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.

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

Journal of Virology ◽

10.1128/jvi.00741-09 ◽

2009 ◽

Vol 83 (17) ◽

pp. 8713-8721 ◽

Cited By ~ 10

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.

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APOBEC3G-Regulated Host Factors Interfere with Measles Virus Replication: Role of REDD1 and Mammalian TORC1 Inhibition

Journal of Virology ◽

10.1128/jvi.00835-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 5

Author(s):

Vishakha Tiwarekar ◽

Julia Wohlfahrt ◽

Markus Fehrholz ◽

Claus-Jürgen Scholz ◽

Susanne Kneitz ◽

...

Keyword(s):

Host Cell ◽

Virus Replication ◽

Peripheral Blood ◽

Measles Virus ◽

Human Peripheral Blood ◽

Vero Cells ◽

Peripheral Blood Lymphocytes ◽

Host Factors ◽

Human Peripheral Blood Lymphocytes

ABSTRACTWe found earlier that ectopic expression of the cytidine deaminase APOBEC3G (A3G) in Vero cells inhibits measles virus (MV), respiratory syncytial virus, and mumps virus, while the mechanism of inhibition remained unclear. A microarray analysis revealed that in A3G-transduced Vero cells, several cellular transcripts were differentially expressed, suggesting that A3G regulates the expression of host factors. One of the most upregulated host cell factors, REDD1 (regulated in development and DNA damage response-1, also called DDIT4), reduced MV replication ∼10-fold upon overexpression in Vero cells. REDD1 is an endogenous inhibitor of mTORC1 (mammalian target of rapamycin complex-1), the central regulator of cellular metabolism. Interestingly, rapamycin reduced the MV replication similarly to REDD1 overexpression, while the combination of both did not lead to further inhibition, suggesting that the same pathway is affected. REDD1 silencing in A3G-expressing Vero cells abolished the inhibitory effect of A3G. In addition, silencing of A3G led to reduced REDD1 expression, confirming that its expression is regulated by A3G. In primary human peripheral blood lymphocytes (PBL), expression of A3G and REDD1 was found to be stimulated by phytohemagglutinin (PHA) and interleukin-2. Small interfering RNA (siRNA)-mediated depletion of A3G in PHA-stimulated PBL reduced REDD1 expression and increased viral titers, which corroborates our findings in Vero cells. Silencing of REDD1 also increased viral titers, confirming the antiviral role of REDD1. Finally, pharmacological inhibition of mTORC1 by rapamycin in PHA-stimulated PBL reduced viral replication to the level found in unstimulated lymphocytes, indicating that mTORC1 activity supports MV replication as a proviral host factor.IMPORTANCEKnowledge about host factors supporting or restricting virus replication is required for a deeper understanding of virus-cell interactions and may eventually provide the basis for therapeutic intervention. This work was undertaken predominantly to explain the mechanism of A3G-mediated inhibition of MV, a negative-strand RNA virus that is not affected by the deaminase activity of A3G acting on single-stranded DNA. We found that A3G regulates the expression of several cellular proteins, which influences the capacity of the host cell to replicate MV. One of these, REDD1, which modulates the cellular metabolism in a central position by regulating the kinase complex mTORC1, was identified as the major cellular factor impairing MV replication. These findings show interesting aspects of the function of A3G and the dependence of the MV replication on the metabolic state of the cell. Interestingly, pharmacological inhibition of mTORC1 can be utilized to inhibit MV replication in Vero cells and primary human peripheral blood lymphocytes.

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Identification of a Second Major Site for CD46 Binding in the Hemagglutinin Protein from a Laboratory Strain of Measles Virus (MV): Potential Consequences for Wild-Type MV Infection

Journal of Virology ◽

10.1128/jvi.76.24.13034-13038.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 13034-13038 ◽

Cited By ~ 30

Author(s):

Nicolas Massé ◽

Thomas Barrett ◽

Claude P. Muller ◽

T. Fabian Wild ◽

Robin Buckland

Keyword(s):

Measles Virus ◽

Laboratory Strain ◽

Cell Receptor ◽

Respiratory Epithelium ◽

Wild Type ◽

Major Site ◽

Hemagglutinin Protein ◽

A Cell ◽

H Protein

ABSTRACT Natural or wild-type (wt) measles virus (MV) infection in vivo which is restricted to humans and certain monkeys represents an enigma in terms of receptor usage. Although wt MV is known to use the protein SLAM (CD150) as a cell receptor, many human tissues, including respiratory epithelium in which the infection initiates, are SLAM negative. These tissues are CD46 positive, but wt MV strains, unlike vaccinal and laboratory MV strains, are not thought to use CD46 as a receptor. We have identified a novel CD46 binding site at residues S548 and F549, in the hemagglutinin (H) protein from a laboratory MV strain, which is also present in wt H proteins. Our results suggest that although wt MV interacts with SLAM with high affinity, it also possesses the capacity to interact with CD46 with low affinity.

