Molecular analysis of structural protein genes of the Yamagata-1 strain of defective subacute sclerosing panencephalitis virus. I. Nucleotide sequence of the nucleoprotein gene

The complete nucleotide sequence of cloned cDNAs corresponding to the fulllength mRNA encoding the NP protein of the Yamagata-1 strain of subacute sclerosing panencephalitis (SSPE) virus was determined. The gene is composed of 1683 nucleotides and contains a single large open reading frame, which is capable of encoding 525 amino acids with a molecular weight of 58,399. Comparison of the nucleotide and predicted amino acid sequences with those of the Edmonston strain of measles virus (MV) showed that the gene and the protein were highly conserved. However, the antigenic sites on the NP protein of the Yamagata-1 strain were found to be changed by an eiptope analysis using monoclonal antibodies against the NP protein of MV. Only 1 of 4 monoclonal antibodies reacted with the NP protein of SSPE virus, and the other three antibodies did not.Almost identical changes in nucleotides and amino acids were found to occur in the NP gene of the Yamagata-1 strain when compared with the IP-3-Ca strain of another SSPE virus. In addition, the deduced secondary structure of the NP protein of the IP-3-Ca strain was similar to that of the Yamagata-1 strain, but differed from the MV. These results suggest that the NP proteins of SSPE viruses have a common property that is different from MV.     

Molecular analysis of structural protein genes of the Yamagata-1 strain of defective subacute sclerosing panencephalitis virus. IV. Nucleotide sequence of the fusion gene

The full-length cDNA corresponding to the mRNA of the fusion (F) protein of the Yamagata-1 strain of subacute sclerosing panencephalitis (SSPE) virus was cloned, and its complete nucleotide sequence was determined. The F gene was composed of 2369 nucleotides and contained a single large coding region, which is located between two noncoding regions. The 5-terminal noncoding region consisted of 584 nucleotides comprising 44.9% cytosine, and had several inverted repetitious sequences. The 3-terminal noncoding region had a relatively low homology of 91.7% with the MV. The coding region was expanded for nucleotides 585–2189, which encoded 534 amino acids with a molecular weight of 57,963. The homology of the amino acid sequence of the F protein between the MV and SSPE virus was 96.27%, and the positions of cysteine and proline were almost identical in the two viruses. The functional domains of SSPE-virus F protein closely resembled those of MV F protein, including the cleavage site, a signal sequence, the fusion-related stretch, the transmembrane region, and four potential glycosylation sites. Four antigenic epitopes on the MV F protein were also conserved on the SSPE-virus F protein.However, deletion of one nucleotide (position 2155) of the SSPE virus was found when compared with the MV, and shifted the coding frame, causing the substitutions of 27 C-terminal amino acids of the MV F protein with 11 different residues. The variations of the C-terminal region of the F protein were observed with two other SSPE viruses, suggesting that this may be a common property of SSPE virus that differs from MV.     

Molecular analysis of structural protein genes of the yamagata-1 strain of defective subacute sclerosing panencephalitis virus. II. Nucleotide sequence of a cDNA corresponding to the P plus M dicistronic mRNA

The nucleotide sequence of a cloned cDNA corresponding to the P+M dicistronic mRNA of a subacute sclerosing panencephalitis (SSPE) virus was determined and compared with data of measles virus (MV). The dicistronic mRNA of the SSPE virus consisted of the 3 proximal 626 nucleotides of P mRNA, intercistronic trinucleotides, a full length of M mRNA, and 75 poly A nucleotides. The part encoding the P protein had a high homology to MV, except at the noncoding region. The terminating consensus sequence of the P gene and the intercistronic trinucleotides of the SSPE virus were CTAC(A)₆ and CCT; in MV they are TTAT(A)₆ and CTT, respectively. In the M gene, the starting consensus sequence was exactly the same as MV, but at the 5 proximal end, one third of this gene was different: The first ATG codon of the MV M gene signaling opening of the reading frame was changed to ACG in the SSPE virus and one long open reading frame started from the third ATG codon. The stop codon (TAG) of the MV M gene was also changed to CAG in the SSPE virus. Thus, the deduced SSPE-virus M protein lacked 50 amino acids at the amino terminal and had 15 extra amino acids at the carboxyl end when compared with the MV M protein.     

