of arabinosyl cytosine in HeLa cells

The object of this study was to examine the effect of inhibition of polyamine biosynthesis on the cell cycle traverse of HeLa cells using α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ornithine decarboxylase. The results of this study indicate that DFMO inhibits HeLa cell growth by causing a decrease in the intracellular levels of putrescine and spermidine without any significant effect on concentration of spermine. The inhibition is readily reversible by exogenous supply of putrescine to the medium. The DFMO treatment also results in an accumulation of cells in S phase. Further, the use of an S phase-specific drug like Ara-C following DFMO treatment results in a synergistic killing of the tumor cells as revealed by the inhibition of cell growth. These observations suggest that exploitation of regulation of the cell cycle by the depletion of polyamines with the use of inhibitors like DFMO might help in designing better therapeutic regimes in combination with other cytotoxic drugs.     

The potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1

Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more serious diseases. It can establish a latent stage in sensory ganglia after primary epithelial infections, and reactivate in response to stress or sunlight. Previous studies have demonstrated that viral immediate-early protein ICP0 plays a key role in regulating the balance between lytic and latent infection. Recently, It has been determined that promyelocytic leukemia (PML) nuclear bodies (NBs), small nuclear sub-structures, contribute to the repression of HSV-1 infection in the absence of functional ICP0. In this review, we discuss the fundamentals of the interaction between ICP0 and PML NBs, suggesting a potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1.     

Chemical interactions with herpes simplex type 2 virus: Enhancement of transformation by hydrazine and 1,2-dimethylhydrazine

Quantitative assays for the morphological transformation of 3T3 Swiss mouse cells by herpes simplex type 2 virus (HSV-2) were employed to examine the effect on cell transformation of chemical carcinogens and suspected carcinogens. Exposure of the cells to the chemical compound, followed by virus infection, resulted in enhancement of transformation when compared to that observed with chemical or virus alone. Enhancement occurred in tests utilizing either UV light-inactivated HSV-2 (strain 333) or a temperature-sensitive (ts) mutant of HSV-2 [A₈(293)]. A series of seven ts-mutants were tested and exhibited varying degrees of transformation. Enhancement of transformation occurred in cells treated with hydrazine (HZ) and 1,2-dimethylhydrazine (SDMH). No enhancement occurred when cells were treated with monomethylhydrazine, 1,1-dimethylhydrazine and the jet fuels JP-5, JP-10, RJ-4 and RJ-5. A strong time dependence after treatment was demonstrated with some enhancement seen at 6 h after chemical treatment but the greatest enhancement appeared when virus infection began after 24 h of chemical exposure.     

Failure of multinucleated giant cell formation in k562 cells infected with newcastle disease virus and human parainfluenza type 2 virus

When K562 cells were infected with Newcastle disease virus (NDV) or human parainfluenza type 2 virus (hPIV-2), polykaryocyte formation could not be detected. Failure of multinucleated giant cell formation in K562 cells infected with either NDV or hPIV-2 is due to disturbance of the viral envelope-cell fusion step or to defect in the cell-cell fusion step, respectively. Especially, NDV completely replicated in K562 cells, and the hemagglutinin-neuraminidase and fusion proteins expressed on the cell surface of NDV-infected K562 cell were fully functional for fusion inducing activity. Therefore, the cell membranes of K562 cells are considered to be resistant to virus-induced cell fusion. Membrane fusion is regulated by many host factors including membrane fluidity, cytoskeletal systems, and fusion regulatory proteins system. An unknown regulatory mechanism of virus-induced cell fusion may function on the cell surface of K562 cells.     

Extracellular matrix metabolism by chondrocytes III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. $\text{Benzyl}\text{-β-}\text{D}\text{-}\text{xyloside}$, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.     

Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV.

Extracts of cultured skin fibroblasts derived from patients with mucolipidosis IV showed a marked increase and altered distribution of GM₃ and GD₃ gangliosides. GD₃ is elevated 1.5–2 times that of normal whereas GM₃ is elevated to a lesser extent. No abnormalities were found in the neutral glycolipids. These two gangliosides apparently comprise most of the accumulated lipid-like material observed on ultrastructural analysis in this disease.     

Herpes simplex virus-1 up-regulates IL-15 gene expression in monocytic cells through the activation of protein tyrosine kinase and PKC zeta/lambda signaling pathways

IL-15 plays a seminal role in innate immunity through enhancing the cytotoxic function as well as cytokine production by NK and T cells. We have previously shown that exposure of PBMC as well as monocytic cells to different viruses results in immediate up-regulation of IL-15 gene expression and subsequent NK cell activation as an innate immune response of those cells to these viruses. However, no signaling pathway involved in this up-regulation has been identified. Here we show for the first time that HSV-1-induced up-regulation of IL-15 gene expression is independent of viral infectivity/replication. IL-15 gene is up-regulated by HSV-1 in human monocytes, but not in CD3+ T cells. HSV-1 induces the phosphorylation of protein tyrosine kinases (PTKs) and protein kinase C (PKC) for inducing IL-15 expression in monocytic cells. Inhibitors for PTKs reduced HSV-1-induced PTK activity, DNA binding activity of NF-kB as well as IL-15 gene expression. In contrast, an inhibitor for membrane-bound tyrosine kinases had no effect on these events. Experiments using PKC inhibitors revealed that phosphorylation of PKC zeta/lambda (PKC zeta/lambda), DNA binding activity of NF-kB and HSV-1-induced up-regulation of IL-15 were all decreased. Furthermore, we found that HSV-1-induced IL-15 up-regulation was also dependent on PTKs regulation of PKC phosphorylation. Thus, we conclude that IL-15 up-regulation in HSV-1-treated monocytic cells is dependent on the activity of both PTKs and PKC zeta/lambda.     

Synthesis of 1′,2′-methano-2′,3′-dideoxynucleosides as potential antivirals

The synthesis of constrained nucleosides has become an important tool to understand the SAR in the interaction between biological and synthetic nucleotides in the context of antisense oligonucleotide therapy. The incorporation of a cyclopropane into a furanose ring of a nucleoside induces some degree of constrain without affecting significantly the steric environment of a nucleoside. Here, we report a new, short and stereocontrolled synthesis of two constrained nucleosides analogues, 1′,2′- methano-2′,3′-dideoxyuridine 9, and the corresponding cytidine analog 12. X-ray crystallography revealed that the furanose ring in the constrained uridine and cytidine analogues was flattened with virtual loss of pseudorotation. The phosphoramidate esters of the novel constrained uridine and cytidine nucleosides, intended as prodrugs, were tested in cell-based assays for viral replication across the herpes virus family and HIV inhibition courtesy of Merck laboratories, Rahway. They were also tested in antiproliferative assays against colorectal and melanoma cell lines. Unfortunately, none of the compounds showed activity in these assays.