C3d and Epstein-Barr Virus (CR2/CD21 Ligands) Stimulate Cells of an HTLV-I Line,MT-2

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT-2 cells. First, we confirmed CR2 expression on MT-2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS-PAGE analysis. When MT-2 lysates were incubated with anti-CR2 mAb HB5 and thereafter with ³²P-labeled ATP, 52- and 74-kDa proteins were phosphorylated, suggesting the activation of MT-2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT-2 cells when incubated with C3d or EBV. The cytotoxicity of the MT-2 supernatant against L929 cells was elevated in a dose- and time-dependent manner. Next, we confirmed EBNA expression on EBV-infected MT-2 cells and attempted to establish an EBV-positive MT-2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT-2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT-2 cells 3 days after EBV treatment gave a 3.0-kb signal which comigrated with the EBV BamHI-W probe. The 3.0-kb signal of genomic EBV-DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2-positive MT-2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT-2 cells might be transient.     

EB病毒及其癌基因LMP1对上皮细胞的转化作用

EB病毒(EBV)是一种人群感染率较高的致病性疱疹病毒,与伯基特氏淋巴瘤、鼻咽癌的关系最为明确。近年来EBV在上皮源性肿瘤发生、发展中的作用受到众多学的关注,而其癌基因LMP1在上皮源性肿瘤发病的病因学方面的作用更成为研究的热点。本综述就EBV进入上皮细胞、永生化上皮细胞的机制、LMP1的基本结构及其对上皮细胞的生物学功能作一较为详细的阐述。     

Herpes Simplex Virus Type 1 Infection of Isogenic Epstein-Barr Virus Genome-Negative and -Positive Burkitt''s Lymphoma-Derived Cell Lines

The Epstein-Barr virus (EBV) genome-negative Burkitt's lymphoma-derived cell lines BJAB and Ramos and their in vitro EBV-converted sublines BJAB-B1, BJAB-A5, BJAB-B95-8, and AW-Ramos were infected with high multiplicities of herpes simplex virus type 1 (HSV-1; 10 to 70 PFU/cell). Cultures were monitored for cell growth and HSV-1 DNA synthesis. EBV-converted BJAB cultures were more permissive for HSV-1 infection than BJAB cultures. Significant cell killing and HSV-1 DNA synthesis were observed during the first 48 h of infection in the EBV-converted BJAB cultures but not in the BJAB cultures. The EBV-converted BJAB-B1 cell line contains an appreciable fraction of EBV-negative cells. Therefore, it was cloned. EBV-positive and -negative cells were identified by using EBV-determined nuclear antigen anti-complement immunofluorescence. Two types of subclones were identified: (i) those which contained both EBV-determined nuclear antigen-positive and -negative cells and (ii) those which contained only EBV-determined nuclear antigen-negative cells. When levels of HSV-1 DNA synthesis were measured in these subclones, it was found that the former were more permissive for HSV-1 infection than the latter. Thus, the presence of the EBV genome in BJAB cells correlates with increased permissiveness of these cells for HSV-1 during the first 48 h of infection. Nonetheless, persistent HSV-1 infections were established in both BJAB and EBV-converted BJAB-B1 cultures. No differences in extent of permissiveness for HSV-1 infection were found for Ramos and EBV-converted AW-Ramos cells.     

Epstein-Barr Virus Promotes Epithelial Cell Growth in the Absence of EBNA2 and LMP1 Expression