Cloning and characterization of the cDNA encoding the HA protein of a hemagglutination-defective measles virus strain

cDNA clones corresponding to the mRNA for the hemagglutinin of the hemagglutination-defective strain AK-1 of measles virus were isolated and characterized. Compared with the prototype Edmonstron strain, 60 nucleotide substitutions that resulted in 18 amino acid changes were detected. An additional potential N-linked glycosylation site was added by point mutation, which was supported by the observation that the hemagglutinin of the AK-1 strain was stained more heavily after NaDodSO₄PAGE and periodic acid-Schiff (PAS) staining than the Edmonston strain. Computer-assisted analysis revealed that three reverse turns in the secondary structure had disappeared in the hemagglutinin of the AK-1 strain. Moreover, one of these structural changes occurred in the closely glycosylated region at amino acid residues 168–240, which appeared to be a biologically important functional domain. The isoelectric point calculated from the predicted amino acid sequence became about 1 pH unit more basic in the AK-1 strain than the Edmonston strain. This present study is the first sequence analysis of the hemagglutinin gene in a hemagglutination-defective strain of the measles virus.     

The nucleotide and predicted amino acid sequence of the attachment protein of canine distemper virus

The nucleotide sequence of the gene coding for the attachment protein of the Convac strain of the canine distemper virus (CDV), corresponding to the haemagglutinin (H) gene of measles virus was determined using a mRNA-derived cDNA clone and genomic viral RNA. The mRNA transcribed from the CDV H gene is 1944 nucleotides long excluding the polyadenylated tail. Only one long open reading frame was found comprising nucleotides 21-1841. The predicted protein has a single hydrophobic region which can serve as a membrane anchoring domain. The deduced 607 amino acids would code for a protein of 68,247 Da, to be compared with an approximate protein molecular weight in SDS-PAGE of the glycosylated protein, which is 85,000 Da. The CDV H protein exhibited seven potential N-linked glycosylation sites. These were concentrated to the carboxyterminal part of the CDV H protein and differed markedly from measles virus (MV) and rinderpest virus (RPV) where the potential sites were mostly conserved and located in the amino-terminal half of the proteins. In spite of the differences in amino acid composition of these three H proteins their hydrophilicity/hydrophobicity plots were closely similar with the major hydrophobic region at an identical location. All the 12 cysteine residues found in the CDV H protein were conserved in MV and RPV. The amino acid homology between CDV and MV H protein was 37% and between CDV and RPV H protein 38%. The fact that the corresponding homology between the MV and RPV proteins is almost 60% shows that the evolutionary separation between CDV and RPV occurred at a much earlier time than the separation between RPV and MV.     

Two species of the phosphoprotein mRNA with differently edited reading frames discovered in measles virus infected Vero cells. Brief report

One cDNA clone representing the phosphoprotein mRNA sequence of the Edmonston strain of measles virus contained 4 G nucleotides at a particular position. Two other clones contained 3 G nucleotides at the same position. Otherwise the nucleotide sequence were identical. The mRNA with 3 G nucleotides codes for the 70 kD phosphoprotein. The mRNA with 4 G nucleotides may code for a putative new peptide with 231 aminoterminal amino acids in common with the P protein whereas the 68 carboxyterminal amino acids are different from any amino acid sequence of the phosphoprotein. Thus the consequence of the insertion of one additional G is a translational shift to a shorter open reading frame. There are several indications that the observation is of biological significance.     

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.     

Molecular cloning and sequence analysis of the rinderpest virus mRNA encoding the hemagglutinin protein

We cloned the full-length cDNAs corresponding to the mRNA for the hemagglutinin (H) protein of rinderpest virus (RV) and determined the nucleotide sequence of RV-H. The gene of RV-H was composed of 1952 nucleotides and contained a single large open reading frame, which was capable of encoding a protein of 609 amino acids with a molecular weight of 68,330 Da. The nucleotide sequence and predicted amino acid sequence were compared with those of the measles virus (MV)-H. The 5' end of the message (nucleotides 1 to 485) was largely conserved, with a homology of 75.1% of the nucleotides and 78.0% of the predicted amino acids. In the middle portion (nucleotides 486-1310), where the potential glycosylation sites exist, 56.6% of the nucleotides and 49.5% of the amino acids were identical. In the 3' end of the message (nucleotides 1311-1850), 63.3% of the nucleotides and 58.1% of the amino acids were identical. Four potential glycosylation sites were found in RV-H protein and three of them were the same as those of MV-H protein. The positions of 13 cysteine residues of RV-H were absolutely identical to those of MV-H. The hydropathy profile of RV-H protein resembled that of MV-H. One major hydrophobic region long enough to be an anchor in the membrane was located near the N-terminus.     

