Identification of interferon-resistant subpopulations in several strains of measles virus: positive selection by growth of the virus in brain tissue.

Subacute sclerosing panencephalitis (SSPE) is a chronic and usually fatal central nervous system disease caused by a persistent infection with measles virus. The pathogenic mechanisms of the disease are poorly understood, but restricted expression of viral antigens within the infected tissue appears to be involved. We have previously proposed that interferon (IFN) plays a role in the pathogenesis of SSPE by interacting with viral subpopulations that are relatively resistant to IFN-mediated inhibition. Such IFN-resistant viral subpopulations have now been identified in six independent strains of measles virus, two derived from patients with measles and four derived from patients with SSPE. By means of a replicative-plating procedure, these IFN-resistant viruses were found to be heterogeneous with respect to their growth in the presence of high levels of IFN. One viral form replicates fully, with complete destruction of the infected-cell culture, whereas the other form induces a restricted, self-limited form of cytopathic effect, similar to that seen with cell-associated strains of measles virus isolated from SSPE patients. Passage of a virus stock containing both of these viral forms through the central nervous system tissue of newborn hamsters strongly selects for the viral form associated with the self-limiting type of cytopathic effect. The presence of this form of IFN-resistant virus coupled with chronic production of IFN within the central nervous system may account for viral persistence in SSPE patients.     

Immune response in subacute sclerosing panencephalitis: reduced antibody response to the matrix protein of measles virus.

Immune precipitation was used to study the humoral immune response of patients with subacute sclerosing panencephalitis (SSPE). Patients with SSPE have a progressive infection of the CNS by measles or a measles variant despite high serum antibody levels to measles virus as measured by standard serologic techniques. However, when the antibody response to individual measles virus proteins was measured, we found a striking reduction in the ability of sera from patients with SSPE to precipitate the matrix (M) protein as compared to the precipitation of the M protein by sera from normal adults who had natural measles infection in childhood, or by convalescent sera obtained 3 to 5 weeks after a naturally occurring measles infection. The decreased antibody response to the M protein in sera from patients with SSPE occurred despite a vigorous antibody response to the other viral proteins, suggesting a selective defect in the production of antibody to a single viral protein. The reduced anti-M antibody in sera from patients with SSPE was demonstrated whether immune precipitation was performed with wild-type measles virus or SSPE virus proteins. These results suggest that in SSPE only small amounts of the M protein are produced. This result may help explain how measles virus persists in the central nervous system of patients with SSPE.     

Complement-dependent cytotoxicity of sera obtained from subacute sclerosing panencephalitis patients

Human lymphoid cells (NC-37) persistently infected with either measles virus (Schwarz and TYCSA strains) or subacute sclerosing panencephalitis (SSPE) virus (Halle and Mantooth strains) were destroyed in the presence of complement by anti-measles sera as well as by sera from SSPE patients. The cytotoxic activity was demonstrated in both IgG and IgM fractions of measles convalescent sera, but only in IgG fraction of SSPE sera. Measles convalescent sera completely lost the cytotoxic activity to all the cell lines, when absorbed with any one of the cell lines, indicating that the viral surface antigens of these cell lines infected with measles or SSPE virus are identical. On the other hand, the cytotoxic activity of SSPE sera could not be readily absorbed with these cells. Thus, the affinity of SSPE sera for the viral surface antigens might be lower than that of measles convalescent sera.     

Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.     

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.     

Localization of measles virus antigens in subacute sclerosing panencephalitis in ferrets

Young adult male ferrets were inoculated intracerebrally (i.c.) with a cell-associated encephalitogenic subacute sclerosing panencephalitis (SSPE) virus strain to study the pathogenesis of the disease at the ultrastructural level. Most became acutely ill in 8-13 days. Areas of the brain were examined with indirect immunoperoxidase labeling techniques to detect measles antigen. None of these animals showed the characteristic viral nucleocapsids or marked inflammatory response associated with SSPE. However, all had positive immunolabeling of unstructured virus antigen, especially in post-synaptic regions in all areas of the brain that were examined. One ferret, immunized with measles vaccine 40 days prior to challenge with SSPE, became ill 18 days post inoculation (p.i.). Perivascular cuffings of inflammatory cells and large cytoplasmic inclusions of fuzzy nucleocapsids were found in the brain and spinal cord. The study indicates that ferrets which become acutely ill after inoculation with cell-associated SSPE virus do so before there is a marked cellular immune response or formation of virus nucleocapsids.     

