Amino Acid Substitutions in Matrix, Fusion and Hemagglutinin Proteins of Wild Measles Virus for Adaptation to Vero Cells (original) (raw)
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Recombinant measles AIK-C strain expressing current wild-type hemagglutinin protein
Vaccine, 2004
We constructed a recombinant measles virus cDNA, pIC-MVAIK-H/87-K, in which the hemagglutinin (H) gene of the AIK-C vaccine strain was replaced by the wild-type (MVi/Tokyo.JPN/87-K: genotype D3) H gene and the remaining genes were the same as the AIK-C vaccine strain. To investigate the feasibility of the recombinant vaccine strain expressing wild-type H protein instead of the AIK-C H protein, we constructed two recombinant measles cDNA, having Leu (small plaque-type) and Phe (large plaque-type) at position 278 of the F protein. Infectious chimeric virus strains, MVAIK-H/87-K/S (small plaque-type) and MVAIK-H/87-K/L (large plaque-type), were recovered, which were designed to induce small (S) and large (L) plaques in Vero cells. The MVAIK-H/87-K/S and MVAIK-H/87-K/L did not grow at 39-40 • C, similar to the original AIK-C strain, and retained the temperature sensitivity (ts) characteristics. They did not induce cytopathic effect (CPE) in Vero cells but produced CPE in B95a cells, similar to the current wild-type measles MVi/Tokyo.JPN/87-K. From the results of Western blotting, the mobility of the H protein of MVAIK-H/87-K/S and MVAIK-H/87-K/L was similar to that of MVi/Tokyo.JPN/87-K. Hyper-immune sera raised by MVAIK-H/87-K/S neutralized all types of current wild strains. Thus, the chimeric measles virus expressing the current wild H protein demonstrated wild-type H properties with ts characteristics of the vaccine strain, indicating that the construction strategy of recombinant measles virus can cope with the hyper-mutated measles virus.
Journal of Virology, 2007
The hemagglutinin (H) protein of measles virus (MV) mediates attachment to cellular receptors. The ectodomain of the H spike is thought to consist of a membrane-proximal stalk and terminal globular head, in which resides the receptor-binding activity. Like other paramyxovirus attachment proteins, MV H also plays a role in fusion promotion, which is mediated through an interaction with the viral fusion (F) protein. The stalk of the hemagglutinin-neuraminidase (HN) protein of several paramyxoviruses determines specificity for the homologous F protein. In addition, mutations in a conserved domain in the Newcastle disease virus (NDV) HN stalk result in a sharp decrease in fusion and an impaired ability to interact with NDV F in a cell surface coimmunoprecipitation (co-IP) assay. The region of MV H that determines specificity for the F protein has not been identified. Here, we have adapted the co-IP assay to detect the MV H-F complex at the surface of transfected HeLa cells. We have also...
Journal of Virology, 1994
The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any signifi...
Journal of Virology, 1982
Since cloning and characterization of DNA complementary to measles virus mRNA encoding for the nucleocapsid protein (M. Gorecki and S. Rozenblatt, Proc. Natl. Acad. Sci. U.S.A. 77:3686-3690, 1980), two additional measlesspecific clones containing different classes of sequences have been characterized. The cloned plasmids contain inserts of 480 and 530 base pairs as shown by agarose gel electrophoresis and electron microscopy. The sizes of the mRNA species complementary to these inserts are 1,700 and 1,550 nucleotides, respectively, as determined by the Northern technique. The cloned DNA fragments were further identified as reverse transcripts of the mRNA coding for the glycoprotein and matrix protein of measles virus. The major cell-free translation products of mRNA selected by hybridization to the individual cloned DNAs comigrated with the 70K in vitro products and matrix proteins. One of the cell-free translation products (70K) was also immunoprecipitated specifically with monoclonal antibodies against measles virus glycoprotein.