Nucleotide sequence of cDNA to the rinderpest virus mRNA encoding the nucleocapsid protein

The full-length cDNA corresponding to the mRNA encoding the nucleocapsid protein (NP) of rinderpest virus (RV) was cloned and its complete nucleotide sequence was determined. The gene of RV-NP was composed of 1683 nucleotides and contained a single large open reading frame, which is capable of encoding a protein of 525 amino acids with a molecular weight of 58,241 Da. The nucleotide sequence and predicted amino acid sequence were compared with those of measles virus (MV) and canine distemper virus (CDV). The nucleotide sequence of the coding region of RV-NP (53–1630) revealed a homology of 68.1% and 63.0% with MV and CDV-NP, respectively. Relatively moderate homologies of 68.7% (MV) and 64.3% (CDV) were found at nucleotides 53–592. The highest homology of 75.3–74.3% was equally present between RV and both MV and CDV in the middle region at nucleotides 593–1312. The homologies of the predicted amino acids in this region were 88.3% (MV) and 86.3% (CDV). Relatively low (MV) or little (CDV) homology was detected in the last 318 nucleotides toward the 3 terminus (1313–1630). The predicted secondary structures of amino acids at the C terminus differed between the three viruses.     

Alterations and diversity in the cytoplasmic tail of the fusion protein of subacute sclerosing panencephalitis virus strains isolated in Osaka,Japan

We determined the nucleotide sequence of the fusion (F) gene of three strains (Osaka-1, -2, and -3) of nonproductive variants of measles virus (MV). These viral strains were isolated in Osaka, Japan, from brain tissues of patients with subacute sclerosing panencephalitis (SSPE). Phylogenetic analysis revealed a close relationship among the three strains of SSPE virus. The cytoplasmic tail of the F protein, predicted from sequence analysis of the gene, is altered in all three SSPE strains when compared to the MV field strains. However, the extent and mode of alteration are different in each strain. The F protein of the Osaka-1 strain has six nonconservative amino acid substitutions and a 29-residue elongation of its cytoplasmic tail. The F protein of the Osaka-3 strain has two nonconservative substitutions and a 5-residue truncation of its C-terminus. Although the termination codon is not altered in the F protein of the Osaka-2 strain, five or six amino acids are changed in the cytoplasmic tail of the F protein of the two sibling viruses of this strain. The significance of the altered cytoplasmic domain of the SSPE viruses in the SSPE pathogenesis is discussed.     

Fusion glycoprotein (F) of rinderpest virus: entire nucleotide sequence of the F mRNA, and several features of the F protein

The full-length cDNA corresponding to the mRNA for the fusion protein of rinderpest virus (RV) was cloned and its complete nucleotide sequence was determined. The mRNA for the F protein was composed of 2359 nucleotides and contained a single large open reading frame which was capable of encoding 566 amino acids with a molecular weight (MW) of 58,929. The RV-F mRNA had a long noncoding region at the 5' end (586 bases) which was C-rich like the measles virus (MV)-F mRNA but they did not appear to be homologous with each other. Their secondary structure with long G-C stems suggested that they are easily folded. The coding region of RV-F mRNA was significantly homologous with that of MV-F; 74% of the nucleotides and 79.0% [corrected] of the amino acids were identical. The predicted RV-F protein had a basic amino acid region (104-108) which may be cleaved by protease to yield an activated form of F1,2. Three regions (1-19, 109-133, 418-513) were highly hydrophobic, and the N-terminal hydrophobic region of F1 or the positions of cysteines were significantly conserved compared with those of the other paramyxovirus F proteins. Three potential sites for glycosylation existed only in the F2 protein. Several features of the predicted RV-F protein were confirmed in polyacrylamide gel electrophoresis.     

Molecular characterization of measles virus strains causing subacute sclerosing panencephalitis in France in 1977 and 2007

Measles virus strains from two subacute sclerosing panencephalitis (SSPE) cases diagnosed in 1977 (Laine strain) and in 2007 (Hoedts strain) were studied. Phylogenetic analysis based on C-terminal part of the nucleoprotein and the entire H gene showed that Hoedts strain, circulating in France presumably in the 1980s, belonged to genotype C2. However, Laine strain, suspected to have circulated between 1940s and 1960s, could not be assigned to any known measles virus genotypes. Sequences analysis of the Laine strain suggested that it originated from a measles virus that may have circulating at the same period as the Edmonston strain. The analysis of the whole genome of both SSPE strains revealed biased hypermutations in M, F, and H gene. Some of these mutations like the L165P found in the M protein sequence of the Laine strain, the amino acid position 94, where a mutation M94V was found in the F protein sequence of the Hoedts strain are known to play an important role in the glycoprotein interaction and to impair the ability of measles virus strain to produce cell-free infectious viral particles.This is the first study on molecular characterization of the entire coding region of measles virus isolated from SSPE cases in France.     

