Detection of antibody to M protein of measles virus in patients with subacute sclerosing panencephalitis: a comparative study on immunoprecipitation

Consistent results have not been obtained yet on the presence of antibody to the M protein of measles virus in the sera of patients with subacute sclerosing panencephalitis (SSPE). We performed a comparative study on various immunoprecipitation systems which appeared in the literature and found that the difference in the composition of the solubilizing buffer produced a large variety of results on the immunoprecipitation. [35S]Methionine-labeled Vero cells infected with the Edmonston strain of measles virus were solubilized by 10 different buffers and reacted with hyperimmune rabbit serum to whole virus, monospecific antisera to H, NP, and M proteins of the virus, normal adults' sera, and the sera from 16 SSPE patients. The immune complex was absorbed by protein A and both solubilization and precipitation rates were compared with each viral protein. Although viral proteins were solubilized by all buffers, the solubilization rate varied considerably. M protein was solubilized and was not coprecipitated nonspecifically with any of the other viral proteins. Purified protein A conjugated to Sepharose was preferable to Staphylococcus aureus for absorption of the immune complex since the latter absorbed both viral and host proteins nonspecifically. The precipitation rates of the viral proteins also varied according to the buffers. Better solubilization of the viral proteins seemed to reduce their rate of precipitation for which the presence of SDS may be responsible, and the presence of the protease inhibitors may also affect the results of immunoprecipitation. Detection of M protein in the immunoprecipitates was largely influenced by the kind of buffer used: some buffers could detect it clearly, but others could not defect it at all. Among the solubilizing buffers tested, Saleh's buffer (Virology 93: 369-376 (1979)),, which contains 0.5% DOC and 0.5% Triton X-100, was most reliable for detection of the anti-M antibody in the rabbit serum, because it showed a high solubilization and high precipitation rates of viral proteins without nonspecific absorption by protein A or coprecipitation of M proteins with any of the other proteins. Using this buffer, we could definitely detect M proteins in the immunoprecipitates from the sera of all six healthy adults and 15 out of 16 patients with SSPE. It was found, however, that the amount of M proteins in SSPE patients was lower than that in healthy adults and varied considerably.     

Humoral and cellular immune responses to matrix protein of measles virus in subacute sclerosing panencephalitis.

The immune response to matrix (M) protein of measles virus was examined in patients with subacute sclerosing panencephalitis (SSPE) and controls. Antibodies specific for M and nucleocapsid (NC) proteins in 11 serum and 8 cerebrospinal fluid (CSF) samples from patients with SSPE were quantitated by enzyme-linked immunosorbent assay by using affinity-purified measles virus proteins. Geometric mean anti-NC antibody titers were higher in the serum (6.58 +/- 0.98 [mean +/- standard deviation]) and CSF (4.38 +/- 0.74) of SSPE patients compared with controls. Anti-M antibodies were present in the serum and CSF of all SSPE samples tested but in titers lower than those of anti-NC antibodies. Geometric mean anti-M antibody titer was 3.35 +/- 0.53 in sera from patients with SSPE compared with 3.05 +/- 0.66 in sera from patients with other neurological diseases and 3.12 +/- 0.74 in sera from healthy individuals. Geometric mean anti-M antibody titer was 2.59 +/- 0.86 in the CSF of eight patients with SSPE compared with a mean less than 1.00 for patients with other neurological disease (controls). Intrathecal synthesis of anti-M or anti-NC antibodies was established in four patients with SSPE. The cellular immune responses to M, F, HA, and NC proteins were examined in four of the patients with SSPE by lymphoproliferation and were not significantly different from those in five healthy controls. The results demonstrate humoral and cellular immune responses to M protein in patients with SSPE and indicate that it is unlikely that a defect in the immune response to this virus component accounts for the disease process in the patients studied.     

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.     

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.     

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.     

Changes in the level of humoral response for measles virus in patients with subacute sclerotic panencephalitis during 1984-1989]

Immunological response to measles virus in serum and cerebro-spinal fluid of 338 patients with subacute sclerotic panencephalitis (SSPE) during the years 1984-1989, was investigated. It was found that mean titer and distribution of results of antibody determination by the hemagglutination inhibition (OZHA) method in patients sera are visibly differing during consecutive years of investigation. Analysis of these parameters in determination by the immunoenzymatic methods (ELISA) has shown their higher stability. Also results of determinations of IgG concentration in sera of patients with SSPE did not demonstrate significant differences between consecutive years of investigation. Systematic decrease of mutual relation between antibodies to measles virus detected by ELISA and OZHA was found. Analysis of local synthesis of antibodies to measles virus in the central nervous system has revealed that it most strongly expressed by hemagglutinin.     

