Measles Viruses Possessing the Polymerase Protein Genes of the Edmonston Vaccine Strain Exhibit Attenuated Gene Expression and Growth in Cultured Cells and SLAM Knock-In Mice

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The P, V, and C proteins of measles virus are encoded in overlapping reading frames of the P gene, which makes it difficult to analyze the functions of the individual proteins in the context of virus infection. We established a system to analyze the C protein independently from the P and V proteins by placing its gene in an additional transcription unit between the H and L genes. Analyses with this system indicated that a highly attenuated Edmonston lineage vaccine strain encodes a fully functional C protein, and the P and/or V protein is involved in the attenuated phenotype.Measles is a highly contagious disease characterized by high fever and a maculopapular rash. About 4% of deaths in children aged under 5 years are caused by measles worldwide (9). The causative agent, measles virus (MV), belongs to the genus Morbillivirus in the family Paramyxoviridae. The genome of MV is a nonsegmented negative-strand RNA of ϳ16 kb in length and contains six genes. Each gene encodes a single structural protein, namely the nucleocapsid (N), phospho (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins (17). The genome forms a helical ribonucleoprotein complex with the N protein and viral RNAdependent RNA polymerase composed of the P and L proteins. The P protein acts as an essential cofactor of RNA-dependent RNA polymerase and tethers the L protein onto the nucleocapsid template (20). The P gene encodes additional gene products, the V and C proteins, by the processes of RNA editing and alternative translational initiation in a different reading frame, respectively (17). Although the V and C proteins are nonessential for MV replication (29, 35), they act as important virulence factors in vivo (11,28,(44)(45)(46). Many lines of evidence have indicated that the C and V proteins of MV antagonize the host interferon (IFN) responses (10,16,22,23,25,26,37,42,47). The V protein directly interferes with pathways of IFN induction (1) and IFN signaling (25,26,42), while the C protein contributes to circumvention of IFN induction by controlling the levels of viral RNA synthesis (22,23,31). Direct interference with IFN signaling by the C protein has also been reported, although its effects are weaker than those of the V protein (16,37).In a recent study, we showed that a recombinant IC-B strain possessing the P gene of the attenuated Edmonston tag strain (IC/EdP) replicates less efficiently than the parental IC-B strain (a virulent strain) (40). The Edmonston tag strain is a recombinant MV derived from the Edmonston B vaccine strain, which has been passaged numerous times in various cultured cells (30). There are many amino acid differences between the P gene products of the IC-B and Edmonston tag strains ( Fig. 1) (27,30,43). Most of the changes found in the Edmonston tag strain are common to the Edmonston lineage MV strains (27). However, owing to two amino acid substitutions at positions 110 and 272, which are not conserved among the Edmonston lineage MV strains (25-27), the V protein of the Edmonston tag strain is de...

“…(Fig. 4C) (40). These data confirm that the attenuated growth of IC/EdP is caused by the P/V protein of the Edmonston tag strain and not by the C protein.…”

supporting

confidence: 76%

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confidence: 78%

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confidence: 99%

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A Highly Attenuated Measles Virus Vaccine Strain Encodes a Fully Functional C Protein

Nakatsu

Takeda

Iwasaki

et al. 2009

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“…This result indicated that the Edmonston H protein does not influence the extent of infection in lymphoid tissues. Proteins other than the H protein, possibly viral polymerase proteins (30), may regulate MV replication in lymphoid tissues.…”

Section: Discussionmentioning

confidence: 99%

Wild-Type Measles Virus with the Hemagglutinin Protein of the Edmonston Vaccine Strain Retains Wild-Type Tropism in Macaques

