scholarly journals Importance of the Cytoplasmic Tails of the Measles Virus Glycoproteins for Fusogenic Activity and the Generation of Recombinant Measles Viruses

2002 ◽  
Vol 76 (14) ◽  
pp. 7174-7186 ◽  
Author(s):  
Markus Moll ◽  
Hans-Dieter Klenk ◽  
Andrea Maisner
Keyword(s):  
Amino Acids ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Measles Virus ◽  
Local Density ◽  
Biologically Active ◽  
Host Cells ◽  
Susceptible Host ◽  
Cytoplasmic Tails ◽  
H Protein

ABSTRACT The generation of replication-competent measles virus (MV) depends on the incorporation of biologically active, fusogenic glycoprotein complexes, which are required for attachment and penetration into susceptible host cells and for direct virus spread by cell-to-cell fusion. Whereas multiple studies have analyzed the importance of the ectodomains of the MV glycoproteins hemagglutinin (H) and fusion protein (F), we have investigated the role of the cytoplasmic tails of the F and H proteins for the formation of fusogenic complexes. Deletions in the cytoplasmic tails of transiently expressed MV glycoproteins were found to have varying effects on receptor binding, fusion, or fusion promotion activity. F tail truncation to only three amino acids did not affect fusion capacity. In contrast, truncation of the H cytoplasmic tail was limited. H protein mutants with cytoplasmic tails of <14 residues no longer supported F-mediated cell fusion, predominantly due to a decrease in surface expression and receptor binding. This indicates that a minimal length of the H protein tail of 14 amino acids is required to ensure a threshold local density to have sufficient accumulation of fusogenic H-F complexes. By using reverse genetics, a recombinant MV with an F tail of three amino acids (rMV-FcΔ30), as well as an MV with an H tail of 14 residues (rMV-HcΔ20), could be rescued, whereas generation of viruses with shorter H tails failed. Thus, glycoprotein truncation does not interfere with the successful generation of recombinant MV if fusion competence is maintained.

scholarly journals Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein

2004 ◽  
Vol 78 (18) ◽  
pp. 9666-9674 ◽  
Author(s):  
G. Grant Welstead ◽  
Eric C. Hsu ◽  
Caterina Iorio ◽  
Shelly Bolotin ◽  
Christopher D. Richardson
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Measles Virus ◽  
Host Cells ◽  
Down Regulation ◽  
Cellular Receptors ◽  
Regulatory Molecule ◽  
Hemagglutinin Protein ◽  
Host Cell Surface ◽  
H Protein

ABSTRACT Measles virus has been reported to enter host cells via either of two cellular receptors, CD46 and CD150 (SLAM). CD46 is found on most cells of higher primates, while SLAM is expressed on activated B, T, and dendritic cells and is an important regulatory molecule of the immune system. Previous reports have shown that measles virus can down regulate expression of its two cellular receptors on the host cell surface during infection. In this study, the process of down regulation of SLAM by measles virus was investigated. We demonstrated that expression of the hemagglutinin (H) protein of measles virus was sufficient for down regulation. Our studies provided evidence that interactions between H and SLAM in the endoplasmic reticulum (ER) can promote the down regulation of SLAM but not CD46. In addition, we demonstrated that interactions between H and SLAM at the host cell surface can also contribute to SLAM down regulation. These results indicate that two mechanisms involving either intracellular interactions between H and SLAM in the ER or receptor-mediated binding to H at the surfaces of host cells can lead to the down regulation of SLAM during measles virus infection.

scholarly journals Previously Unrecognized Amino Acid Substitutions in the Hemagglutinin and Fusion Proteins of Measles Virus Modulate Cell-Cell Fusion, Hemadsorption, Virus Growth, and Penetration Rate

2009 ◽  
Vol 83 (17) ◽  
pp. 8713-8721 ◽  
Author(s):  
Hiromi Okada ◽  
Masae Itoh ◽  
Kyosuke Nagata ◽  
Kaoru Takeuchi
Keyword(s):  
Amino Acid ◽  
Cell Fusion ◽  
Measles Virus ◽  
Vero Cells ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Virus Growth ◽  
F Protein ◽  
H Protein ◽  
Cell Cell

ABSTRACT Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.

scholarly journals Recombinant Measles Viruses Expressing Altered Hemagglutinin (H) Genes: Functional Separation of Mutations Determining H Antibody Escape from Neurovirulence

