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 sequence 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.
Nucleotide sequence analysis was carried out to study genes encoding the matrix (M) protein of measles virus (MV) from several regions of the brain of a case of subacute sclerosing panencephalitis. ...This analysis revealed the presence of MV with "wild-type" sequences as well as variants which had undergone at least five biased hypermutation events (U to C and A to G in the positive strand sequences). Despite the presence of MV variants with genes encoding the intact matrix protein open reading frame, M protein could not be detected in any of the brain regions. The distribution of virus variants was studied by cDNA cloning and sequence analysis and by in situ hybridization. The hypermutated viruses appeared to expand clonally throughout the brain of patient B.
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.
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.
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.
Measles virus (MV) replication in brain tissue of Lewis rats with acute (AE) and subacute (SAME) encephalitis was characterized by biochemical techniques. Messenger RNAs specific for measles virus ...nucleocapsid (N), phospho (P)-, matrix (M), fusion (F), and haemagglutinin (H) protein were detected in all brain extracts examined. The quantity of the individual MV mRNA species was quite different in comparison to lytically infected Vero cells. A steep gradient of MV transcripts was found in brain tissue which is most likely due to strongly attenuated transcription of mRNAs along the viral genome, representing particularly low transcription of the glycoprotein genes. In addition, in vitro translation assays only revealed synthesis of N and P protein in consistent fashion. The mRNAs for the glycoproteins did not direct the synthesis of detectable viral proteins whereas the M mRNA revealed some activity in animals with AE. The data indicate a strong restriction of the MV envelope gene expression in infected brain tissue, which is independent of the incubation time and type of the central nervous system (CNS) disease. This phenomenon which is similar to the findings observed in measles inclusion body encephalitis and subacute sclerosing panencephalitis suggest that host factors may initially be responsible for the initiation of transcriptional and translational alterations.
Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, ...subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon.
The measles virus (MV) phosphoprotein (P) gene encodes two known proteins, P (Mr approximately 70,000), involved in viral transcription, and, in a different reading frame, C (Mr approximately ...20,000). By a combination of cDNA cloning, cDNA and RNA sequencing, and in vitro translation, we demonstrate here that the MV P gene also expresses a third product (Mr approximately 46,000) containing the amino-terminal region of P but a different, cysteine-rich carboxy-terminal motif. This third protein is translated from mRNAs in which one G residue has been inserted after three genomically encoded Gs, a modification found in about 50% of the P mRNAs. A smaller fraction of transcripts contain several additional G residues.
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.
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