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.     

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.     

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.     

Virus entry is a major determinant of cell tropism of Edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins

The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVDeltaG*). MV glycoproteins were efficiently incorporated into VSVDeltaG*, producing the VSV pseudotypes. VSVDeltaG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVDeltaG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVDeltaG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVDeltaG*-EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role.     

Slow development of measles virus (Edmonston strain) infection in the brain of nude mice

The Edmonston strain of measles virus caused neurologic disease in athymic nude mice by intracerebral inoculation. The incubation periods of the disease, however, were extremely long, ranging from 59 to 140 days when the mice were inoculated with 10(4) plaque forming units (PFU) of the virus. The Edmonston strain was highly infectious in the nude mouse brain since virus infection was established even with 1 PFU of the virus. Virus titers in the brains of infected mice increased with the time of incubation. These results indicate that the extremely long incubation period of the disease is ascribed to very slow development of virus infection in the mouse brain. On the other hand, the incubation periods of the Biken strain of SSPE virus were very short (generally within 2 weeks) even with inoculations of 1 PFU of the virus. However, the extent of the dissemination of infection in brains was not significantly different between the two viruses as examined by immunofluorescent staining.     

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.     

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.     

Differential type I IFN-inducing abilities of wild-type versus vaccine strains of measles virus

Laboratory adapted and vaccine strains of measles virus (MV) induced type I IFN in infected cells. The wild-type strains in contrast induced it to a far lesser extent. We have investigated the mechanism for this differential type I IFN induction in monocyte-derived dendritic cells infected with representative MV strains. Laboratory adapted strains Nagahata and Edmonston infected monocyte-derived dendritic cells and activated IRF-3 followed by IFN-beta production, while wild-type MS failed to activate IRF-3. The viral IRF-3 activation is induced within 2 h, an early response occurring before protein synthesis. Receptor usage of CD46 or CD150 and nucleocapsid (N) protein variations barely affected the strain-to-strain difference in IFN-inducing abilities. Strikingly, most of the IFN-inducing strains possessed defective interference (DI) RNAs of varying sizes. In addition, an artificially produced DI RNA consisting of stem (the leader and trailer of MV) and loop (the GFP sequence) exhibited potential IFN-inducing ability. In this case, however, cytoplasmic introduction was needed for DI RNA to induce type I IFN in target cells. By gene-silencing analysis, DI RNA activated the RIG-I/MDA5-mitochondria antiviral signaling pathway, but not the TLR3-TICAM-1 pathway. DI RNA-containing strains induced IFN-beta mRNA within 2 h while the same recombinant strains with no DI RNA required >12 h postinfection to attain similar levels of IFN-beta mRNA. Thus, the stem-loop structure, rather than full genome replication or specific internal sequences of the MV genome, is required for an early phase of type I IFN induction by MV in host cells.     

Identification of a nonproductive, cell-associated form of measles virus by its resistance to inhibition by recombinant human interferon

Subacute sclerosing panencephalitis (SSPE) is a fatal disease in children and young adults that is caused by persistent infection of the central nervous system (CNS) by a nonproductive, cell-associated form of measles virus. Using an experimental model for SSPE (LEC viral strain in newborn hamsters), we have shown previously that establishment of such CNS infections involves selective elimination from the CNS of productively infected cells by host defensive mechanisms, coupled with the selective sparing of cells carrying nonproductive viral forms. That interferon (IFN) may play a role in this process was suggested by the disappearance of productively infected cells from the CNS tissues prior to the appearance of antiviral antibodies and by the demonstration of cell-associated, IFN-resistant viral variants in the virus stocks that were used. Results of this study support these conclusions by showing that similar IFN-resistant viral variants are present in the HBS strain of SSPE-derived measles virus and that these variants, in the presence of IFN, have properties that are similar to those of naturally occurring cell-associated strains of SSPE viruses, e.g., DR, IP3, and Biken. These IFN-resistant forms of HBS virus were isolated and were shown to maintain their resistance to inhibition by IFN after cloning. However, on removal of IFN, they reverted to productive forms similar to the parental HBS virus. The potential role of such viral forms in the pathogenesis of SSPE is discussed.     