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Morbillivirus Downregulation of CD46

Journal of Virology ◽

10.1128/jvi.72.12.10292-10297.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 10292-10297 ◽

Cited By ~ 28

Author(s):

Sareen E. Galbraith ◽

Ashok Tiwari ◽

Michael D. Baron ◽

Brett T. Lund ◽

Thomas Barrett ◽

...

Keyword(s):

Measles Virus ◽

Adenovirus Vector ◽

Vero Cells ◽

Wild Type ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Human Monocytic Cell Line ◽

Dolphin Morbillivirus ◽

Human Monocytic Cell ◽

H Protein

ABSTRACT There is evidence that CD46 (membrane cofactor protein) is a cellular receptor for vaccine and laboratory-passaged strains of measles virus (MV). Following infection with these MV strains, CD46 is downregulated from the cell surface, and consequent complement-mediated lysis has been shown to occur upon infection of a human monocytic cell line. The MV hemagglutinin (H) protein alone is capable of inducing this downregulation. Some wild-type strains of MV fail to downregulate CD46, despite infection being prevented by anti-CD46 antibodies. In this study we show that CD46 is also downregulated to the same extent by wild-type, vaccine, and laboratory-passaged strains of rinderpest virus (RPV), although CD46 did not appear to be the receptor for RPV. Expression of the RPV H protein by a nonreplicating adenovirus vector was also found to cause this downregulation. A vaccine strain of peste des petits ruminants virus caused slight downregulation of CD46 in infected Vero cells, while wild-type and vaccine strains of canine distemper virus and a wild-type strain of dolphin morbillivirus failed to downregulate CD46. Downregulation of CD46 can, therefore, be a function independent of the use of this protein as a virus receptor.

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

Journal of Virology ◽

10.1128/jvi.00248-07 ◽

2007 ◽

Vol 81 (13) ◽

pp. 6827-6836 ◽

Cited By ~ 52

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.

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Multiple Amino Acid Substitutions in Hemagglutinin Are Necessary for Wild-Type MeaslesVirus To Acquire the Ability To Use Receptor CD46 Efficiently

Journal of Virology ◽

10.1128/jvi.02449-06 ◽

2006 ◽

Vol 81 (6) ◽

pp. 2564-2572 ◽

Cited By ~ 49

Author(s):

Maino Tahara ◽

Makoto Takeda ◽

Fumio Seki ◽

Takao Hashiguchi ◽

Yusuke Yanagi

Keyword(s):

Fusion Proteins ◽

Measles Virus ◽

Lymphocyte Activation ◽

Cellular Receptor ◽

Wild Type ◽

Recombinant Viruses ◽

B Lymphoid ◽

Multiple Amino Acid ◽

H Protein ◽

Lymphoid Cell Lines

ABSTRACT Measles virus (MV) possesses two envelope glycoproteins, namely, the receptor-binding hemagglutinin (H) and fusion proteins. Wild-type MV strains isolated in B-lymphoid cell lines use signaling lymphocyte activation molecule (SLAM), but not CD46, as a cellular receptor, whereas MV vaccine strains of the Edmonston lineage use both SLAM and CD46 as receptors. Studies have shown that the residue at position 481 of the H protein is critical in determining the use of CD46 as a receptor. However, the wild-type IC-B strain with a single N481Y substitution in the H protein utilizes CD46 rather inefficiently. In this study, a number of chimeric and mutant H proteins, and recombinant viruses harboring them, were generated to determine which residues of the Edmonston H protein are responsible for its efficient use of CD46. Our results show that three substitutions (N390I and E492G plus N416D or T446S), in addition to N481Y, are necessary for the IC-B H protein to use CD46 efficiently as a receptor. The N390I, N416D, and T446S substitutions are present in the H proteins of all strains of the Edmonston lineage, whereas the E492G substitution is found only in the H protein of the Edmonston tag strain generated from cDNAs. The T484N substitution, found in some of the Edmonston-lineage strains, resulted in a similar effect on the use of CD46 to that caused by the E492G substitution. Thus, multiple residues in the H protein that have not previously been implicated have important roles in the interaction with CD46.

