Volker ter Meulen

Hemagglutinin Protein of Wild-Type Measles Virus Activates Toll-Like Receptor 2 Signaling

ABSTRACT Pattern recognition via Toll-like receptors (TLR) by antigen-presenting cells is an important element of innate immunity. We report that wild-type measles virus but not vaccine strains activate cells via both human and murine TLR2, and this is a property of the hemagglutinin (H) protein. The ability to activate cells via TLR2 by wild-type MV H protein is abolished by mutation of a single amino acid, asparagine at position 481 to tyrosine, as is found in attenuated strains, which is important for interaction with CD46, the receptor for these strains. TLR2 activation by MV wild-type H protein stimulates induction of proinflammatory cytokines such as interleukin-6 (IL-6) in human monocytic cells and surface expression of CD150, the receptor for all MV strains. Confirming the specificity of this interaction, wild-type H protein did not induce IL-6 release in macrophages from TLR2−/− mice. Thus, the unique property of MV wild-type strains to activate TLR2-dependent signals might essentially contribute not only to immune activation but also to viral spread and pathogenicity by upregulating the MV receptor on monocytes.

Biased hypermutation and other genetic changes in defective measles viruses in human brain infections.

Abstract We assessed the alterations of viral gene expression occurring during persistent infections by cloning full-length transcripts of measles virus (MV) genes from brain autopsies of two subacute sclerosing panencephalitis patients and one measles inclusion body encephalitis (MIBE) patient. The suquence of these MV genes revealed that, most likely, almost 2% of the nucleotides were mutated during persistence, and 35% of these differences resulted in amino acid changes. One of these nucleotide substitutions and one deletion resulted in alteration of the reading frames of two fusion genes, as confirmed by in vitro translation of synthetic mRNAs. One cluster of mutations was exceptional; in the matrix gene of the MIBE case, 50% of the U residues were changed to C, which might result from a highly biased copying event exclusively affecting this gene. We propose that the cluster of mutations in the MIBE case, and other combinations of mutations in other cases, favored propagation of MV infections in brain cells by conferring a selective advantage to the mutated genomes.

Adoptive transfer of EAE-like lesions from rats with coronavirus-induced demyelinating encephalomyelitis

Viruses have been found to induce inflammatory demyelinating lesions in central nervous system (CNS) tissue of both animal and man, either by natural infections or after vaccination1,2. At least two different pathogenic mechanisms have been proposed for these changes, a cytopathic viral infection of oligodendroglia cells with subsequent cell death, and a host immune reaction against virus and brain antigens. We now report the occurrence of cell-mediated immune reactions against basic myelin proteins in the course of coronavirus infections in Lewis rats. Infection of rats with the murine coronavirus JHM leads to demyelinating encephalomyelitis developing several weeks to months post-infection3–7. Lymphocytes from these diseased Lewis rats can be restimulated with basic myelin protein (BMP) and adoptive transfer of these cells leads to lesions resembling those of experimental allergic encephalomyelitis (EAE) in recipients, which can be accompanied by a mild clinical disease. This model demonstrates that a virus infection in CNS tissue is capable of initiating an autoimmune response which may be of pathogenic importance.

Mutated and hypermutated genes of persistent measles viruses which caused lethal human brain diseases

Persistent measles viruses (MVs) causing lethal human brain diseases are defective, and the structure of several mutated matrix genes has been elucidated previously. The present study of four persistent MVs revealed a high number of differences from a consensus sequence also in other genes. Amino acid changes accumulated in the carboxyl terminus of the nucleocapsid protein and in the amino terminus of the phosphoprotein, but did not significantly alter these products, which are implicated in viral replication and transcription. The contrary is true for the envelope glycoproteins: In three of four cases, mutations caused partial deletion of the short intracellular domain of the fusion protein, most likely compromising efficient viral budding. Moreover, in the hemagglutinin gene of a strain showing strongly reduced hemadsorption, 20 clustered A to G mutations, resulting in 16 amino acid changes, were detected. This hypermutation might be due to unwinding modification of a part of the MV RNA genome accidentally present in a double-stranded form. Finally, we classified four lytic and seven persistent MV strains on the basis of their sequences. Surprisingly, the four lytic viruses considered belong to the same class. The persistent viruses form more loosely defined groups, which all differ from the vaccine strain Edmonston.

Altered ratios of measles virus transcripts in diseased human brains

In rare cases measles virus (MV) induces subacute sclerosing panencephalitis (SSPE) or measles inclusion body encephalitis (MIBE), two lethal diseases of the human central nervous system. MV transcripts present in the brains of two SSPE patients and one MIBE patient were analyzed by quantitative Northern blots. In all three cases the transcripts from the first MV gene were relatively abundant, amounting to about one-tenth of that in lytically infected cells. However, the quantity of transcripts decreased sharply for each subsequent MV gene, arriving at 200-fold lower levels for the fifth MV gene. In comparison gradients of transcript levels are more shallow in either lytically or persistently infected cultured cells, where the transcripts of the fifth MV gene are only about five times less abundant than those of the first. These altered ratios of mRNAs appear to be typical for persistent MV brain infections and most likely lead to reduced expression of the viral envelope proteins, encoded by distal MV genes, at the surface of brain cells. This could account for the lack of viral budding and allow persistent MV infections to elude immune surveillance.

