Suppression of viral RNA polymerase activity is necessary for persistent infection during the transformation of measles virus into SSPE virus.

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by measles virus (MV), which typically develops 7 to 10 years after acute measles. During the incubation period, MV establishes a persistent infection in the brain and accumulates mutations that generate neuropathogenic SSPE virus. The neuropathogenicity is closely associated with enhanced propagation mediated by cell-to-cell fusion in the brain, which is principally regulated by hyperfusogenic mutations of the viral F protein. The molecular mechanisms underlying establishment and maintenance of persistent infection are unclear because it is impractical to isolate viruses before the appearance of clinical signs. In this study, we found that the L and P proteins, components of viral RNA-dependent RNA polymerase (RdRp), of an SSPE virus Kobe-1 strain did not promote but rather attenuated viral neuropathogenicity. Viral RdRp activity corresponded to F protein expression; the suppression of RdRp activity in the Kobe-1 strain because of mutations in the L and P proteins led to restriction of the F protein level, thereby reducing cell-to-cell fusion mediated propagation in neuronal cells and decreasing neuropathogenicity. Therefore, the L and P proteins of Kobe-1 did not contribute to progression of SSPE. Three mutations in the L protein strongly suppressed RdRp activity. Recombinant MV harboring the three mutations limited viral spread in neuronal cells while preventing the release of infectious progeny particles; these changes could support persistent infection by enabling host immune escape and preventing host cell lysis. Therefore, the suppression of RdRp activity is necessary for the persistent infection of the parental MV on the way to transform into Kobe-1 SSPE virus. Because mutations in the genome of an SSPE virus reflect the process of SSPE development, mutation analysis will provide insight into the mechanisms underlying persistent infection.

Upregulation of viral RNA polymerase activity promotes adaptation of SSPE virus to neuronal cells.

Subacute sclerosing panencephalitis (SSPE) is a rare progressive neurodegenerative disease caused by measles virus variants (SSPE viruses) that results in eventual death. Amino acid substitution(s) in the viral fusion (F) protein are key for viral propagation in the brain in a cell-to-cell manner, a specific trait of SSPE viruses, leading to neuropathogenicity. In this study, we passaged an SSPE virus in cultured human neuronal cells and isolated an adapted virus that propagated more efficiently in neuronal cells and exhibited increased cell-to-cell fusion. Contrary to our expectation, the virus harbored mutations in the large protein, a viral RNA-dependent RNA polymerase, and in the phosphoprotein, its co-factor, rather than in the F protein. Our results imply that upregulated RNA polymerase activity, which increases F protein expression and cell-to-cell fusion, could be a viral factor that provides a growth advantage and contributes to the adaptation of SSPE viruses to neuronal cells.

Generation of recombinant adenovirus expressing siRNA against the L mRNA of measles virus and subacute sclerosing panencephalitis virus.

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by prolonged persistent infection of the central nervous system with a measles virus (MV) mutant called SSPE virus. At present, there is no effective treatment to completely cure SSPE and development of a new therapeutic measure(s) against this fatal slow virus infection is needed. We previously reported that replication of MV and SSPE virus was effectively inhibited by small interfering RNA (siRNA), either chemically synthetic or plasmid-driven ones, that were targeted against different sequences of the mRNA for the L protein of MV. In this study, we have generated recombinant adenovirus expressing the siRNAs (rAd-siRNA-MV-L2, -L4 and -L5) and demonstrated that these rAd-siRNAs efficiently inhibited replication of MV and SSPE virus in a dose-dependent manner. Due to their high capacity for gene delivery to nerve cells and the potential to inhibit SSPE virus replication, the rAd-siRNAs could be a good candidate for a novel therapeutic measure against SSPE.

Evidence that the hypermutated M protein of a subacute sclerosing panencephalitis measles virus actively contributes to the chronic progressive CNS disease.

Subacute sclerosing panencephalitis (SSPE) is a progressive degenerative disease of the brain uniformly leading to death. Although caused by measles virus (MV), the virus recovered from patients with SSPE differs from wild-type MV; biologically SSPE virus is defective and its genome displays a variety of mutations among which biased replacements of many uridine by cytidine resides primarily in the matrix (M) gene. To address the question of whether the SSPE MVs with M mutations are passive in that they are not infectious, cannot spread within the CNS, and basically represent an end-stage result of a progressive infection or alternatively SSPE viruses are infectious, and their mutations enable them to persist and thereby cause a prolonged neurodegenerative disease, we utilized reverse genetics to generate an infectious virus in which the M gene of MV was replaced with the M gene of Biken strain SSPE MV and inoculated the recombinant virus into transgenic mice bearing the MV receptor. Our results indicate that despite biased hypermutations in the M gene, the virus is infectious in vivo and produces a protracted progressive infection with death occurring as long as 30 to 50 days after that caused by MV. In primary neuron cultures, the mutated M protein is not essential for MV replication, prevents colocalization of the viral N with membrane glycoproteins, and is associated with accumulation of nucleocapsids in cells' cytoplasm and nucleus.

The matrix proteins of neurovirulent subacute sclerosing panencephalitis virus and its acute measles virus progenitor are functionally different.

Persistence of measles virus in the brains of patients with subacute sclerosing panencephalitis (SSPE) is accompanied by changes in the viral matrix (M) protein. To understand the significance of these changes, cell culture and cell-free assays were developed to compare the functions of the M proteins of an SSPE virus Biken strain and its acute measles virus progenitor Nagahata strain. The Nagahata viral M protein is associated with the intracellular viral nucleocapsids and the plasma membrane, whereas the Biken viral M protein is localized mainly in the cytosol. The lack of M protein in the Biken viral nucleocapsids is due to a failure of the Biken M protein to bind to the viral nucleocapsids. The Biken M protein also fails to bind to the Nagahata viral nucleocapsids. Conversely, the Nagahata M protein can bind to the Biken viral nucleocapsids, although this association is not as stable at physiological salt concentration. These results offer concrete evidence that the M protein of an SSPE virus is functionally different from that of its progenitor acute measles virus.

