Naoko Miyajima

Measles virus V protein blocks interferon (IFN)-α/β but not IFN-γ signaling by inhibiting STAT1 and STAT2 phosphorylation

Measles virus (MV), a member of the family Paramyxoviridae, encodes C and V non‐structural proteins. To clarify the functions of MV C and V proteins, HeLa cell lines constitutively expressing C or V protein were established. We found that expression of V protein inhibited interferon (IFN)‐α/β signaling but not IFN‐γ signaling. C protein had no inhibitory effect on IFN signaling in our experimental condition. Degradation of selective signal transducers and activators of transcription (STAT) proteins was not observed in HeLa cells expressing V protein. In contrast, tyrosine phosphorylation of both STAT1 and STAT2 was inhibited in these cells after IFN‐β stimulation.

Recovery of Pathogenic Measles Virus from Cloned cDNA

ABSTRACT Reverse genetics technology so far established for measles virus (MeV) is based on the Edmonston strain, which was isolated several decades ago, has been passaged in nonlymphoid cell lines, and is no longer pathogenic in monkey models. On the other hand, MeVs isolated and passaged in the Epstein-Barr virus-transformed marmoset B-lymphoblastoid cell line B95a would retain their original pathogenicity (F. Kobune et al., J. Virol. 64:700–705, 1990). Here we have developed MeV reverse genetics systems based on the highly pathogenic IC-B strain isolated in B95a cells. Infectious viruses were successfully recovered from the cloned cDNA of IC-B strain by two different approaches. One was simple cotransfection of B95a cells, with three plasmids each encoding the nucleocapsid (N), phospho (P), or large (L) protein, respectively, and their expression was driven by the bacteriophage T7 RNA polymerase supplied by coinfecting recombinant vaccinia virus vTF7-3. The second approach was transfection with the L-encoding plasmid of a helper cell line constitutively expressing the MeV N and P proteins and the T7 polymerase (F. Radecke et al., EMBO J. 14:5773–5784, 1995) on which B95a cells were overlaid. Virus clones recovered by both methods possessed RNA genomes identical to that of the parental IC-B strain and were indistinguishable from the IC-B strain with respect to growth phenotypes in vitro and the clinical course and histopathology of experimentally infected cynomolgus monkeys. Thus, the systems developed here could be useful for studying viral gene functions in the context of the natural course of MeV pathogenesis.

Stringent Requirement for the C Protein of Wild-Type Measles Virus for Growth both In Vitro and in Macaques

ABSTRACT The P gene of measles virus (MV) encodes the P protein and three accessory proteins (C, V, and R). However, the role of these accessory proteins in the natural course of MV infection remains unclear. For this study, we generated a recombinant wild-type MV lacking the C protein, called wtMV(C−), by using a reverse genetics system (M. Takeda, K. Takeuchi, N. Miyajima, F. Kobune, Y. Ami, N. Nagata, Y. Suzaki, Y. Nagai, and M. Tashiro, J. Virol. 74:6643-6647). When 293 cells expressing the MV receptor SLAM (293/hSLAM) were infected with wtMV(C−) or parental wild-type MV (wtMV), the growth of wtMV(C−) was restricted, particularly during late stages. Enhanced green fluorescent protein-expressing wtMV(C−) consistently induced late-stage cell rounding and cell death in the presence of a fusion-inhibiting peptide, suggesting that the C protein can prevent cell death and is required for long-term MV infection. Neutralizing antibodies against alpha/beta interferon did not restore the growth restriction of wtMV(C−) in 293/hSLAM cells. When cynomolgus monkeys were infected with wtMV(C−) or wtMV, the number of MV-infected cells in the thymus was >1,000-fold smaller for wtMV(C−) than for wtMV. Immunohistochemical analyses showed strong expression of an MV antigen in the spleen, lymph nodes, tonsils, and larynx of a cynomolgus monkey infected with wtMV but dramatically reduced expression in the same tissues in a cynomolgus monkey infected with wtMV(C−). These data indicate that the MV C protein is necessary for efficient MV replication both in vitro and in cynomolgus monkeys.

Wild-type measles virus induces large syncytium formation in primary human small airway epithelial cells by a SLAM(CD150)-independent mechanism

In the natural course of measles virus (MV) infection, epithelial cells are primary targets of MV. However, it has been shown that wild-type MV utilizes signaling lymphocyte activation molecule (SLAM or CD150) as a cellular receptor, which is expressed only in some T and B cells, thymocytes, and dendritic cells. To understand how wild-type MV infects non-lymphoid cells, several non-lymphoid cells were examined for their susceptibility to wild-type MV. Here, we show that wild-type MV can infect primary human small airway epithelial cells (SAEC) and induce formation of large syncytia in vitro. mRNA specific for SLAM was not detected in SAEC, indicating that wild-type MV infects SAEC and induces syncytia formation via a SLAM-independent mechanism.

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.