2001 ◽  
Vol 75 (16) ◽  
pp. 7612-7620 ◽  
Author(s):  
Kerstin Moeller ◽  
Iain Duffy ◽  
Paul Duprex ◽  
Bert Rima ◽  
Rudi Beschorner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Basis ◽  
Measles Virus ◽  
Escape Mutant ◽  
Amino Acid Substitutions ◽  
Structural Alterations ◽  
Recombinant Viruses ◽  
The Brain ◽  
H Protein

ABSTRACT Measles virus (MV) strain CAM/RB, which was adapted to growth in the brain of newborn rodents, is highly neurovirulent. It has been reported earlier that experimentally selected virus variants escaping from the monoclonal antibodies (MAbs) Nc32 and L77 to hemagglutinin (H) preserved their neurovirulence, whereas mutants escaping MAbs K71 and K29 were found to be strongly attenuated (U. G. Liebert et al., J. Virol. 68:1486–1493, 1994). To investigate the molecular basis of these findings, we have generated a panel of recombinant MVs expressing the H protein from CAM/RB and introduced the amino acid substitutions thought to be responsible for antibody escape and/or neurovirulence. Using these recombinant viruses, we identified the amino acid changes conferring escape from the MAbs L77 (377R→Q and 378M→K), Nc32 (388G→S), K71 (492E→K and 550S→P), and K29 (535E→G). When the corresponding recombinant viruses were tested in brains of newborn rodents, we found that the mutations mediating antibody escape did not confer differential neurovirulence. In contrast, however, replacement of two different amino acids, at positions 195G→R and 200S→N, which had been described for the escape mutant set, caused the change in neurovirulence. Thus, antibody escape and neurovirulence appear not to be associated with the same structural alterations of the MV H protein.

scholarly journals Identification of Hendra Virus G Glycoprotein Residues That Are Critical for Receptor Binding

2007 ◽  
Vol 81 (11) ◽  
pp. 5893-5901 ◽  
Author(s):  
Kimberly A. Bishop ◽  
Tzanko S. Stantchev ◽  
Andrew C. Hickey ◽  
Dimple Khetawat ◽  
Katharine N. Bossart ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Measles Virus ◽  
Mammalian Species ◽  
Hendra Virus ◽  
Host Cells ◽  
Single Amino Acid ◽  
Receptor Interaction ◽  
Alanine Scanning Mutagenesis ◽  
Receptor Binding Site ◽  
Protein Receptor

ABSTRACT Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformation-dependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.

scholarly journals Characterization of a Region of the Measles Virus Hemagglutinin Sufficient for Its Dimerization

2000 ◽  
Vol 74 (14) ◽  
pp. 6485-6493 ◽  
Author(s):  
Richard K. Plemper ◽  
Anthea L. Hammond ◽  
Roberto Cattaneo
Keyword(s):  
Cell Surface ◽  
Intermolecular Interactions ◽  
Measles Virus ◽  
Viral Envelope ◽  
Full Length ◽  
Biologically Active ◽  
Covalent Dimerization ◽  
Mediate Cell ◽  
Reduced Rate ◽  
H Protein

ABSTRACT Attachment of measles virus (MV) to its cellular receptor is mediated by the viral envelope glycoprotein hemagglutinin (H). H exists at the viral surface as a disulfide-linked dimer which may associate into a tetramer. We aimed to define regions of H essential for its homo-oligomerization. To delineate these more precisely, we have generated a series of H ectodomain truncation mutants and studied their abilities to form both homotypic complexes and heterotypic complexes with full-length H. We define a “minimal unit” which is sufficient for MV H dimerization as that encompassing residues 1 to 151. This unit forms both homodimers and heterodimers with full-length H protein, although neither is transported to the cell surface even in the presence of other MV proteins. We show that cysteine residues at positions 139 and 154 are both critical in mediating covalent dimerization, not only of the truncated H mutants but also of full-length MV H protein. Even those cysteine mutants unable to form covalent intermolecular interactions are biologically active, mediating the formation of syncytia, albeit at a reduced rate. We demonstrate that this impaired capacity to mediate cell-to-cell fusion is based mainly on a reduced transport rate of the mutant molecules to the cell surface, indicating a role for covalent intermolecular interactions in efficient transport of MV H dimers to the cell surface.

scholarly journals Cleavage of K-FGF produces a truncated molecule with increased biological activity and receptor binding affinity.