Takeuchi

Nagata

Kato

et al. 2012

A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro, MV-EdH replicated in SLAM ؉ as well as CD46 ؉ cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM ؉ cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM ؉ lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo. They also suggest that the vaccine H protein attenuates MV growth in vivo. Measles remains a major cause of childhood morbidity and mortality worldwide especially in developing countries in spite of significant progress in global measles control programs. Measles virus (MV), belonging to the genus Morbillivirus of the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome (11). The MV genome encodes 6 structural proteins: the nucleocapsid (N), phospho (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins. Two envelope glycoproteins, the F and H proteins, initiate infection of the target cells via binding of the H protein to its cellular receptors. Therefore, the H protein is of primary importance for determining the cell specificity of MV (22).The Edmonston strain of MV was isolated in 1954 by using a primary culture of human kidney cells (7). The Edmonston strain was subsequently adapted in a variety of cells, including chicken embryo fibroblasts, to enable the production of attenuated live vaccines, which are currently used worldwide (27). These live, attenuated MV strains are safe and induce strong cellular and humoral immune responses against MV. The Edmonston vaccine strain is no longer pathogenic in monkey models (2, 7, 37, 39). In contrast, wild-type MV strains isolated and passaged in B95a cells induce clinical signs resembling those of human measles in experimentally infected cynomolgus and rhesus monkeys (15, 16).A major difference betwe...

“…Even in the presence of 2F4, rMVs with F483A, Y543S, or S544A/ Y541S were able to spread in SLAM ϩ B95a cells, and rMV with R533 was able to spread in nectin4 ϩ II-18 cells, whereas the wildtype MV infection was completely neutralized by 2F4. Similar experiments were performed using luciferase-expressing rMVs (21). Two rMVs with mutations at SLAM-interacting residues (D505S and R533A) (17,19) (Fig.…”

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confidence: 89%

The Receptor-Binding Site of the Measles Virus Hemagglutinin Protein Itself Constitutes a Conserved Neutralizing Epitope

Tahara

Ohno

Sakai

et al. 2013

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e Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature. Measles virus (MV) is an enveloped virus that belongs to the genus Morbillivirus of the family Paramyxoviridae and possesses two types of surface glycoprotein spikes, the hemagglutinin (H) and fusion (F) proteins. MV infection is initiated by binding of the H protein to cellular receptors on the target host cells. The binding triggers membrane fusion between the virus envelope and the host cell plasma membrane mediated by the F protein. The signaling lymphocyte activation molecule (SLAM) on a subset of immune cells and nectin4 at adherens junctions are the principal receptors for MV (1-4). The H and F proteins are both neutralizing targets, but greater amounts of antibodies (Abs) are elicited against the H protein than the F protein, and the H protein-specific antibodies mainly contribute to neutralization of MV infection in vivo (5-8). All the available data suggest that measles eradication is biologically feasible (9, 10), and one of the major factors that would ensure measles eradication is the single-serotype nature of MV. Our previous paper suggested that the receptor-binding site (RBS) on the H protein, which is exposed outside the heavy N-glycan shields, may constitute a major neutralizing epitope that contributes to the single-serotype nature of MV (11). However, our recent paper (12) showing the detailed locations of five epitopes (I, II, iv, v, and vi) on the H protein provided insufficient evidence that the RBS acts as a neutralizing epitope. Here, we provide direct and concrete evidence that the RBS itself forms an effective conserved neutralizing epitope (CNE) which provides a strong functional constraint against change.In the present study, we characterized a new mouse monoclonal antibody (MAb), 2F4. MAb 2F4 was produced using a cell line expressing the H protein of the wild-type IC-B strain as an antigen. For competitive binding enzyme-linked immunosorbent assays (cELISAs), MV antigens precoated on cELISA plates (Denka Seiken) were incubated with previously reported MAbs (E81, E128, E185, E39, and E103) (12, 13), 2F4, or phosphate-buffered saline for 1 h and then incubated with MAb 2F4 labeled with biotin using a biotin labeling kit (NH2; Dojindo Laboratories). After three washes, the MV antigens bound by the MAbs were incubated with a streptavidin-horseradish peroxidase (HRP) conjugate (Thermo Scientific) for 1 h before addition of the TMB substrate (3,3=5,5=-tetramethylbenzidine; Denka Seiken). The plates were incubated for 25 min, and the colorimetric...