Measles virus encephalitis in ferrets as a model for subacute sclerosing panencephalitis

Young adult ferrets were used as experimental animals to study subacute sclerosing panencephalitis (SSPE). When cells infected with cell-associated measles virus strains isolated from SSPE patients were inoculated intracerebrally (i.c.) into ferrets, they developed an acute encephalitis and died within 1 to 3 weeks without detectable antibody formation. Immunization with live measles vaccine 5 weeks before i.c. inoculation changed the course of the infection in about 50% of the ferrets. These animals developed a subacute encephalitis within weeks or months after inoculation. Cell-associated measles virus was isolated from their brains and high measles antibody titers were found in their sera, comparable to those in sera of SSPE patients. Measles virus specific immunoglobulins (IgG) were present in their brains and determination of IgG/albumin ratios indicated that antibodies were synthesized in the brain in response to the persistent measles virus infection. Measles specific oligoclonal IgG bands were found in the sera and spinal fluids of these animals. Therefore, subacute ferret encephalitis has virological and immunological characteristics in common with SSPE, indicating that it may serve as a model for the human disease. Other animal models of SSPE are described briefly.     

Comparison of Subacute Sclerosing Panencephalitis and Measles Viruses: an Electron Microscope Study

The ultrastructure of CV-1 cells infected with subacute sclerosing panencephalitis (SSPE) viruses was compared with that of CV-1 cells infected with the wild or Edmonston strain of measles virus. Both SSPE viruses and the measles viruses produced two types of nucleocapsid structures: smooth filaments, 15 to 17 nm in diameter, and granular filaments, 22 to 25 nm. The smooth and granular filaments produced by SSPE and measles virus did not differ in appearance. In CV-1 cells infected with SSPE viruses, smooth filaments formed large intranuclear inclusions and granular filaments occupied a large area of the cytoplasm, but always spared the area under the cell membrane. Particles budding from the surface of these cells contained no nucleocapsids. In CV-1 cells infected with measles virus, only small aggregates of smooth filaments were seen in the nuclei. Granular filaments in the cytoplasm predominantly occupied the area under the cell membrane, and were aligned beneath the cell membrane in a parallel fashion and assembled into budding particles. These differences between SSPE and measles virus may be regarded as quantitative, but they do distinguish SSPE viruses from measles virus. Moreover, the formation of large nuclear inclusions filled with smooth filaments appears to be a characteristic process of SSPE, but not of measles, since this type of inclusion is invariably seen in SSPE brain tissues, brain cultures derived from them, and CV-1 cells infected with SSPE viruses.     

Elimination of hepatitis C virus from hepatocytes by a selective activation of therapeutic molecules

To eliminate hepatitis C virus (HCV) from infected hepatocytes, we generated two therapeutic molecules specifically activated in cells infected with HCV. A dominant active mutant of interferon (IFN) regulatory factor 7 (IRF7) and a negative regulator of HCV replication, VAP-C (Vesicle-associated membrane protein-associated protein subtype C), were fused with the C-terminal region of IPS-1 (IFNβ promoter stimulator-1), which includes an HCV protease cleavage site that was modified to be localized on the ER membrane, and designated cIRF7 and cVAP-C, respectively. In cells expressing the HCV protease, cIRF7 was cleaved and the processed fragment was migrated into the nucleus, where it activated various IFN promoters, including promoters of IFNα6, IFNβ, and IFN stimulated response element. Activation of the IFN promoters and suppression of viral RNA replication were observed in the HCV replicon cells and in cells infected with the JFH1 strain of HCV (HCVcc) by expression of cIRF7. Suppression of viral RNA replication was observed even in the IFN-resistant replicon cells by the expression of cIRF7. Expression of the cVAP-C also resulted in suppression of HCV replication in both the replicon and HCVcc infected cells. These results suggest that delivery of the therapeutic molecules into the liver of hepatitis C patients, followed by selective activation of the molecules in HCV-infected hepatocytes, is a feasible method for eliminating HCV.     

Characterization of Virus-Specific RNAs from Subacute Sclerosing Panencephalitis Virus-Infected CV-1 Cells

CV-1 cells infected with subacute sclerosing panencephalitis (SSPE) virus incorporated uridine-(3)H into at least four virus-specific RNA components in the presence of actinomycin D. The component sedimenting fastest had a sedimentation coefficient of 50s corresponding to a molecular weight of 6 x 10(6). The other three RNA components have sedimentation constants of 35s, 22s, and 18s corresponding to molecular weights of 2.5 x 10(6), 1.0 x 10(6), and 0.75 x 10(6), respectively. The base composition of the 50s RNA is distinct from that of cellular RNA and comparable with base compositions of viral RNAs of other paramyxoviruses. The base composition of the 18s RNA shows approximate complementarity with the 50s RNA. RNA-RNA annealing experiments using unlabeled 50s SSPE RNA with labeled 18s RNA from cells infected with SSPE virus or measles virus show 100% annealing with 18s SSPE RNA but only 60% annealing with 18s measles RNA. These experiments suggest some differences between the 18s RNAs of SSPE virus-infected cells and measles virus-infected cells.     