Tyrosine phosphorylation of measles virus nucleocapsid protein in persistently infected neuroblastoma cells.

Subacute sclerosing panencephalitis is a slowly progressing fatal human disease of the central nervous system which is a delayed sequel of measles virus (MV) infection. A typical pathological feature of this disease is the presence of viral ribonucleocapsid structures in the form of inclusion bodies and the absence of infectious virus or budding viral particles. The mechanisms governing the establishment and maintenance of a persistent MV infection in brain cells are still largely unknown. To understand the mechanisms underlying MV persistence in neuronal cells, a tissue culture model was studied. Clone NS20Y/MS of the murine neuroblastoma C1300 persistently infected with the wild-type Edmonston strain of MV secretes relatively high levels of alpha/beta interferon (IFN). As shown previously, treatment of the persistently infected cultures with anti-IFN serum converted the persistent state into a productive infection indicated by the appearance of multinucleated giant cells. In this study, we have investigated whether alpha/beta IFN produced by NS20Y/MS cells activates cellular protein tyrosine kinases which will induce tyrosine phosphorylating activity specific to virus-infected cells. We present data to show augmented protein tyrosine kinase activity in the persistently infected cells. We demonstrate that the MV N protein is phosphorylated on tyrosine in addition to serine and threonine in the persistent state but not in NS20Y cells acutely infected with MV.     

Measles viruses possessing the polymerase protein genes of the Edmonston vaccine strain exhibit attenuated gene expression and growth in cultured cells and SLAM knock-in mice

Live attenuated vaccines against measles have been developed through adaptation of clinical isolates of measles virus (MV) in various cultured cells. Analyses using recombinant MVs with chimeric genomes between wild-type and Edmonston vaccine strains indicated that viruses possessing the polymerase protein genes of the Edmonston strain exhibited attenuated viral gene expression and growth in cultured cells as well as in mice expressing an MV receptor, signaling lymphocyte activation molecule, regardless of whether the virus genome had the wild-type or vaccine-type promoter sequence. These data demonstrate that the polymerase protein genes of the Edmonston strain contribute to its attenuated phenotype.     

A Recombinant Measles Vaccine Virus Expressing Wild-Type Glycoproteins: Consequences for Viral Spread and Cell Tropism

Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism.     

Multiple amino acid substitutions in hemagglutinin are necessary for wild-type measles virus to acquire the ability to use receptor CD46 efficiently

Measles virus (MV) possesses two envelope glycoproteins, namely, the receptor-binding hemagglutinin (H) and fusion proteins. Wild-type MV strains isolated in B-lymphoid cell lines use signaling lymphocyte activation molecule (SLAM), but not CD46, as a cellular receptor, whereas MV vaccine strains of the Edmonston lineage use both SLAM and CD46 as receptors. Studies have shown that the residue at position 481 of the H protein is critical in determining the use of CD46 as a receptor. However, the wild-type IC-B strain with a single N481Y substitution in the H protein utilizes CD46 rather inefficiently. In this study, a number of chimeric and mutant H proteins, and recombinant viruses harboring them, were generated to determine which residues of the Edmonston H protein are responsible for its efficient use of CD46. Our results show that three substitutions (N390I and E492G plus N416D or T446S), in addition to N481Y, are necessary for the IC-B H protein to use CD46 efficiently as a receptor. The N390I, N416D, and T446S substitutions are present in the H proteins of all strains of the Edmonston lineage, whereas the E492G substitution is found only in the H protein of the Edmonston tag strain generated from cDNAs. The T484N substitution, found in some of the Edmonston-lineage strains, resulted in a similar effect on the use of CD46 to that caused by the E492G substitution. Thus, multiple residues in the H protein that have not previously been implicated have important roles in the interaction with CD46.     