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Measles Virus Infects and Suppresses Proliferation of T Lymphocytes from Transgenic Mice Bearing Human Signaling Lymphocytic Activation Molecule

Journal of Virology ◽

10.1128/jvi.77.6.3505-3515.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3505-3515 ◽

Cited By ~ 47

Author(s):

Bumsuk Hahm ◽

Nathalie Arbour ◽

Denise Naniche ◽

Dirk Homann ◽

Marianne Manchester ◽

...

Keyword(s):

T Cells ◽

Cell Division ◽

Transgenic Mice ◽

Measles Virus ◽

Laboratory Strain ◽

Proximal Promoter ◽

Wild Type ◽

Natural Reservoir ◽

Signaling Lymphocytic Activation Molecule

ABSTRACT Humans are the only natural reservoir of measles virus (MV), one of the most contagious viruses known. MV infection and the profound immunosuppression it causes are currently responsible for nearly one million deaths annually. Human signaling lymphocytic activation molecule (hSLAM) was identified as a receptor for wild-type MV as well as for MV strains prepared as vaccines. To better evaluate the role of hSLAM in MV pathogenesis and MV-induced immunosuppression, we created transgenic (tg) mice that expressed the hSLAM molecule under the control of the lck proximal promoter. hSLAM was expressed on CD4+ and CD8+ T cells in the blood and spleen and also on CD4+, CD8+, CD4+ CD8+, and CD4− CD8− thymocytes. Wild-type MV, after limited passage on B95-8 marmoset B cells, and the Edmonston laboratory strain of MV infected hSLAM-expressing cells. There was a direct correlation between the amount of hSLAM expressed on the cells' surface and the degree of viral infection. Additionally, MV infection induced downregulation of receptor hSLAM and inhibited cell division and proliferation of hSLAM+ but not hSLAM− T cells. Therefore, these tg mice provide the opportunity for analyzing and comparing MV-T cell interactions and MV pathogenesis in cells expressing only the hSLAM MV receptor with those of tg mice whose T cells selectively express another MV receptor, CD46.

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Recombinant wild-type measles virus containing a single N481Y substitution in its haemagglutinin cannot use receptor CD46 as efficiently as that having the haemagglutinin of the Edmonston laboratory strain

Journal of General Virology ◽

10.1099/vir.0.81682-0 ◽

2006 ◽

Vol 87 (6) ◽

pp. 1643-1648 ◽

Cited By ~ 21

Author(s):

Fumio Seki ◽

Makoto Takeda ◽

Hiroko Minagawa ◽

Yusuke Yanagi

Keyword(s):

Clinical Isolate ◽

Cell Lines ◽

Recombinant Virus ◽

Measles Virus ◽

Lymphocyte Activation ◽

Laboratory Strain ◽

Cellular Receptor ◽

Wild Type

Signalling lymphocyte activation molecule (SLAM) acts as a cellular receptor for Measles virus (MV). The recombinant MV, based on a SLAM-using clinical isolate in which asparagine at position 481 of the haemagglutinin was replaced with tyrosine, was generated. Characterization of this recombinant virus revealed that the N481Y substitution in the haemagglutinin allowed it to utilize CD46 as an alternative receptor, but that its ability to use CD46 was rather low in CD46+ SLAM− cell lines compared with that of the recombinant virus possessing the haemagglutinin of the Edmonston laboratory strain. Thus, an N481Y substitution alone may not be sufficient to make SLAM-using MVs use CD46 efficiently, suggesting that further substitutions in the haemagglutinin are required for them to grow efficiently in CD46+ cells like the Edmonston strain. This may be a reason why few CD46-using MVs are detected in vivo.

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A Recombinant Measles Vaccine Virus Expressing Wild-Type Glycoproteins: Consequences for Viral Spread and Cell Tropism

Journal of Virology ◽

10.1128/jvi.73.8.6903-6915.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6903-6915 ◽

Cited By ~ 45

Author(s):

Ian C. D. Johnston ◽

V. ter Meulen ◽

Jürgen Schneider-Schaulies ◽

Sibylle Schneider-Schaulies

Keyword(s):

Hela Cells ◽

Measles Virus ◽

Cell Types ◽

Vero Cells ◽

Vaccine Virus ◽

Cell Tropism ◽

Wild Type ◽

Recombinant Viruses ◽

Viral Spread

ABSTRACT Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism.

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