We attempted to infect primary gastric epithelia (PGE) with recombinant Epstein-Barr virus (EBV) carrying a selectable marker that made it possible to select EBV-infected cells. Cells dually positive for EBV-determined nuclear antigen (EBNA) and cytokeratin were detected in 3 of 21 primary cultures after 3 days of EBV inoculation. From one culture, EBV-infected cell clones were repeatedly obtained at a frequency of 3 to 5 cell clones per 10⁶ cells. EBV-infected clones had enhanced population doubling and grew to attain a highly increased saturation density, together with acquisition of marked anchorage independence. The infected clones retained the ultrastructural morphology characteristic of gastric mucosal epithelium and have been growing stably for more than 18 months (corresponding to at least 300 generations) so far, in clear contrast to the parental PGE cells, which ceased growth after 60 generations. The p53 gene of the parental PGE cells was found to be overexpressed, perhaps thereby conferring the basal potential for long-term survival in vitro. Moreover, EBV infection accelerated, to a significant extent, the growth rate and agar clonability of NU-GC-3 cells, an established EBV-negative but EBV-susceptible human gastric carcinoma cell line. Both EBV-converted PGE and NU-GC-3 clones, like EBV-positive gastric carcinoma biopsy specimens, expressed a restricted set of EBV latent infection genes characterized by the absence of EBNA2 and latent membrane protein 1 (LMP1) expression. These results indicate that EBV infection causes a transformed phenotype on PGE in the setting of possible unregulated cell cycling and renders even established gastric carcinoma cells more malignant via a limited spectrum of viral latent-gene expression. This study may reflect an in vivo scenario illustrating multiphasic involvement of EBV in carcinogenesis of gastric or other epithelial cancers.     

Stable Polarized Expression of hCAT-1 in an Epithelial Cell Line

Our laboratory has recently identified and cloned three cationic amino-acid transporters of human placenta. We have now examined the plasma membrane domain localization and functional expression of one of these transporters, hCAT-1, in a polarized epithelial cell line (MDCK). To facilitate identification of expressed protein we first transferred the hCAT-1 cDNA to a vector with C-terminal green fluorescent protein (GFP). The resultant hCAT-1-CT-GFP fusion protein stimulated L-[3H] lysine uptake in Xenopus oocytes. In confluent monolayers of stably transfected cells grown on porous nitrocellulose filters, saturable uptake of L-[3H] lysine from the basolateral surface was stimulated 7-fold over that of untransfected cells. Concentration-dependence studies in Na+-free medium at pH 7.4 demonstrated a Km of approximately 68 +/- 13 microM and a Vmax of 970 +/-170 pmol/mg protein/min. Uptake from the apical plasma membrane surface was negligible in both transfected and untransfected cells. Consistent with these results, confocal microscopy of confluent monolayers of hCAT-1-CT-GFP-expressing cells revealed localization of the transporter solely on the basolateral domain of the cell. This is apparently the first report of a cultured polarized epithelial cell model for stable expression of a cationic amino-acid transporter. It has the potential to aid in the identification of targeting signals for transport protein localization.     

A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR,from-7 to 1012).The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR.One clone,designated BFVL,was selected from ten neomycin-res...     

hsp70-Dependent Antiviral Immunity against Cytopathic Neuronal Infection by Vesicular Stomatitis Virus

The major inducible 70-kDa heat shock protein (hsp70) protects against measles virus (MeV) neurovirulence in the mouse that is caused by a cell-associated noncytolytic neuronal infection. Protection is type I interferon (IFN) dependent, and we have established a novel axis of antiviral immunity in which hsp70 is released from virus-infected neurons to induce IFN-β in macrophages. The present work used vesicular stomatitis virus (VSV) to establish the relevance of hsp70-dependent antiviral immunity to fulminant cytopathic neuronal infections. In vitro, hsp70 that was constitutively expressed in mouse neuronal cells caused a modest increase in VSV replication. Infection induced an early extracellular release of hsp70 from viable cells, and the release was progressive, increasing with virus-induced apoptosis and cell lysis. The impact of this VSV-hsp70 interaction on neurovirulence was established in weanling male hsp70 transgenic and nontransgenic mice. Constitutive expression of hsp70 in neurons of transgenic mice enhanced viral clearance from brain and reduced mortality, and it was correlated with enhanced expression of type I IFN mRNA. Nontransgenic mice were also protected against neurovirulence and expressed increased type I IFN mRNA in brain when hsp70 was expressed by a recombinant VSV (rVSV-hsp70), indicating that hsp70 in the virus-infected cell is sufficient for host protection. In vitro data confirmed extracellular release of hsp70 from cells infected with rVSV-hsp70 and also showed that viral replication is not enhanced when hsp70 is expressed in this manner, suggesting that hsp70-mediated protection in vivo is not dependent on stimulatory effects of hsp70 on virus gene expression.     