1993 ◽  
Vol 121 (3) ◽  
pp. 705-713 ◽  
Author(s):  
P Bellosta ◽  
D Talarico ◽  
D Rogers ◽  
C Basilico
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Growth Factor ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Biologically Active ◽  
Heparin Binding ◽  
Wild Type ◽  
Mammalian Expression

The K-FGF/HST (FGF-4) growth factor is a member of the FGF family which is efficiently secreted and contains a single N-linked glycosylation signal. To study the role of glycosylation in the secretion of K-FGF, we mutated the human K-fgf cDNA to eliminate the glycosylation signal and the mutated cDNA was cloned into a mammalian expression vector. Studies of immunoprecipitation from the conditioned medium of cells expressing this plasmid revealed that the lack of glycosylation did not impair secretion, however the unglycosylated protein was immediately cleaved into two NH2-terminally truncated peptides of 13 and 15 kD, which appeared to be more biologically active than the wild-type protein. These two proteins also showed higher heparin binding affinity than that of wt K-FGF. We have expressed in bacteria the larger of these two proteins (K140), in which the NH2-terminal 36 amino acids present in the mature form of K-FGF have been deleted. Mitogenicity assays on several cell lines showed that purified recombinant K140 had approximately five times higher biological activity than wild-type recombinant K-FGF. Studies of receptor binding showed that K140 had higher affinity than wt K-FGF for two of the four members of FGF receptor's family, specifically for FGFR-1 (flg) and FGFR-2 (bek). K140 also had increased heparin binding ability, but this property does not appear to be responsible for the increased affinity for FGF receptors. Thus removal of the NH2-terminal 36 amino acids from the mature K-FGF produces growth factor molecules with an altered conformation, resulting in higher heparin affinity, and more efficient binding to FGF receptors. Although it is not clear whether cleavage of K-FGF to generate K140 occurs in vivo, this could represent a novel mechanism of modulation of growth factor activity.

scholarly journals Measles Viruses with Altered Envelope Protein Cytoplasmic Tails Gain Cell Fusion Competence

1998 ◽  
Vol 72 (2) ◽  
pp. 1224-1234 ◽  
Author(s):  
Toni Cathomen ◽  
Hussein Y. Naim ◽  
Roberto Cattaneo
Keyword(s):  
Cell Fusion ◽  
Measles Virus ◽  
Subacute Sclerosing Panencephalitis ◽  
Protein Composition ◽  
M Protein ◽  
Viral Glycoprotein ◽  
Virus Envelope ◽  
Coding Regions ◽  
Viral Particles ◽  
Cytoplasmic Tails

ABSTRACT The cytoplasmic tail of the measles virus (MV) fusion (F) protein is often altered in viruses which spread through the brain of patients suffering from subacute sclerosing panencephalitis (SSPE). We transferred the coding regions of F tails from SSPE viruses in an MV genomic cDNA. Similarly, we constructed and transferred mutated tail-encoding regions of the other viral glycoprotein hemagglutinin (H) gene. From the mutated genomic cDNAs, we achieved rescue of viruses that harbor different alterations of the F tail, deletions in the membrane-distal half of the H tail, and combinations of these mutations. Viruses with alterations in any of the tails spread rapidly through the monolayer via enhanced cell-cell fusion. Double-tail mutants had even higher fusion competence but slightly decreased infectivity. Analysis of the protein composition of released mutant viral particles indicated that the tails are necessary for accurate virus envelope assembly and suggested a direct F tail-matrix (M) protein interaction. Since even tail-altered glycoproteins colocalized with M protein in intracellular patches, additional interactions may exist. We conclude that in MV infections, including SSPE, the glycoprotein tails are involved not only in virus envelope assembly but also in the control of virus-induced cell fusion.

scholarly journals Altered Interaction of the Matrix Protein with the Cytoplasmic Tail of Hemagglutinin Modulates Measles Virus Growth by Affecting Virus Assembly and Cell-Cell Fusion

2007 ◽  
Vol 81 (13) ◽  
pp. 6827-6836 ◽  
Author(s):  
Maino Tahara ◽  
Makoto Takeda ◽  
Yusuke Yanagi
Keyword(s):  
Cell Fusion ◽  
Measles Virus ◽  
Matrix Protein ◽  
Cytoplasmic Tail ◽  
Vero Cells ◽  
M Protein ◽  
Virus Growth ◽  
The Matrix ◽  
H Protein ◽  
Cell Cell

