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. 1999 Aug;73(8):6903-15.
doi: 10.1128/JVI.73.8.6903-6915.1999.

A recombinant measles vaccine virus expressing wild-type glycoproteins: consequences for viral spread and cell tropism

I C Johnston  1 , V ter Meulen, J Schneider-Schaulies, S Schneider-Schaulies
Affiliations

A recombinant measles vaccine virus expressing wild-type glycoproteins: consequences for viral spread and cell tropism

I C Johnston et al. J Virol. 1999 Aug.

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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Figures

FIG. 1
FIG. 1
Cell-cell fusion induced following transfection of HeLa cells with plasmids expressing vaccine or wild-type glycoproteins. HeLa cells were transfected in duplicate wells as detailed in Materials and Methods with EdH plus EdF (A and G), WTFF plus WTFH (B and H), WTFF plus EdH (C and I), EdF plus WTFH (D and J), and pCG vector (E). (F) Vero cells transfected with EdF + WTFH. Transfected HeLa cells (A to E) or transfected Vero cells (F) were photographed 48 h posttransfection. (G to J) A total of 2 × 105 transfected HeLa cells were plated onto a monolayer of B95a cells 48 h posttransfection. Following transfection, the HeLa cells had been treated with 50 μg of FIP per ml, which completely inhibited syncytium formation (data not shown). Cells were photographed 8 h after cocultivation (G to J). Bar in panel F, 100 μm (A to F); bar in panel I, 50 μm (G to J).
FIG. 1
FIG. 1
Cell-cell fusion induced following transfection of HeLa cells with plasmids expressing vaccine or wild-type glycoproteins. HeLa cells were transfected in duplicate wells as detailed in Materials and Methods with EdH plus EdF (A and G), WTFF plus WTFH (B and H), WTFF plus EdH (C and I), EdF plus WTFH (D and J), and pCG vector (E). (F) Vero cells transfected with EdF + WTFH. Transfected HeLa cells (A to E) or transfected Vero cells (F) were photographed 48 h posttransfection. (G to J) A total of 2 × 105 transfected HeLa cells were plated onto a monolayer of B95a cells 48 h posttransfection. Following transfection, the HeLa cells had been treated with 50 μg of FIP per ml, which completely inhibited syncytium formation (data not shown). Cells were photographed 8 h after cocultivation (G to J). Bar in panel F, 100 μm (A to F); bar in panel I, 50 μm (G to J).
FIG. 2
FIG. 2
Schematic representation of the MV genomic plasmid construct p(+)MVNSe and the wild-type recombinant constructs. The unique restriction sites used for the introduction of the wild-type glycoprotein sequences are indicated. The F and H glycoprotein genes of WTF were cloned by RT-PCR as detailed in Materials and Methods. The WTF sequence is indicated by the shaded boxes.
FIG. 3
FIG. 3
Characterization of recombinant MVs with an antibody that distinguishes between Ed and WTF H glycoproteins. BJAB cells were infected with an MOI of 0.1 and 3 days postinfection were labelled for FACS analysis by using, as a control, a standard isotype control antibody or the mouse MAbs specific for the MV-F protein (A504) and MV-H protein (L77 and K53). MAb K53 is unable to bind to the WTFH protein, whereas L77 recognizes both EdH and WTFH efficiently.
FIG. 4
FIG. 4
Growth of recombinant viruses in human lymphoid cells. (A) BJAB cells were infected at an MOI of 3, and 5 × 104 cells were then incubated at 37°C in 200 μl of medium. At various time points after infection samples were removed and stored at −80°C. Cell lysates were then titrated on B95a cells, and virus titers were determined as log10 TCID50/ml. The means from two experiments are shown. (B) BJAB cells were infected at an MOI of 0.1 and were analyzed at 24-h intervals, beginning at day 0 (0d), following infection for the expression of the MV-N protein by FACS analysis. In this particular experiment a higher MOI for the MV(WTF H)Ed virus (0.3 MOI) was used.
FIG. 5
FIG. 5
Spread and cytopathic effect of the recombinant viruses in cell culture. B95a cells (a to e), Vero cells (f to m), and HeLa cells (n to u) were infected with Ed-tag (a, f, and n), WTF (b, g, k, o, and s), MV(WTF H)Ed (c, h, l, p, and t), MV(WTF F/WTF H)Ed (d, i, m, q, and u), or MV(WTF F)Ed (e, j, and r) at an MOI of 0.1 in suspension and were then plated out on glass coverslips. At 1, 2, and 4 days postinfection (dpi) the cells were fixed and permeabilized, and the expression of the MV-H protein was studied by immunohistochemistry. Magnification, ×80 (all panels).
FIG. 6
FIG. 6
Growth and release of recombinant virus in cell culture. B95a cells (A to E), Vero cells (F to J), and HeLa cells (K to O) were infected with Ed-tag (A, F, and K), WTF (B, G, and L), MV(WTF H)Ed (C, H, and M), MV(WTF F/WTF H)Ed (D, I, and N), or MV(WTF F)Ed (E, J, and O) at an MOI of 0.1 in suspension. A total of 105 cells were then plated out in 48-well plates in 0.5 ml of medium, and cell-associated (squares) and cell-free (circles) virus were titrated on B95a cells at various time points postinfection as detailed in Materials and Methods. Virus titers were calculated as log10 TCID50/ml.
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