Growth of measles and subacute sclerosing panencephalitis viruses in human neural cell lines

Growth of cell-free subacute sclerosing panencephalitis (SSPE) virus was compared with that of measles virus in three human neural cell lines; neuroblastoma, oligodendroglioma, and glioblastoma. The Edmonston strain of measles virus replicated in these neural cells as efficiently as in Vero cells. In contrast, the growth of the Mantooth strain of SSPE virus was suppressed moderately in neuroblastoma cells and markedly in oligodendroglioma and glioblastoma cells in spite of the induction of apparent cytopathic effects in these cells. Virus adsorption, defective interfering particles, interferon, and temperature sensitivity were not responsible for this low yield of SSPE virus in neural cell lines. Synthesis of viral proteins of SSPE virus was slower than that of measles virus in oligodendroglioma and glioblastoma cells. These results suggest that the slow rate of synthesis of viral proteins may be relevant to the low yield of SSPE virus in neural cells.     

Cell-associated immunity to measles (rubeola): The demonstration of in vitro lymphocyte tritiated thymidine incorporation in response to measles complement fixation antigen

Lack of a reliable in vitro assay for lymphocyte responsiveness to measles (rubeola) has hampered our understanding of the cell-associated response in diseases caused by, or related to, the measles virus. We report a reliable and reproducible system for demonstrating specific lymphocyte incorporation of ³H-thymidine in response to measles complement fixation antigen (CFA). Seventeen patients with positive histories of measles as children demonstrated a dose-response curve that varied between individuals but was constant for each individual. Kinetic data disclosed maximal responsiveness on day 7, and viral inactivation experiments disclosed that live virus was neither necessary for nor inhibitory to the reaction. The implications of this assay in terms of our understanding of the cell-associated response to measles virus in clinical measles and SSPE are discussed. The concept is explored that membrane-associated antigen is crucial in demonstrating the host's cellular immune response to viruses that can grow by cell-to-contiguous cell spread.     

Isolation and characterization of interferon-resistant variants from S49 mouse lymphoma

S49 mouse lymphoma cells were found to be extremely sensitive to the antiproliferative activity of interferon. These characteristics were studied to select for IFN-resistant cell variants. Some 0.6% of the parental S49 cell population were resistant to the antiproliferative and cytotoxic activities of IFN. The resistant cells were cloned and analyzed for their responses to several of the activities of IFN, namely, inhibition of encephalomyocarditis (EMC) virus, murine leukemia virus (MuLV) replications, and the induction of (2'-5') oligoadenylate synthetase. Among the clones selected some were highly resistant while others demonstrated only partial responsiveness to IFN. S49 cells demonstrate tubular structures in the cytoplasm. These structures were previously reported to be antigenically related to mouse mammary tumor virus (MMTV). We report here that IFN treatment decreases the expression of these cytoplasmic viral structures as revealed by electron microscopy. To correlate this novel antiviral activity to the more established functions of IFN we utilized the above mentioned S49 IFN-resistant variants. The anti-MMTV activity of IFN correlated with the other effects of IFN in both the highly resistant and partially responsive S49 clones. Our findings indicate that a relatively high proportion of S49 cells vary in their response to IFN. The defect in the resistant cells appears to affect a primary response to IFN which is common to its diverse activities. Furthermore, the effect of IFN on MMTV-related structures involves the usual pathway of IFN action.     

Detection of IgG antibody to measles virus by radioimmunoassay technique

A highly sensitive procedure of solid-phase radioimmunoassay (RIA) was developed for the detection of measles IgG antibody. HeLa cells persistently infected with measles virus were used as a solid-phase antigen. This technique was applied to the detection of measles IgG antibody in patients with subacute sclerosing panencephalitis (SSPE) and multiple sclerosis. Normal subjects having experienced natural measles or measles vaccination and patients with various neurological diseases of non-virus nature were also examined as control groups. Measles antibody was detected at high titers in both the sera and cerebrospinal fluid of SSPE patients. Moreover, RIA/HI ratios of SSPE patients were significantly higher than those of normal subjects, suggesting the presence in the formers of antibodies to nucleocapsids at high titers as well as to viral envelopes. On the other hand, no significant difference was found in both RIA and HI titers between the sera of multiple sclerosis and those of various neurological diseases.     

