Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Receptor (CD46) modulation and complement-mediated lysis of uninfected cells after contact with measles virus-infected cells.

Recently, it has been observed that the infection of human target cells with certain measles virus (MV) strains leads to the downregulation of the major MV receptor CD46. Here we report that CD46 downregulation can be rapidly induced in uninfected cells after surface contact with MV particles or MV-infected cells. Receptor modulation is detectable after 30 min of cocultivation of uninfected cells with MV-infected cells and is complete after 2 to 4 h, a time after which newly synthesized MV hemagglutinin (MV-H) cannot be detected in freshly infected target cells. This contact-mediated receptor modulation is also induced by recombinant MV-H expressed by vaccinia virus and is inhibitable with antibodies against CD46 and MV-H. By titrating the effect with MV Edmonston strain-infected cells, a significant contact-mediated CD46 modulation was detectable up to a ratio of 1 infected to 64 uninfected cells. As a result of CD46 downregulation, an increased susceptibility of uninfected cells for complement-mediated lysis was observed. This phenomenon, however, is MV strain dependent, as observed for the downregulation of CD46 after MV infection. These data suggest that in acute measles or following measles vaccination, uninfected cells might also be destroyed by complement after contacting an MV-infected cell.     

Persistent Infection of Cells in Culture by Measles Virus III. Comparison of Virus-Specific RNA Synthesized in Primary and Persistent Infection in HeLa Cells

The pattern of actinomycin D-resistant RNA synthesis was examined during primary infection of HeLa cells by virulent Edmonston measles virus and in two HeLa clones persistently infected by the same strain of virus. One of these clones, K11, produces infectious virus of low virulence for HeLa cells, and the other, K11A-HG-1, has thus far failed to yield infectious virus. The patterns of virus-specific RNA synthesized in these three types of infection are qualitatively similar to each other and to the patterns of virus-specific RNA synthesis in other paramyxovirus infections. There were, however, quantitative differences. In addition, virions of the virulent Edmonston strain of measles virus were found to contain high-molecular-weight RNA with a sedimentation constant identical to that of Newcastle disease virus.     

Measles virus spread by cell-cell contacts: uncoupling of contact-mediated receptor (CD46) downregulation from virus uptake.

CD46, which serves as a receptor for measles virus (MV; strain Edmonston), is rapidly downregulated from the cell surface after contact with viral particles or infected cells. We show here that the same two CD46 complement control protein (CCP) domains responsible for primary MV attachment mediate its downregulation. Optimal downregulation efficiency was obtained with CD46 recombinants containing CCP domains 1 and 2, whereas CCP 1, alone and duplicated, induced a slight downregulation. Using persistently infected monocytic/promyelocytic U937 cells which release very small amounts of infectious virus, and uninfected HeLa cells as contact partners, we then showed that during contact the formation of CD46-containing patches and caps precedes CD46 internalization. Nevertheless, neither substances inhibiting capping nor the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly-OH (FIP) blocked CD46 downregulation. Thus, CD46 downregulation can be uncoupled from fusion and subsequent virus uptake. Interestingly, in that system cell-cell contacts lead to a remarkably efficient infection of the target cells which is only partially inhibited by FIP. The finding that the contact of an infected with uninfected cells results in transfer of infectious viral material without significant (complete) fusion of the donor with the recipient cell suggests that microfusion events and/or FIP-independent mechanisms may mediate the transfer of MV infectivity from cell to cell.     

Measles-specific T cell clones derived from a twin with multiple sclerosis: genetic restriction studies

The association between multiple sclerosis (MS) and HLA-DR2 suggests that the disease may be associated with an aberrant immune response, likely directed against an antigen of either viral or host origin. We have used measles virus-specific T cell clones derived from a patient with MS to study genetic restriction patterns of antigen presentation by macrophage-enriched (E-) populations. Twenty-two clones proliferated in response to measles-infected Vero cells but not to mumps-infected or uninfected Veros. E- cells from both the autologous subject and her healthy, measles nonresponder identical twin were capable of presenting antigen to all clones. Studies with E- cells obtained from a panel of cell donors demonstrated clones which recognized antigen in association with D2/DR2, DR4, subgroups of DR4, and SB3. Three clones recognized antigen only in association with the autologous or twin's cells, but not with other sets of HLA-matched E-cells obtained from healthy donors or from other patients with MS. These studies indicate that the differing responses to measles virus demonstrated by these two identical twins are not explained by alterations in the interactions between antigen-presenting cells and T cells. Furthermore, at the clonal level, no preferential role is seen for HLA-DR2 as the restricting element for presentation of measles virus to these clones.     