Mode of Subacute Sclerosing Panencephalitis (SSPE) Virus Infection in Tissue Culture Cells: III. Neurovirulence of Cell-Free SSPE Viruses of Niigata-1, Kitaken-1, and Biken Strains

Cell-free viruses recovered from virus-carrying cultures of the Niigata-1, Kitaken-1, and Biken strains of SSPE virus were examined for neurovirulence. The cell-free viruses were prepared by freezing and thawing or by EDTA treatment of the virus-carrying cultures and inoculated into adult mice intracerebrally. A considerable number of the inoculated mice showed clinical signs about 1 to 5 weeks after the inoculation. The first symptom was hyperreactivity, which was followed by paresis and myoclonus. All of the affected mice fell in paralysis and finally died. The virus could be recovered from the moribund mice by cocultivation of the brain cells with Vero cells. Immunofluorescence staining of the brain tissue revealed that infected cells containing viral antigens were distributed sparsely. No inflammatory feature, however, was observed in the brain as far as examined and neutralizing antibody against SSPE virus was not detected in sera from the mice inoculated with the cell-free SSPE viruses.     

Evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of Alpha/Beta interferon production

Measles is a highly contagious disease currently responsible for over one million childhood deaths, particularly in the developing world. Since alpha/beta interferons (IFNs) are pivotal players both in nonspecific antiviral immunity and in specific cellular responses, their induction or suppression by measles virus (MV) could influence the outcome of a viral infection. In this study we compare the IFN induction and sensitivity of laboratory-passaged attenuated MV strains Edmonston and Moraten with those of recent wild-type viruses isolated and passaged solely on human peripheral blood mononuclear cells (PBMC) or on the B958 marmoset B-cell line. We report that two PBMC-grown wild-type measles isolates and two B958-grown strains of MV induce 10- to 80-fold-lower production of IFN by phytohemagglutinin-stimulated peripheral blood lymphocytes (PBL) compared to Edmonston and Moraten strains of measles. Preinfection of PBL with these non-IFN-inducing MV isolates prevents Edmonston-induced but not double-stranded-RNA-induced IFN production. This suggests that the wild-type viruses can actively inhibit Edmonston-induced IFN synthesis and that this is not occurring by double-stranded RNA. Furthermore, the wild-type MV is more sensitive than Edmonston MV to the effect of IFN. MV is thus able to suppress the synthesis of the earliest mediator of antiviral immunity, IFN-alpha/beta. This could have important implications in the virulence and spread of MV.     

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 virus spread between neurons requires cell contact but not CD46 expression, syncytium formation, or extracellular virus production

In patients with subacute sclerosing panencephalitis (SSPE), which is associated with persistent measles virus (MV) infection in the brain, little infectious virus can be recovered despite the presence of viral RNA and protein. Based on studies of brain tissue from SSPE patients and our work with MV-infected NSE-CD46(+) mice, which express the measles receptor CD46 on neurons, several lines of evidence suggest that the mechanism of viral spread in the central nervous system differs from that in nonneuronal cells. To examine this alternate mechanism of viral spread, as well as the basis for the loss of normal transmission mechanisms, infection and spread of MV Edmonston was evaluated in primary CD46(+) neurons from transgenic mice and differentiated human NT2 neurons. As expected, unlike that between fibroblasts, viral spread between neurons occurred in the absence of syncytium formation and with minimal extracellular virus. Electron microscopy analysis showed that viral budding did not occur from the neuronal surface, although nucleocapsids were present in the cytoplasm and aligned at the cell membrane. We observed many examples of nucleocapsids present in the neuronal processes and aligned at presynaptic neuronal membranes. Cocultures of CD46(+) and CD46(-) neurons showed that cell contact but not CD46 expression is required for MV spread between neurons. Collectively, these results suggest that the neuronal environment prevents the normal mechanisms of MV spread between neurons at the level of viral assembly but allows an alternate, CD46-independent mechanism of viral transmission, possibly through the synapse.     

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.