Journal of Virology, 2008
The glycoprotein complex of paramyxoviruses mediates receptor binding and membrane fusion. In particular, the measles virus (MV) fusion (F) protein executes membrane fusion, after receptor binding by the hemagglutinin (H) protein. Structures and single amino acids influencing fusion function have been identified in the F-protein ectodomain and cytoplasmic tail, but not in its transmembrane (TM) region. Since this region influences function of the envelope proteins of other viruses, we examined its role in the MV F protein. Alanine-scanning mutagenesis revealed that an F protein with a single mutation of a central TM region leucine (L507A) was more fusogenic than the unmodified F protein while retaining similar kinetics of proteolytic processing. In contrast, substitution of residues located near the edges of the lipid bilayer reduced fusion activity. This was true not only when the mutated F proteins were coexpressed with H but also in the context of infections with recombinant viruses. Analysis of the H-F complexes with reduced fusion activities revealed that more precursor (F 0) than activated (F 1؉2) protein coprecipitated with H. In contrast, in complexes with enhanced fusion activity, including H-F L507A , the F 0 /F 1؉2 ratio shifted toward F 1؉2. Thus, fusion activity correlated with an active F-H protein complex, and the MV F protein TM region modulated availability of this complex.
Measles virus: Evolution of a persistent infection in BGM cells
Archives of Virology, 1981
An African green monkey kidney cell line (BGM) persistently infected with measles virus (BGM/Hall5 cells) has been studied during 3 years in culture. The early cell passages were characterized by slow growing cultures producing high yields of infections virus (106-107 PFU/ml). These cells were gTadually replaced by a population of cells multiplying at a similar rate as non-infected cells. During this evolution, the virus released ~rom the cells changed from a large plaque variant, to a small plaque and eventually unlysed foci. Despite >~95 per cent of the cells being infected, the virus yield fell to the limits of detection. [s5S]-methionine labelling of BGM/Hall5 cultures at the 20th and 140th passage showed that in both cases all the measles virus structural proteins were synthesized. There were no changes in the apparent molecular sizes of the viral proteins during passage. Cell surface [125I]-labelling of BGM/Halld cells indicates that viral envelope antigens are inserted into the membrane despite the diminution in virus yield. Several cell proteins not labelled in non-infected BGM cells are also labelled. These proteins could also be [125I]-]abelled in clones of BGM/Halld cells with had been "cured" of virus. Chase experiments of [t25I]-pre-labelled BGM/Halld cultures showed the radiolabelled antigen to be incorporated into virus particles. Non-infections virus particles released from the cells contained the same polypeptides as those released from a lyric infection.
Journal of virology, 1998
An understanding of the determinants of measles virus (MV) virulence has been hampered by the lack of an experimental model of infection. We have previously demonstrated that virulence phenotypes in human infections are faithfully reproduced by infection of human thymus/liver (thy/liv) implants engrafted into SCID mice, where the virus grows primarily in stromal cells but induces thymocyte apoptosis (P. G. Auwaerter et al., J. Virol. 70:3734-3740, 1996). To begin to elucidate the roles of the C protein, V protein, and the 5' untranslated region of the F gene (F 5'UTR) in MV infection in vivo, the replication of strains bearing mutations of these genes was compared to that of the parent sequence-tagged Edmonston strain (EdTag). Growth curves show that mutants fall into two phenotypic classes. One class of mutants demonstrated kinetics of growth similar to that of EdTag, with decreased peak titers. The second class of mutants manifested peak titers similar to that of EdTag but...
APMIS, 1988
Several cDNA libraries have been generated from poly(A)RNA from Vero cells infected for 24 hours with measles virus. Different protocols for cDNA library construction were compared and some critical steps were evaluated. From these libraries, a measles virus specific sequence corresponding to 885 of 1600 nucleotides of the measles virus phosphoprotein gene has been cloned. The phosphoprotein gene accounts for 1% of the total cDNA library after 24 hours of infection at 37 "C. The technique of differential colony hybridization was used to analyze the distribution and change ofthe poly(A)-RNA expression in uninfected Vero cells and in cells infected with measles virus for 24 hours.
Purification of Measles Virus with Preservation of Infectivity and Antigenicity
Journal of General Virology, 1979
Measles virus Edmonston strain was purified by ultrafiltration followed by two successive redimentations through sucrose. Purified virus retained infectivity and, when used as an immunogen, elicited high titred antibody to measles antigens by conventional serology. The measles preparations were examined by SDS-PAGE followed by staining. In addition, following PAGE, the purity of these preparations was assessed immunochemically using antisera directed to measles and host cell antigens. The results of these studies demonstrate the utility of the purification method for the preparation of milligram quantities of relatively pure measles virus.