Immunoperoxidase Stain of Measles Antigen in Tissue Culture

A specific electron microscopy staining technique for measles antigen has been developed by using Vero cells infected with a subacute sclerosing panencephalitis (SSPE) measles virus strain and fixed in glutaraldehyde or formaldehyde. Peroxidase-labeled antibody was prepared according to the method of Avrameas (4). Sera from SSPE patients with high measles antibody titer as well as normal human sera with and without measles antibody were used. With both fixatives, specific labeling was obtained on the surface of infected cells, on the budding site, and on complete viral particles. The cell membrane staining sometimes had a patchy distribution in that the reaction was most intense on the surface projections in front of each nucleocapsid. This suggests modification of the cell membrane in association with the nucleocapsids. In contrast, no label was detected on the membranes of the cells during the latent period from penetration through maturation of the virus. In formaldehyde-fixed cultures, cytoplasmic inclusions were stained, and this label was located on the "fuzzy" material around the nucleocapsids. The smooth type of nucleocapsids, mainly seen in the nucleus, were never labeled. These findings suggest that the antigenic nature of the "fuzzy" nucleocapsids in the cytoplasm may be different from that of the "smooth" nucleocapsids. The immunoperoxidase method gives good resolution of viral antigenic sites at high magnifications under electron microscopy and may be of value in studies on the immunopathogenesis of SSPE and other chronic viral infections.     

Subacute sclerosing panencephalitis virus dominantly interferes with replication of wild-type measles virus in a mixed infection: implication for viral persistence.

Interaction between the Edmonston or Nagahata strain of acute measles virus (MV) and the defective Biken strain of MV isolated from a patient with subacute sclerosing panencephalitis (SSPE) was examined by a cell fusion protocol. Biken-CV-1 cells nonproductively infected with Biken strain SSPE virus were fused with neomycin-resistant CV-1 cells. All the fused cells selected with the neomycin analog G418 expressed Biken viral proteins, as determined by an immunofluorescence assay. This procedure enabled the transfer of Biken viral genomes into cells previously infected with MV. In the fused cells coinfected by Biken strain SSPE virus and Edmonston or Nagahata strain MV, early MV gene expression was suppressed, as determined by immunoprecipitation with strain-specific antibodies. Maturation of Edmonston strain MV was also suppressed. When the coinfected fused cells were selected with G418, Biken viral proteins remained at a constant level for up to 7 weeks. Wild-type MV proteins gradually decreased to a barely detectable level after 4 weeks and became undetectable after 7 weeks. Immunofluorescence studies showed a steady decline in cells expressing wild-type MV proteins in the coinfected cultures. These results suggest that Biken strain SSPE virus dominantly interferes with the replication of wild-type MV. The possible mechanisms of dominant interference and the implication for evolution of a persistent MV infection are discussed.     

Expression of defective measles virus genes in brain tissues of patients with subacute sclerosing panencephalitis.

The persistence of measles virus in selected areas of the brains of four patients with subacute sclerosing panencephalitis (SSPE) was characterized by immunohistological and biochemical techniques. The five measles virus structural proteins were never simultaneously detectable in any of the brain sections. Nucleocapsid proteins and phosphoproteins were found in every diseased brain area, whereas hemagglutinin protein was detected in two cases, fusion protein was detected in three cases, and matrix protein was detected in only one case. Also, it could be shown that the amounts of measles virus RNA in the brains differed from patient to patient and in the different regions investigated. In all patients, plus-strand RNAs specific for these five viral genes could be detected. However, the amounts of fusion and hemagglutinin mRNAs were low compared with the amounts in lytically infected cells. The presence of particular measles virus RNAs in SSPE-infected brains did not always correlate with mRNA activity. In in vitro translations, the matrix protein was produced in only one case, and the hemagglutinin protein was produced in none. These results indicate that measles virus persistence in SSPE is correlated with different defects of several genes which probably prevent assembly of viral particles in SSPE-infected brain tissue.     

Cloning the antibody response in humans with chronic inflammatory disease: immunopanning of subacute sclerosing panencephalitis (SSPE) brain sections with antibody phage libraries prepared from SSPE brain enriches for antibody recognizing measles virus antigens in situ

In central nervous system (CNS) infectious and inflammatory diseases of known cause, oligoclonal bands represent antibody directed against the causative agent. To determine whether disease-relevant antibodies can be cloned from diseased brain, we prepared an antibody phage display library from the brain of a human with subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus, and selected the library against SSPE brain sections. Antibodies that were retrieved reacted strongly with measles virus cell extracts by enzyme-linked immunosorbent assay and were specific for the measles virus nucleocapsid protein. These antibodies immunostained cells in different SSPE brains but not in control brain. Our data provide the first demonstration that diseased brain can be used to select in situ for antibodies directed against the causative agent of disease and point to the potential usefulness of this approach in identifying relevant antibodies in chronic CNS or systemic inflammatory diseases of unknown cause.