Human T cell leukemia/lymphoma virus-infected antigen-specific T cell clones: indiscriminant helper function and lymphokine production

The ability of human T cell leukemia/lymphoma virus (HTLV)-I to alter the function of infected T lymphocytes was examined directly by investigating the properties of an antigen-specific T cell clone before and after transformation with HTLV-I. Following infection, the T4 antigen-specific clone manifested a tenfold increase in its surface interleukin 2 (IL 2) receptor (Tac) density and acquired the viral determinants p19, p24, and 4D12 not present in the uninfected clone. Prior to infection, the T cell clone responded to antigen stimulation in the presence of histocompatible antigen-presenting cells with proliferation and secretion of multiple lymphokines, including IL 2, B cell growth factor (BCGF), B cell differentiation factor (BCDF), and interferon-gamma (IFN-gamma). Following infection, the T cell clone both proliferated and produced constitutively three of these lymphokines (BCGF, BCDF, and IFN-gamma) in the absence of accessory cells or antigen. Co-cultivation with any accessory cells regardless of histocompatibility resulted in increased proliferation and lymphokine production. IL 2 production by the HTLV-I-transformed cell, however, could not be detected. Similarly, the uninfected clone was able to provide B cell help for Ig production only when stimulated with both histocompatible cells and antigen. In contrast, the infected cell provided T cell help to B cells in an unregulated manner, independent of antigen or histocompatibility. Thus, functions such as the induction of proliferation, B cell help, and lymphokine production, which are finely regulated in uninfected antigen-specific T cell clones, became indiscriminant after HTLV-I infection.     

Lymphocyte adherence in multiple sclerosis: Lack of virus specificity

The virus specificity of adherence of peripheral blood mononuclear leukocytes from patients with multiple sclerosis and from age- and sex-matched healthy volunteers to tissue culture cells infected with measles virus, Newcastle disease virus, and vesicular stomatitis virus was studied. Lymphocyte adherence to uninfected cells is uniformly low (5–15% tissue culture cells with > 3 lymphocytes adhered). Adherence to cells infected with virus is enhanced 2- to 4-fold in controls and 2- to 10-fold in patients with multiple sclerosis. Virus-specific antigen, antiserum, and receptor, at least in part, inhibited adherence to all cells tested. It is concluded from these studies that increased lymphocyte adherence in multiple sclerosis is not measles virus specific.     

Selective inhibition of translation of the mRNA coding for measles virus membrane protein at elevated temperatures.

The elevation of culture temperatures of C6 cells that were persistently infected with the Lec strain of the subacute sclerosing panencephalitis (SSPE) virus (C6/SSPE) resulted in immediate selective inhibition of membrane (M) protein synthesis. This phenomenon was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cytoplasmic lysates and immunoprecipitation with monoclonal antibody against the M protein in short-time labeling experiments. The synthesis of various viral mRNAs in the presence of actinomycin D decreased gradually at similar rates after a shift to 39 degrees C. No specific disappearance of the mRNA coding for the M protein was observed when viral RNAs isolated from the infected cells were compared before and after a shift up by Northern blot analysis. Results of pulse-chase experiments did not show any significant difference in M protein stability between 35 and 39 degrees C. This rapid block of M protein synthesis was observed not only in Vero cells that were lytically infected with plaque-purified clones from the Lec strain, clones isolated from C6/SSPE cells and the standard Edmonston strain of measles virus but also in CV1, MA160, and HeLa cells that were lytically infected with the Edmonston strain. Poly(A)+ RNAs that were extracted from C6/SSPE cells before and after a shift to 39 degrees C produced detectable phospho, nucleocapsid, and M proteins in cell-free translation systems at 32 degrees C. Even higher incubation temperatures did not demonstrate the selective depression of M protein synthesis described above in vitro. All these data indicate that M protein synthesis of measles virus is selectively suppressed at elevated temperatures because of an inability of the translation apparatus to interact with the M protein-encoded mRNA.     