Recombinant Wild-Type and Edmonston Strain Measles Viruses Bearing Heterologous H Proteins: Role of H Protein in Cell Fusion and Host Cell Specificity

ABSTRACT Wild-type measles virus (MV) isolated from B95a cells has a restricted host cell specificity and hardly replicates in Vero cells, whereas the laboratory strain Edmonston (Ed) replicates in a variety of cell types including Vero cells. To investigate the role of H protein in the differential MV host cell specificity and cell fusion activity, H proteins of wild-type MV (IC-B) and Ed were coexpressed with the F protein in Vero cells. Cell-cell fusion occurred in Vero cells when Ed H protein, but not IC-B H protein, was expressed. To analyze the role of H protein in the context of viral infection, a recombinant IC-B virus bearing Ed H protein (IC/Ed-H) and a recombinant Ed virus bearing IC-B H protein (Ed/IC-H) were generated from cloned cDNAs. IC/Ed-H replicated efficiently in Vero cells and induced small syncytia in Vero cells, indicating that Ed H protein conferred replication ability in Vero cells on IC/Ed-H. On the other hand, Ed/IC-H also replicated well in Vero cells and induced small syncytia, although parental Ed induced large syncytia in Vero cells. These results indicated that an MV protein(s) other than H protein was likely involved in determining cell fusion and host cell specificity of MV in the case of our recombinants. SLAM (CDw150), a recently identified cellular receptor for wild-type MV, was not expressed in Vero cells, and a monoclonal antibody against CD46, a cellular receptor for Ed, did not block replication or syncytium formation of Ed/IC-H in Vero cells. It is therefore suggested that Ed/IC-H entered Vero cells through another cellular receptor.

A defective non-LTR retrotransposon is dispersed throughout the genome of the silkworm, Bombyx mori

The presence of long repetitive sequences is demonstrated in the genome of the silkworm, Bombyx mori. Members of this BMC1 family reveal several features typical of the L1 (long interspersed sequence one) family of mammals, except for species specific elements. The number of BMC1 elements is estimated to be approximately 3500 per haploid genome. Elements containing the full length unit of 5.1 kb are dispersed throughout the genome and their restriction sites are conserved, although most members are preferentially truncated to varying extents at their 5′ ends. DNA sequencing indicates that this element contains six tandem repeats of 15 bp CpG-rich sequence in the 5′ proximal region. It terminates with a 3′ oligo(A) stretch, and is flanked at both ends by a 7–10 bp target sequence duplication. In addition, there is significant evidence for amino acid sequence homology with reverse transcriptase domains of other L1 families, especially F, Doc and lockey of Drosophila melanogaster. No large open reading frame is present. The BMC1 element is suggested to be dispersed in the genome by a transposition mechanism involving RNA intermediates.

Suppression of Interferon-Induced Antiviral Activity in Cells Expressing Hepatitis C Virus Proteins

To elucidate the mechanism of the persistent nature of hepatitis C virus (HCV) infection, we examined whether the expression of HCV proteins affect the antiviral activity of interferon (IFN). Antiviral activity of IFN in HepG2 cells expressing all HCV (type 1b) proteins was much lower than vector control (VC) HepG2 cells when encephalomyocarditis virus (EMCV) was used as a challenge virus. Lesser sensitivity to IFN was also observed in cells expressing NS3, NS4, and NS5 and in cells expressing only NS5A. In contrast, HepG2 cells expressing core, E1, E2, NS2, and NS3 proteins were equally sensitive to IFN as VC cells. We then tested the antiviral activity by IFN in two human amnion-derived FL cell lines expressing NS5A from two different clones, one with an intact sequence of IFN sensitivity-determining region (ISDR) and the other with a mutated ISDR sequence. They were almost equally insensitive to IFN treatment when EMCV was challenged. HCV thus has functional protein(s), possibly NS5A, to suppress IFN-induced antiviral activity and plays an important role in virus-cell interaction and regulation of viral replication.

Nucleolus organizers in the wild silkworm Bombyx mandarina and the domesticated silkworm B. mori

Two types (Ra1 and Ra2) of nucleolus organizers were identified in the genome of Bombyx mandarina (Japan) which occurs in Japan. Genetical analysis of a hybrid with B. mori suggested that the loci of both nucleolus organizers are allelic and correspond to the R0 locus of B. mori. These nucleolus organizers segregated and were inherited by the progeny in a Mendelian fashion. The majority of the Ra1 rDNA units were 10.6 kb in length and had an additional EcoRI site in the transcribed spacer region when compared with the same size unit of R0. On the other hand, the KpnI site present in the non-transcribed spacer region of the R0 rDNA was not detected in the Ra1 unit. The 15.1 kb unit observed in the Ra2 locus was the same as the unit with the type II intron of R0. The four major components of Ra2 rDNA, with lengths of 10.6, 15.1, 15.7 and 20.8 kb, were also found in the R0 locus, and thus the Ra2 and the R0 loci were considered to be closely related. Usually the functional unit of rDNA in the nucleolus organizers of homologous or non-homologous chromosomes cannot be easily distinguished. However, in the case of B. mandarina (Japan), distinct functional 10.6 kb units were observed in the allelic Ra1 and Ra2 loci. Therefore the existence of the two distinct units suggest the possibility of introducing the chromosomes of the interspecies in the genus Bombyx.

BASIC CONDITIONS FOR THE DRUG SELECTION AND TRANSIENT GENE EXPRESSION IN THE CULTURED CELL LINE OF BOMBYX MORI

SummaryWe established basic conditions for transient gene expression and selection of antibiotics in the cultured cell line of silkworm,Bombyx mori, by use of the promoter of the heat shock protein (hsp70) gene ofDrosophila melanogaster. The control promoter (hsp70) promoted the expression of chloramphenicol acetyltransferase (CAT) gene ligated at the downstream, dependent on the orientation of the promoter in the silkworm cell. The cell line is able to be supplied for the promoter assay of the silkworm genes. The concentration for the drug selection was determined as 0.75 mg/ml on neomycin analog, G418 (geneticin).