Resistance of a measles virus mutant to fusion inhibitory oligopeptides is not associated with mutations in the fusion peptide

The nucleotide sequence and predicted amino acid sequence has been obtained for the fusion (F) protein gene of the R93 strain of measles virus and compared to that of the parental strain, Edmonston B. The R93 strain is a mutant measles virus which is able to grow and induce cell fusion in the presence of the fusion inhibiting oligopeptide, Z-D-Phe-L-Phe-L-(NO2)Arg (SV4814). Primer extension sequencing on isolated R93 mRNA demonstrated the presence of three nucleotide changes leading to three amino acid changes, none of which are in the hydrophobic NH2-terminal region of the F1 polypeptide.     

Effect of the alterations in the fusion protein of measles virus isolated from brains of patients with subacute sclerosing panencephalitis on syncytium formation

Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE) and viruses isolated from brains of the patients contain numerous mutations. We have previously demonstrated that the hemagglutinin (H) protein of MV SSPE strains can interact with the signaling lymphocyte activation molecule (SLAM) and an unidentified molecule on Vero cells, but not with CD46, as a receptor. The mechanism by which MV SSPE strains can induce cell–cell fusion in SLAM-negative Vero cells is not understood. We report here on the effect of mutations in the fusion (F) proteins of three MV SSPE strains on syncytium formation. The F proteins of the three SSPE strains were functional and co-expression with H protein from the MV wild-type or SSPE strains in this study induced formation of large syncytia in Vero cells as well as in cell lines expressing SLAM or CD46. Expression of chimeric F proteins of SSPE strains showed that amino acid substitutions in the F protein extracellular as well as cytoplasmic domain contributed to enhanced cell–cell fusion in Vero cells. These findings suggest a common molecular mechanism and a key role of the F protein for syncytium formation in cells expressing an unidentified third receptor for MV.     

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.     

Molecular cloning and complete nucleotide sequence of genomic RNA of the AIK-C strain of attenuated measles virus

Twelve cDNA clones covering the entire genome of the AIK-C strain of a seed for live measles vaccine were obtained, and the nucleotide sequences were determined. The full viral genomic RNA consists of 15,894 nucleotides. Comparisons of the nucleotide sequence and the deduced amino acid sequence between the AIK-C and other Edmonston strains revealed the following changes: 56 nucleotide differences and one C residue insertion, 31 amino acid changes, and 19 silent mutations.     

Nucleotide sequence of the gene encoding the matrix protein of mumps virus (Miyahara strain)

The nucleotide sequence of the gene encoding the matrix (M) protein of mumps virus (MuV), Miyahara strain, has been determined from several overlapping cDNA clones. The M protein mRNA is 1248 nucleotides in length, exclusive of the poly(A) tail, and codes for a protein of 375 amino acids (Mr41,556). Comparison of the deduced amino acid sequence of the M protein of the Miyahara strain with that of the SBL-1 strain revealed that the M proteins of both strains are highly conserved. A significantly lower rate of nucleotide differences conducive to amino acid differences in the M gene compared with other genes appeared to indicate the importance of the conserved primary structure of the M protein for its function.Requests for reprints should be addressed to Kiyoshi Tanabayashi, Department of Measles Virus, National Institute of Health, 4-7-1 Gakuen, Musashimurayama, Tokyo 190-12, Japan.     

Comparative Nucleotide Sequence Analyses of the Entire Genomes of B95a Cell-Isolated and Vero Cell-Isolated Measles Viruses from the same Patient

Experimental infection of monkeys with the IC-B strain of measles virus (MV), which was isolated in marmoset B lymphoblastoid B95a cells from an acute measles patient, caused clinical signs typical for measles, while infection by the IC-V strain isolated in African green monkey kidney Vero cells from the same patient did not cause any clinical signs in infected monkeys. The IC-B strain replicated only in B95a cells, whereas the IC-V strain replicated in both B95a and Vero cells (3,6). To clarify which gene or mutation(s) was responsible for the difference in these phenotypes, the nucleotide sequences of the entire genomes of the IC-B and IC-V strains were determined. Comparative nucleotide sequence analyses revealed only two nucleotide differences, one in the P/V/C gene and the other in the M gene, predicting amino acid differences in the P, V and M proteins and a 19 amino acid deletion in the C protein of the IC-V strain. The truncation in the C protein was confirmed for the IC-V strain by immunoprecipitation using the C protein specific antiserum. No nucleotide difference was found in the envelope H gene. These results indicated that nucleotide difference(s) in the P/V/C or/and M gene, and not H gene, was responsible for the different cell tropism and pathogenicity of MV in this case.