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.     

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.     

Diversity of matrix protein in subacute sclerosing panencephalitis and measles virus-infected cells.

Expression of the viral matrix (M) proteins in Vero cells infected with 18 strains of subacute sclerosing panencephalitis (SSPE) virus and measles virus was examined by immunocytochemistry and Western blot analysis using an anti-M monospecific serum and two sera against the M protein specific synthetic peptides. By immunocytochemistry using the anti-M monospecific serum, M protein was detected in all of the virus-infected cells regardless of cell-free virus production. M proteins of the seven non-productive strains were found to vary significantly in their epitope, in their reactivity to different assay systems, and in their molecular weight, whereas M proteins of the other 11 productive strains were detected consistently. These results suggest diversification of M protein of the non-productive strains.     

Protective effect of measles virus inoculation on subacute sclerosing panencephalitis virus-infected mice

The Biken strain of subacute sclerosing panencephalitis (SSPE) virus caused a fatal neurologic disease in adult mice after intracerebral inoculation. However, the mice were completely protected from the disease when a high dose of measles virus was given intracerebrally after the SSPE virus infection. The measles virus inoculation induced interferon production and immune responses. An experiment with athymic nude mice showed that interferon and anti-measles antibody were able to prolong the incubation period of the disease but not to protect the SSPE virus-infected nude mice from death. For complete protection, T lymphocytes appeared to be essential. The present study suggested that the protective effect of measles virus inoculation is basically due to the induction of immune responses and that SSPE virus infection in mice is susceptible to immune reactions.     

Mice immunized with measles virus develop antibodies to a cell surface receptor for binding virus.

Mice were immunized with measles virus to determine whether an auto-anti-idiotypic antireceptor response could be generated as a probe for measles virus receptors. Mice initially responded to viral antigens (days 11 to 18) and subsequently developed antibodies to a putative measles virus receptor (peak at day 30 to 35) by three criteria: the sera (1) agglutinated erythrocytes which virus agglutinates, (2) reacted with Vero cells, and (3) inhibited virus attachment to Vero cells. Additionally, select sera inhibited virus infection of Vero cells. The cell-reactive activity was identified as immunoglobulin G antibody and was neutralized by sera reacting with virus (idiotype). The application of this anti-idiotypic antibody to identify measles virus-binding sites on Vero cells was revealed by the ability of sera to immunoprecipitate 20- and 30.5-kilodalton proteins from metabolically labeled ([35S]methionine) Vero cells.     

Development of antibody to measles virus polypeptides during complicated and uncomplicated measles virus infections.

Immune precipitation of 181 sera from 152 patients with natural measles was studied to determine the temporal course and frequency of antibody responses to nucleocapsid, fusion, hemagglutinin, and matrix proteins of measles virus. Large amounts of antibody to nucleocapsid protein developed in all patients by day one of the rash. Antibody to hemagglutinin and fusion proteins developed in all patients over the next 3 weeks, the former to high levels and the latter to low levels. Antibody to matrix protein developed to very low levels and was detectable in only 41% of the patients; this poor response to matrix protein was not correlated with the age of the patient or the acute neurological complications of measles.     

Functional analysis of matrix proteins expressed from cloned genes of measles virus variants that cause subacute sclerosing panencephalitis reveals a common defect in nucleocapsid binding.

We have developed an in vitro nucleocapsid-binding assay for studying the function of the matrix (M) protein of measles virus (MV) (A. Hirano, A. H. Wang, A. F. Gombart, and T. C. Wong, Proc. Natl. Acad. Sci. USA, 89:8745-8749, 1992). In this communication we show that the M proteins of three MV strains that cause acute infection (Nagahata, Edmonston, and YN) bind efficiently to the viral nucleocapsids whereas the M proteins of four MV strains isolated from patients with subacute sclerosing panencephalitis (SSPE) (Biken, IP-3, Niigata, and Yamagata) fail to bind to the viral nucleocapsids. MV Biken (an SSPE-related virus) produces variant M sequences which encode two antigenically distinct forms of M protein. A serine-versus-leucine difference is responsible for the antigenic variation. MV IP-3 (an SSPE-related virus) also produces variant M sequences, some of which have been postulated to encode a functional M protein responsible for the production of an infectious revertant virus. However, the variant M proteins of Biken and IP-3 strains show no nucleocapsid-binding activity. These results demonstrate that the nucleocapsid-binding function is conserved in the M proteins of MV strains that cause acute infection and that the M proteins of MV strains that cause SSPE exhibit a common defect in this function. Analysis of chimeric M proteins indicates that mutations in the amino-terminal, carboxy-proximal, or carboxy-terminal region of the M protein all abrogate nucleocapsid binding, suggesting that the M protein conformation is important for interaction with the viral nucleocapsid.     