Expression of Epstein-Barr Virus Nuclear Antigen 1 Is Associated with Enhanced Expression of CD25 in the Hodgkin Cell Line L428

Epstein-Barr virus is associated with several human malignancies including Burkitt’s lymphoma, nasopharyngeal carcinoma, and Hodgkin’s disease (HD). To examine the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1) in the pathogenesis of HD, we transfected the gene into the HD cell line L428. EBNA-1 expression was associated with significantly enhanced CD25 expression (interleukin 2 [IL-2]-receptor α chain) in transient and stably transfected L428 cells but did not affect the expression of IL-2 receptor β and γ chains. There was no up-regulation of the B-cell activation molecules CD23, CD30, CD39, CD40, CD44, CD71, and CD54 (intercellular adhesion molecule 1) or enhanced production of IL-6, IL-10, lymphotoxin alpha, and the soluble form of CD25. Stable EBNA-1-expressing L428 cells were nontumorigenic in SCID mice but showed enhanced lymphoma development in nonobese diabetic-SCID mice compared to mock-transfected cells.     

Isolation of an Insulin-Responsive Preadipose Cell Line and a Mammary Tumor Virus-Producing, Dome-Forming Epithelial Cell Line from a Mouse Mammary Tumor

Two functional tissue culture cell lines, MTD and MTF cell lines, have been isolated from a mouse mammary tumor. MTD cells are epithelial and retain the ability to transport fluid leading to the formation of three-dimensional fluid-filled multicellular structures called "domes" or "hemicysts". Another property of MTD cells is the production of murine mammary tumor virus (MTV). Release of MTV into the culture medium was verified by immunological, electrophoretic and enzymatic analyses. Addition of dexamethasone in the culture medium enhanced both the formation of domes and the production of MTV. Thus, MTD cells retain the morphological and functional properties of the original mammary tumor cells.
MTF cells show the fibroblastic morphology in subconfluent cultures. After reaching confluence, however, these cells gradually accumulated triglycerides in the cytoplasm and eventually assumed the morphology of fat cells. This adipose conversion was greatly enhanced by the presence of insulin in the culture medium. The morphological resemblance of adipose-converted MTF cells to the mammary fat cells suggests that the MTF cell line was derived from the mammary fat pad stroma. These functional cell lines will be useful to study cell differentiation as well as cell-to-cell interactions in the mammary gland.     

Host Cell Regulation of Induction of Epstein-Barr Virus

When Epstein-Barr virus (EBV) negative cells (Raji) were treated with iododeoxyuridine, only the early antigen (EA) component was induced. There was no significant increase in EBV DNA and no virus particles were observed. Somatic-cell hybrids were prepared from the fusion of Raji and D98 cells (D98/Raji). When these cells were treated with iododeoxyuridine, early antigen EBV DNA, and virus particles were synthesized. These data suggest cellular control over the expression of the EBV genome.     

细胞间接触是EB病毒自发感染人类上皮细胞的有效途径

选用产EB病毒的绒猴淋巴细胞B95-8系和补体受体2型(complement receptor 2, CR2)与多聚免疫球蛋白受体(polymeric immunoglobulin receptor, pIgR)表达阴性的人永生化上皮细胞Hacat系共培养,进行细胞接触感染实验.一周后去除B95-8细胞,仅留Hacat细胞,并以自行改进的方法鉴定前者是否得以彻底去除.在证实没有B95-8残留后,PCR和原位杂交分别检验剩余Hacat细胞中EB病毒的感染结果.实验结果表明改进的方法能够灵敏和简便地判断B95-8细胞的污染与否,并且与B95-8细胞接触共培养的Hacat细胞能被EB病毒有效地感染,后者暗示了EB病毒对上皮细胞可能存在细胞融合和CR2或pIgR介导之外新的感染途径.本研究在一定程度上简化了前人的细胞接触感染方法,也为建立天然的EB病毒自发有效地感染上皮细胞的模型奠定了基础.