Expression and properties of the V protein in acute measles virus and subacute sclerosing panencephalitis virus strains.

Measles virus (MV) inserts one guanosine (G) residue at a specific site in a subpopulation of the mRNA transcribed from the phosphoprotein (P) gene to produce V mRNA. Using an antiserum against the unique carboxyl-terminal region of the predicted V protein, we found that a phosphorylated V protein was expressed in two acute MV strains (Edmonston and Nagahata) and three SSPE virus strains (Biken, Yamagata, and Niigata). The V protein of Biken strain SSPE virus was electrophoretically and antigenically indistinguishable from the V protein of Nagahata strain acute MV, the likely progenitor of the Biken strain. The V protein of these two viruses was not present in the intracellular viral nucleocapsids, but was found only in the cytosolic free protein pool. Pulse-chase experiments failed to show transport of the V protein to the plasma membrane. The V protein was also absent in the extracellular virions. The P protein synthesized from the cloned gene associated with the MV nucleocapsids in vitro, but the V protein had no affinity to the MV nucleocapsids. These results suggest that expression and properties of the V protein are conserved in chronic MV infection.

Complete nucleotide sequence of the phosphoprotein of the Yamagata-1 strain of a defective subacute sclerosing panencephalitis (SSPE) virus.

The complete nucleotide sequence of the phosphoprotein (P) gene of the Yamagata-1 strain of a defective subacute sclerosing panencephalitis (SSPE) virus was determined. Comparison with the P gene of the Edmonston strain of measles virus (MV) revealed 44 differences of which 23 nucleotides substitutions were identical with those revealed between other SSPE viruses and MV (Cattaneo et al. (1989) Virology 173, 415-425). The consensus sequence of the G insertion site was completely conserved, whereas mRNAs with one or three non-templated G residue insertions were found in addition to the mRNA of the exact genome copy. As a result of the frameshift downstream of the site of G insertion, the cysteine-rich V protein was predicted from the one G-inserted mRNA besides the P and C proteins predicted from the genome-copied 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.

Effect of measles virus antibodies on a measles SSPE virus persistently infected C6 rat glioma cell line.

Maintenance of measles (SSPE-Lec) virus persistently infected C6 rat glioma cells in medium containing polyclonal measles antiserum resulted in the loss of detectable expression of all measles virus proteins. Removal of these cells from antiserum, however, led to a re-expression of virus proteins and the production of infectious virus. Cloning of antibody-modulated non-expressing cells in the presence of antiserum showed that re-expression of virus proteins was not due to an incomplete curing process following the addition of antiserum, as a large number of non-expressing cell clones developed the capacity to express measles virus antigen at different periods after removal of antiserum. Irradiation of persistently infected cells to give a non-growing culture showed that modulation was not mediated by a selection and outgrowth of a small percentage of non-expressing cells originally present in the culture. Antibody directed against C6 membrane proteins did not lead to modulation and it was also shown that only monoclonal antibodies with neutralizing activity could affect intracellular antigen expression. Immunoglobulin Fab fragments with neutralizing activity also had modulating activity. Although all modulated cell clones were more susceptible to homologous virus infection than control C6 cells, it was not possible to rescue any defective measles virus which may have been maintained in the culture.

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.

The measles virus-specific protein synthesis of persistently and lytically infected cells studied in vivo and in vitro.

The virus-specific protein synthesis of the hamster brain cells persistently infected with a wild-type measles virus (M-MB/MVB) was investigated in vivo and in vitro and compared to the protein synthesis of the Vero-cells lytically infected with the wild-type measles virus and the SSPE-measles virus strain LEC. The virus-specific protein synthesis in the M-HB/MVB-cells in vivo was weak. Only two virus-specific polypeptides, corresponding to polypeptide G and nucleocapsid polypeptide NP, were detected. On the other hand, the in vitro translation products, coded by the RNA extracted from the actinomycin D-treated M-HB/MVB-cells, were similar compared to those products coded by the RNAs from the lytically infected Vero-cells. The polypeptides with MWs of 75 000, 61 000, 40 000, 37 000 and 18 000 were coded with all three RNA-extracts. So the similar mRNAs were induced in the actinomycin D-treated M-HB/MVB-cells, but their translation in vivo was limited. In addition to those polypeptides mentioned above the polypeptide with a MW of 42 000 was detected among the in vitro translation products of the M-HB/MVB-cells.

Persistent and lytic infections with SSPE virus: a comparison of the synthesis of virus-specific polypeptides.

The synthesis of virus-specific polypeptides and messenger RNA in cell cultures persistently infected with an isolate of measles virus from a patient with subacute sclerosing panencephalitis (SSPE) has been compared to that found in a lytic infection with the homologous virus. The persistent infection described here was chosen as its biological characteristics reflect those of virus-infected brain cells from SSPE patients. The synthesis of H, N and possibly F protein was seen in both lytic and persistent infections, but the synthesis of M protein was only detected in the lytic infection. However, messenger RNA isolated from either the lytic or persistent infection directed the synthesis in a cell-free translation system of all structural polypeptides, including M, and also three non-structural polypeptides, with mol. wt. of 34 000, 30 000 and 18 000. Messenger RNAs coding for the virus-specific polypeptides were also shown to be polyadenylated. In addition, those polypeptides made in vitro which were antigenically related to the haemagglutinin, demonstrated structural changes after passage through a persistent infection.