麻疹病毒沪191对HeLa肿瘤细胞抑制作用的体内外实验研究

研究麻疹病毒减毒疫苗沪191株(MV沪191)在组织培养中和裸鼠体内对HeLa肿瘤细胞的抑制作用.用空斑实验测定MV沪191感染HeLa细胞后细胞裂解液中病毒量;用MTF试验测定MV沪191感染对细胞活性的影响;用流式细胞仪分析测定MV沪191感染引起的细胞凋亡和对细胞周期的影响;HeLa肿瘤细胞背部皮下接种BALB/C裸鼠引起的肿瘤,评估MV沪191体内抑瘤作用.MV沪191感染HeLa细胞后可引起广泛的CPE,感染的HeLa细胞与对照组相比细胞活性明显降低.MV沪191感染HeLa细胞后随着时间延长,G1/G0细胞率明显增多,S期率明显减少,细胞凋亡率明显增加(P＜0.01).给药第60天时瘤内治疗组、静脉治疗组和对照组肿瘤体积平均分别为15.5、64.6、156.4 mm3.瘤内治疗组与对照组相比有显著差异(P＜0.01);静脉治疗组与对照组相比有明显差异(P＜0.05).MV沪191减毒株在组织培养中和裸鼠体内对HeLa肿瘤有明显的杀伤作用.     

Rescue of a positive stranded RNA virus from antigen negative neuroblastoma cells.

Single cell clones of latently infected mouse neuroblastoma cells were isolated from a culture chronically infected with mouse hepatitis virus in the presence of an antiviral antibody. These cell clones did not produce infections virus or exhibit viral cytopathic effects during cultivation at 32, 37, or 39°C. Infectious virus was isolated from single cell clones via fusion with permissive cells using polyethylene glycol, but not after fusion with inactivated Sendai virus or following treatment with metabolic inhibitors. One cell clone (S-3) from which virus was rescued was negative for viral antigen by immunofluorescence. The S-3 cell clone and no demonstrable virus antigen by complement-fixation tests using cytoplasmic extracts or virus-specified proteins detectable by polyacrylamide gel electrophoresis. The rescued viruses exhibited a temperature dependent growth defect at 32°C and have been classified as cold sensitive mutants. This study suggests that a complete genome of a positive stranded RNA virus can remain latent in infected cells without the expression of detectable virus antigen.     

Clonal analysis of mammalian cell cultures persistently infected with Japanese encephalitis virus.

More than 200 cells were cloned from populations of mammalian cells persistently infected with Japanese encephalitis virus. Only four cloned cultures contained cells that had viral antigen measurable by immunofluorescence and that released infectious virus, yet all clones harbored virus-specific RNA. Superinfection of cloned cells with wild-type Japanese encephalitis virus did not produce cytopathic effects, but resulted in production of viral antigen and infectious virus in formerly nonproducing clones. Cocultivation of nonproducer clone cells with normally permissive cells did not induce virus production, nor did treatment of nonproducer clones with various inhibitors of DNA, RNA, or protein synthesis. It is suggested that the cloning procedure may have selected for a particular subpopulation of cells and that defective virus is also involved in establishment and maintenance of persistent infection.     

麻疹病毒血凝素基因工程抗原及其抗原性检测(英文)

将麻疹病毒 (Nepal株 )的血凝素 (hemagglutinin)基因插入真核表达载体pIRES EGFP ,并在HeLa细胞中表达 .因其较低的表达量 ,所以将其截短 ,去除跨膜区 .使这个截短的HA基因与绿色荧光蛋白基因融合 ,并克隆至原核表达载体pET 2 8b中 .将重组质粒转入大肠杆菌中表达 ,产生了分子量约为 90kD的融合蛋白 .通过ELISA和Western印迹来检测这个基因工程蛋白的抗原性 .在检测一系列的血凝素阳性或阴性的人血清中 ,这个融合蛋白的阳性检出率为 90 % ,阴性检出率为 10 0 % (与市售麻疹病毒诊断试剂盒相比较 ) .由于此HA蛋白是原核表达产物 ,回避了真核表达系统复杂的操作过程和昂贵的费用 ,所以 ,这个麻疹病毒血凝素基因工程抗原有望成为一种新型、便捷的麻疹病毒诊断试剂     

Infection of human peripheral blood mononuclear cells with a temperature-sensitive mutant of measles virus.