ABSTRACT Clinical isolates of measles virus (MV) use signaling lymphocyte activation molecule (SLAM) as a cellular receptor, whereas vaccine and laboratory strains may utilize the ubiquitously expressed CD46 as an additional receptor. MVs also infect, albeit inefficiently, SLAM− cells, via a SLAM- and CD46-independent pathway. Our previous study with recombinant chimeric viruses revealed that not only the receptor-binding hemagglutinin (H) but also the matrix (M) protein of the Edmonston vaccine strain can confer on an MV clinical isolate the ability to grow well in SLAM− Vero cells. Two substitutions (P64S and E89K) in the M protein which are present in many vaccine strains were found to be responsible for the efficient growth of recombinant virus in Vero cells. Here we show that the P64S and E89K substitutions allow a strong interaction of the M protein with the cytoplasmic tail of the H protein, thereby enhancing the assembly of infectious particles in Vero cells. These substitutions, however, are not necessarily advantageous for MVs, as they inhibit SLAM-dependent cell-cell fusion, thus reducing virus growth in SLAM+ B-lymphoblastoid B95a cells. When the cytoplasmic tail of the H protein is deleted, a virus with an M protein possessing the P64S and E89K substitutions no longer grows well in Vero cells yet causes cell-cell fusion and replicates efficiently in B95a cells. These results reveal a novel mechanism of adaptation and attenuation of MV in which the altered interaction of the M protein with the cytoplasmic tail of the H protein modulates MV growth in different cell types.

scholarly journals Selectively Receptor-Blind Measles Viruses: Identification of Residues Necessary for SLAM- or CD46-Induced Fusion and Their Localization on a New Hemagglutinin Structural Model

2004 ◽  
Vol 78 (1) ◽  
pp. 302-313 ◽  
Author(s):  
Sompong Vongpunsawad ◽  
Numan Oezgun ◽  
Werner Braun ◽  
Roberto Cattaneo
Keyword(s):  
Amino Acids ◽  
Conformational Changes ◽  
Measles Virus ◽  
Structural Model ◽  
Three Dimensional ◽  
Lymphocyte Activation ◽  
Cell Receptor ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
H Protein

ABSTRACT Measles virus (MV) enters cells either through the signaling lymphocyte activation molecule SLAM (CD150) expressed only in immune cells or through the ubiquitously expressed regulator of complement activation, CD46. To identify residues on the attachment protein hemagglutinin (H) essential for fusion support through either receptor, we devised a strategy based on analysis of morbillivirus H-protein sequences, iterative cycles of mutant protein production followed by receptor-based functional assays, and a novel MV H three-dimensional model. This model uses the Newcastle disease virus hemagglutinin-neuraminidase protein structure as a template. We identified seven amino acids important for SLAM- and nine for CD46 (Vero cell receptor)-induced fusion. The MV H three-dimensional model suggests (i) that SLAM- and CD46-relevant residues are located in contiguous areas in propeller β-sheets 5 and 4, respectively; (ii) that two clusters of SLAM-relevant residues exist and that they are accessible for receptor contact; and (iii) that several CD46-relevant amino acids may be shielded from direct receptor contacts. It appears likely that certain residues support receptor-specific H-protein conformational changes. To verify the importance of the H residues identified with the cell-cell fusion assays for virus entry into cells, we transferred the relevant mutations into genomic MV cDNAs. Indeed, we were able to recover recombinant viruses, and we showed that these replicate selectively in cells expressing SLAM or CD46. Selectively receptor-blind viruses will be used to study MV pathogenesis and may have applications for the production of novel vaccines and therapeutics.

scholarly journals Role of the Mutation Q252R in Activating Membrane Fusion in the Murine Leukemia Virus Surface Envelope Protein

2003 ◽  
Vol 77 (20) ◽  
pp. 10841-10849 ◽  
Author(s):  
Chi-Wei Lu ◽  
Monica J. Roth
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Envelope Protein ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Host Cells ◽  
Cell Binding ◽  
Surface Envelope ◽  
Murine Leukemia ◽  
Cell Cell

ABSTRACT Entry of retroviruses into host cells requires the fusion between the viral and cellular membranes. It is unclear how receptor binding induces conformational changes within the surface envelope protein (SU) that activate the fusion machinery residing in the transmembrane envelope protein (TM). In this report, we have isolated a point mutation, Q252R, within the proline-rich region of the 4070A murine leukemia virus SU that altered the virus-cell binding characteristics and induced cell-cell fusion. Q252R displays a SU shedding-sensitive phenotype. Cell-cell fusion is receptor dependent and is observed only in the presence of MuLV Gag-Pol. Both cellular binding and fusion by Q252R are greatly enhanced in conjunction of G100R, a mutation within the SU variable region A which increases viral binding through an independent mechanism. Deletion of a conserved histidine (His36) at the SU N terminus abolished cell-cell fusion by G100R/Q252R Env without compromising virus-cell binding. Although G100R/Q252R virus has no detectable titer, replacement of the N-terminal nine 4070A SU amino acids with the equivalent ecotropic MuLV sequence restored viral infectivity. These studies provide insights into the functional cooperation between multiple elements of SU required to signal receptor binding and activate the fusion machinery.

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