Accumulated measles virus mutations in a case of subacute sclerosing panencephalitis: interrupted matrix protein reading frame and transcription alteration

Subacute sclerosing panencephalitis (SSPE) is a fatal disease affecting the human central nervous system several years after acute measles infection. Measles virus (MV) genomes replicating in SSPE brains do not give rise to budding particles and present various defects in gene expression, mostly concerning the matrix (M) protein. For one SSPE case (K), shown previously to be devoid of M protein expression, we examined here in detail the features involved in this defect. In the brain of patient K the normal, monocistronic MV M mRNA was completely substituted by a bicistronic RNA containing the coding sequence of the preceding phosphoprotein (P) gene in addition to the M coding sequence. Analysis of the P-M intercistronic region by direct cDNA sequencing showed that the consensus sequence at this RNA processing site was unaltered but revealed several distant point mutations. cDNA cloning and sequencing of the entire M coding region established that one of the point mutations leads to a stop codon at triplet 12 of the M reading frame. It is unknown whether this defect, explaining by itself the lack of M protein, is related also to the block of M mRNA formation. In addition we note that as much as 1% of the nucleotides differed between two overlapping clones from the same brain. This high sequence variability could possibly account for the diversity of defects observed in MV gene expression in SSPE brains and may be a general phenomenon associated with RNA virus persistence.

Disruption of Akt kinase activation is important for immunosuppression induced by measles virus

Surface-contact–mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2–dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

Successful Vaccine-Induced Seroconversion by Single-Dose Immunization in the Presence of Measles Virus-Specific Maternal Antibodies

ABSTRACT In humans, maternal antibodies inhibit successful immunization against measles, because they interfere with vaccine-induced seroconversion. We have investigated this problem using the cotton rat model (Sigmodon hispidus). As in humans, passively transferred antibodies inhibit the induction of measles virus (MV)-neutralizing antibodies and protection after immunization with MV. In contrast, a recombinant vesicular stomatitis virus (VSV) expressing the MV hemagglutinin (VSV-H) induces high titers of neutralizing antibodies to MV in the presence of MV-specific antibodies. The induction of neutralizing antibodies increased with increasing virus dose, and all doses gave good protection from subsequent challenge with MV. Induction of antibodies by VSV-H was observed in the presence of passively transferred human or cotton rat antibodies, which were used as the models of maternal antibodies. Because MV hemagglutinin is not a functional part of the VSV-H envelope, MV-specific antibodies only slightly inhibit VSV-H replication in vitro. This dissociation of function and antigenicity is probably key to the induction of a neutralizing antibody in the presence of a maternal antibody.

Subacute sclerosing panencephalitis is typically characterized by alterations in the fusion protein cytoplasmic domain of the persisting measles virus

Our recent extensive analysis of three cases of subacute sclerosing panencephalitis (SSPE) revealed intriguing genetic defects in the persisting measles virus (MV): the fusion (F) genes encoded truncated cytoplasmic F protein domains (Cattaneo et al., Virology 173, 415-425, 1989). Now this MV genomic region has been investigated in eight additional SSPE cases by PCR amplification, replacement cloning into a vector containing the F gene of a lytic MV, in vitro expression, and sequencing. In all cases at least part of the clones showed mutations leading to F protein truncations, elongation, or nonconservative amino acid replacements. It is proposed that alteration of the F protein cytoplasmic domain may play a critical role in the development of SSPE.

Generation and Properties of Measles Virus Mutations Typically Associated with Subacute Sclerosing Panencephalitis

Subacute sclerosing panencephalitis (SSPE), a very rare but lethal disease caused by measles viruses (MV) persisting in the human central nervous system (CNS) is characterized by lack of viral budding, reduced expression of the viral envelope proteins and spread of MV genomes through the CNS despite massive immune responses. The five major MV genes from several SSPE cases were cloned and sequenced, the two transmembrane envelope glycoproteins hemagglutinin (H) and fusion protein (F) were expressed and their maturation, cellular localization and functionality analyzed. We conclude that 1) mutations in the MV genes arise not only individually, by errors of the MV polymerase, but also in clusters as hypermutations, presumably due to RNA unwinding/modifying activity altering accidentally formed double-stranded RNA regions, 2) MVs spread in SSPE brains after clonal selection, 3) the MV matrix (M) gene is most heavily mutated and dispensable, 4) the two genes encoding envelope transmembrane proteins give rise to functional but altered proteins (typically F is heavily altered in its cytoplasmic domain), 5) H protein is transported poorly to the cell surface, 6) F and H proteins maintain tightly interdepending fusion functions, presumably to allow local cell fusion and MV ribonucleoprotein (RNP) spread through the CNS.