Role of biased hypermutation in evolution of subacute sclerosing panencephalitis virus from progenitor acute measles virus.

We identified an acute measles virus (Nagahata strain) closely related to a defective virus (Biken strain) isolated from a patient with subacute sclerosing panencephalitis (SSPE). The proteins of Nagahata strain measles virus are antigenically and electrophoretically similar to the proteins of Edmonston strain measles virus. However, the nucleotide sequence of the Nagahata matrix (M) gene is significantly different from the M genes of all the acute measles virus strains studied to date. The Nagahata M gene is strikingly similar to the M gene of Biken strain SSPE virus isolated several years later in the same locale. Eighty percent of the nucleotide differences between the Nagahata and Biken M genes are uridine-to-cytosine transitions known as biased hypermutation, which has been postulated to be caused by a cellular RNA-modifying activity. These biased mutations account for all but one of the numerous missense genetic changes predicted to cause amino acid substitutions. As a result, the Biken virus M protein loses conformation-specific epitopes that are conserved in the M proteins of Nagahata and Edmonston strain acute measles viruses. These conformation-specific epitopes are also absent in the cryptic M proteins encoded by the hypermutated M genes of two other defective SSPE viruses (Niigata and Yamagata strains). Nagahata-like sequences are found in the M genes of at least five other SSPE viruses isolated from three continents. These data indicate that Biken strain SSPE virus is derived from a progenitor closely resembling Nagahata strain acute measles virus and that biased hypermutation is largely responsible for the structural defects in the Biken virus M protein.     

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.     

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.     

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.     

Functional and nonfunctional measles virus matrix genes from lethal human brain infections.

Subacute sclerosing panencephalitis (SSPE) is a lethal disease induced by the persistence of measles virus in the human brain. In many SSPE cases, the viral matrix (M) protein cannot be detected; in others, M proteins of the expected size are found and sequence analysis of M cDNAs has confirmed that the reading frames are intact, showing only several missense mutations. To determine whether these alterations result in nonfunctional proteins, we have replaced the M gene of an infectious full-length genomic cDNA (from vaccine strain Edmonston) with different M genes derived from four patients with SSPE. One of the SSPE M genes tested proved to be functionally competent, giving rise to a virus yielding titers similar to those of viruses containing the M gene from control lytic strains. The other three SSPE M genes were not functionally competent in the same test. In all three cases, the inactivating changes resided in the carboxyl-terminal half of the M protein, as shown by the exchange of either of the two genes halves. In summary, mutational M gene alterations, which either prevent synthesis of M protein altogether or only allow synthesis of nonfunctional M protein, have been detected by us and by others in 9 of 10 SSPE cases. The one functional M gene appears to be an exception to the rule, indicating that M gene alteration might not be an absolute requirement for disease development.     

Immunoglobulin g antibody-mediated enhancement of measles virus infection can bypass the protective antiviral immune response

Antibodies to viral surface glycoproteins play a crucial role in immunity to measles by blocking both virus attachment and subsequent fusion with the host cell membrane. Here, we demonstrate that certain immunoglobulin G (IgG) antibodies can also enhance the entry of measles virus (MV) into monocytes and macrophages. Antibody-dependent enhancement of infectivity was observed in mouse and human macrophages using virions opsonized by a murine monoclonal antibody against the MV hemagglutinin (H) glycoprotein, polyclonal mouse anti-MV IgG, or diluted measles-immune human sera. Neither H-specific Fab fragments nor H-specific IgM could enhance MV entry in monocytes or macrophages, indicating involvement of a Fc γ receptor (FcγR)-mediated mechanism. Preincubation with an anti-fusion protein (anti-F) monoclonal antibody or a fusion-inhibitory peptide blocked infection, indicating that a functional F protein was required for viral internalization. Classical complement pathway activation did not promote infection through complement receptors and inhibited anti-H IgG-mediated enhancement. In vivo, antibody-enhanced infection allowed MV to overcome a highly protective systemic immune response in preimmunized IfnarKo-Ge46 transgenic mice. These data demonstrate a previously unidentified mechanism that may contribute to morbillivirus pathogenesis where H-specific IgG antibodies promote the spread of MV infection among FcγR-expressing host cells. The findings point to a new model for the pathogenesis of atypical MV infection observed after immunization with formalin-inactivated MV vaccine and underscore the importance of the anti-F response after vaccination.     

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.