A stable temperature-sensitive mutant of measles virus (MV ts38) was used to study the mechanism of virus-mediated immune suppression of peripheral blood mononuclear cells in vitro. Both unstimulated and phytohemagglutinin-stimulated cultures released infectious virus at 32 degrees C, whereas no virus was released at 37 degrees C, although both viral RNA and viral proteins were synthesized. However, the response of the lymphoid cells to phytohemagglutinin, concanavalin A, and herpes simplex virus antigen was decreased in the presence of MV ts38 at 37 degrees C. The viability of infected cells was not diminished, therefore excluding cell death as a reason for immunosuppression. Interleukin 2 did not play a role in the inhibitory effect of MV ts38. Antibodies to alpha interferon partially reversed the inhibitory effect of the virus infection on lymphocyte mitogenesis, thus implying that alpha interferon plays a role in the immunosuppression. Depletion experiments indicated that adherent cells play a greater role in the measles virus-induced immunosuppression than nonadherent cells. However, monocyte maturation to macrophages had no effect on the degree of immunosuppression.     

Effect of Vesicular Stomatitis Virus Infection on the Histocompatibility Antigen of L Cells

When mouse L cells are infected for 22 hr with vesicular stomatitis virus (VSV), a ribonucleic acid-containing enveloped virus, greater than 70% of the major histocompatibility antigen (H-2), is no longer detectable by the method of inhibition of immune cytolysis. Infected cells prelabeled with (14)C-glucosamine also show a correspondingly greater loss of trichloroacetic acid-insoluble radioactivity than uninfected cells. The loss of H-2 antigenic activity is not due to the viral inhibition of host cell protein synthesis since cells cultured for 18 hr in the presence of cycloheximide have the same amount of H-2 activity as untreated controls. Also, cells infected with encephalomyocarditis virus, a picornavirus, show no loss of H-2 activity at a time when host cell protein synthesis is completely inhibited. VSV structural proteins associated in vitro with uninfected L-cell plasma membranes do not render H-2 sites inaccessible to the assay. Although antibodies may not combine with all the H-2 antigenic sites on the plasma membrane, anti-H-2 serum reacted with L cells before infection does not prevent a normal infection with VSV. H-2 activity can be detected in virus samples purified from the medium of infected L cells; this virus purified after being mixed with L-cell homogenates shows greater H-2 activity than virus purified after being mixed with HeLa cell homogenates. However, VSV made in HeLa cells shows no H-2 activity when mixed with L-cell homogenates.     

Predominant infection of CD150+ lymphocytes and dendritic cells during measles virus infection of macaques

Measles virus (MV) is hypothesized to enter the host by infecting epithelial cells of the respiratory tract, followed by viremia mediated by infected monocytes. However, neither of these cell types express signaling lymphocyte activation molecule (CD150), which has been identified as the receptor for wild-type MV. We have infected rhesus and cynomolgus macaques with a recombinant MV strain expressing enhanced green fluorescent protein (EGFP); thus bringing together the optimal animal model for measles and a virus that can be detected with unprecedented sensitivity. Blood samples and broncho-alveolar lavages were collected every 3 d, and necropsies were performed upon euthanasia 9 or 15 d after infection. EGFP production by MV-infected cells was visualized macroscopically, in both living and sacrificed animals, and microscopically by confocal microscopy and FACS analysis. At the peak of viremia, EGFP fluorescence was detected in skin, respiratory and digestive tract, but most intensely in all lymphoid tissues. B- and T-lymphocytes expressing CD150 were the major target cells for MV infection. Highest percentages (up to 30%) of infected lymphocytes were detected in lymphoid tissues, and the virus preferentially targeted cells with a memory phenotype. Unexpectedly, circulating monocytes did not sustain productive MV infection. In peripheral tissues, large numbers of MV-infected CD11c+ MHC class-II+ myeloid dendritic cells were detected in conjunction with infected T-lymphocytes, suggesting transmission of MV between these cell types. Fluorescent imaging of MV infection in non-human primates demonstrated a crucial role for lymphocytes and dendritic cells in the pathogenesis of measles and measles-associated immunosuppression.     

Establishment of a persistent measles virus infection in HEp-2 cells.

A productive measles virus persistent infection has been established in HEp-2 cells. Greater than 90% of the persistently infected HEp-2 cells (H2MV) exhibited measles specific immunofluorescence and haemadsorption. Although most of the H2MV cells contained measles specific antigens, only a small percentage (less than 1%) actually produced infectious measles virus as determined by infectious centre assays. The measles virus produced by H2MV cells exhibited properties different from the initiating parent Edmonston strain virus, being reduced in virulence and also temperature sensitive for replication at 39 degrees C. The role of these altered virus properties in the establishment of persistence is considered.     

Engineered serine protease inhibitor prevents furin-catalyzed activation of the fusion glycoprotein and production of infectious measles virus.

We have identified the major cellular endoprotease that activates the fusion (F) glycoprotein of measles virus (MV) and have engineered a serine protease inhibitor (serpin) to target the endoprotease and inhibit the production of infectious MV. The F-protein precursor of MV was not cleaved efficiently into the mature F protein in human colon carcinoma cells lacking functional furin, indicating that furin is the major enzyme responsible for activation of the MV F protein. A human serpin alpha 1-antitrypsin variant was engineered to specifically inhibit furin. When expressed from a recombinant vaccinia virus in primate cells infected by MV, the engineered serpin (alpha 1-PDX) specifically inhibited furin-catalyzed cleavage of the F-protein precursor without affecting synthesis of other MV proteins. We generated human glioma cells stably expressing alpha 1-PDX. MV infection in these cells did not result in syncytia. The infected cells produced all the MV proteins, but the F-protein precursor remained largely uncleaved. This did not prevent virus assembly. However, the released virions contained inactive F-protein precursor rather than mature F protein, and infectious-virus titers were reduced by 3 to 4 orders of magnitude. These results show that a mature F protein is not required for the assembly of MV but is crucial for virus infectivity. The engineered serpin may offer a novel molecular antiviral approach against MV.     

Binding sites for measles virus antigens on human B and T lymphocytes

The present study examined the differences in the binding of measles virus antigens to human peripheral blood lymphocyte (PBL) subpopulations. PBL binding sites for measles antigens were detected by an assay involving the rosetting of PBL to measles-infected HeLa cells (HeLa-K11). Three approaches were employed to examine whether measles virus antigen binding sites were present on restricted subpopulations of PBL. First, no significant difference in the proportion of HeLa-K11 forming clusters was observed with unfractionated cells in comparison with enriched B- or T-lymphocyte suspensions. Second, the profile of lymphocyte surface markers before and after adherence of PBL suspensions to HeLa-K11 cells was measured. No difference in the proportion of PBL forming E-rosettes or lymphocytes with Fc-IgG receptors, surface immunoglobulin, or complement receptors was observed. Finally, the percentage of B (Raji, B-35M, Bristow-7B) and T (Molt-3) cell human lymphoid cells which adhered to HeLa-K11 versus noninfected HeLa cells was compared. In all cases, a highly significant adherence of the lymphoid cell suspensions to HeLa-K11 cells was observed in comparison with uninfected HeLa cells. This is the first direct demonstration of binding sites for measles virus antigens present on both human B and T lymphocytes.     

Antigen discovery in chronic human inflammatory central nervous system disease: panning phage-displayed antigen libraries identifies the targets of central nervous system-derived IgG in subacute sclerosing panencephalitis.

The presence of increased IgG in the brains of humans with infectious and inflammatory CNS diseases of unknown etiology such as multiple sclerosis may be a clue to the cause of disease. For example, the intrathecally synthesized oligoclonal bands in diseases such as subacute sclerosing panencephalitis (SSPE) or cryptococcal meningitis have been shown to represent Ab directed against the causative agents, measles virus (MV), or Cryptococcus neoformans, respectively. Using SSPE as a model system, we developed a strategy to identify the antigenic targets of the intrathecal disease-relevant IgG in chronic human inflammatory and demyelinating diseases of the CNS. Libraries of cDNA Ags were displayed on the surface of T7Select bacteriophage and biopanned on IgG extracted from the brain of an SSPE patient, or on a monospecific recombinant Fab identified from SSPE brain. After three or six rounds of biopanning on either Ab, positive phage-displayed Ags reacting with IgG were enriched to 35-77% of all panned clones. Sequence analysis of the positive clones identified fragments of the nucleocapsid protein of MV, the cause of SSPE. The sensitivity of the system was determined by diluting the positive clones from this SSPE phage-displayed library at a ratio of 10(-6) into another phage-displayed library that did not contain any detectable MV Ags; after six rounds of panning, the positive clones comprised 34% of all phage and were also shown to be MV nucleocapsid specific. This strategy will be useful to identify potentially rare Ags in diseases of unknown cause.