Stringent requirement for the C protein of wild-type measles virus for growth both in vitro and in macaques

Abstract

The P gene of measles virus (MV) encodes the P protein and three accessory proteins (C, V, and R). However, the role of these accessory proteins in the natural course of MV infection remains unclear. For this study, we generated a recombinant wild-type MV lacking the C protein, called wtMV(C-), by using a reverse genetics system (M. Takeda, K. Takeuchi, N. Miyajima, F. Kobune, Y. Ami, N. Nagata, Y. Suzaki, Y. Nagai, and M. Tashiro, J. Virol. 74:6643-6647). When 293 cells expressing the MV receptor SLAM (293/hSLAM) were infected with wtMV(C-) or parental wild-type MV (wtMV), the growth of wtMV(C-) was restricted, particularly during late stages. Enhanced green fluorescent protein-expressing wtMV(C-) consistently induced late-stage cell rounding and cell death in the presence of a fusion-inhibiting peptide, suggesting that the C protein can prevent cell death and is required for long-term MV infection. Neutralizing antibodies against alpha/beta interferon did not restore the growth restriction of wtMV(C-) in 293/hSLAM cells. When cynomolgus monkeys were infected with wtMV(C-) or wtMV, the number of MV-infected cells in the thymus was >1,000-fold smaller for wtMV(C-) than for wtMV. Immunohistochemical analyses showed strong expression of an MV antigen in the spleen, lymph nodes, tonsils, and larynx of a cynomolgus monkey infected with wtMV but dramatically reduced expression in the same tissues in a cynomolgus monkey infected with wtMV(C-). These data indicate that the MV C protein is necessary for efficient MV replication both in vitro and in cynomolgus monkeys.

FIG. 1.

Construction and generation of recombinant wtMV lacking expression of the C protein. (A) Schematic diagram of genomic organization of wtMV showing the P cistron encoding the P, C, and V proteins and the mutations in the C open reading frame. Altered nucleotides are underlined. The expression of the C protein was eliminated by placing two stop codons, UGA [p(+)322 position 1832] and UAG [p(+)MV322 position 1844], downstream in the C open reading frame. (B) Western blot analysis using rabbit antisera against the MV C protein and MV virions. Lysates from cells infected with wtMV or wtMV(C−) or from mock-infected cells were probed with antisera against the MV C protein and the MV Toyoshima strain.

FIG. 2.

Replication kinetics of wtMV and wtMV(C−). B95a cells (A) and 293/hSLAM cells (B) were infected with wtMV (circles) or wtMV(C−) (triangles) at an MOI of 0.01 TCID₅₀/cell. Cells and media were harvested at 1, 2, 3, and 4 dpi, and infectivity titers were assessed as TCID₅₀ using B95a cells.

FIG. 3.

Cytopathic effects of wtMV and wtMV(C−) in 293/hSLAM cells. 293/hSLAM cells were infected with wtMV (A and B) or wtMV(C−) (C and D), and cells were photographed under a microscope at 1 dpi (A and C) and 3 dpi (B and D).

FIG. 4.

Cell rounding and cell death observed in 293/hSLAM cells infected with wtMV(C−)-EGFP. 293/hSLAM cells were infected with wtMV-EGFP (A and B) or wtMV(C−)-EGFP (C and D) and incubated in the presence of the FIP. The expression of EGFP was monitored by using a fluorescence microscope at 3 dpi (A and C) and 7 dpi (B and D).

FIG. 5.

Effect of IFN on wtMV(C−) replication in 293/hSLAM cells and effect of the MV C protein on IFN-responsive reporter gene assay. (A) 293/hSLAM cells were infected with wtMV(C−) and incubated in the presence of a mixture of monoclonal antibodies against IFN-α and IFN-β (ΜοΑb) or a mixture of polyclonal antisera against IFN-α and IFN-β (PoAb). The cells were harvested at 3 dpi, and virus titers were assessed as TCID₅₀ for B95a cells. Black bars represent the average titers obtained from triplicate samples. Standard deviations are also indicated. (B) 293/hSLAM cells in a 24-well plate were infected with wtMV or wtMV(C−) at an MOI of 0.01. After incubation at 37°C for 1 h, the cells were washed three times and incubated with complete medium containing 1,000 IU/ml of IFN-β. The cells were harvested at 2 dpi, and virus growth was assessed as the TCID₅₀ for B95a cells. Black bars represent the average titers obtained from triplicate samples. Standard deviations are also indicated. (C) HepG2 cells were transfected with a plasmid containing an IFN-responsive luciferase reporter gene, the control pSEAP plasmid, and the pKS336 plasmid (pKS), the pKS336 plasmid expressing the MV V (pKS-V) or C (pKS-C) protein, the pCAGGS plasmid (pCA), or the pCAGGS plasmid expressing the MV C protein (pCA-C). After transfection, the cells were stimulated with 1,000 IU/ml of IFN-β or were left unstimulated. Relative expression levels were normalized to the SEAP activity and expressed as changes in activation. Black bars represent the average titers obtained from triplicate samples. Standard deviations are also indicated.

FIG. 6.

Spread of MV antigen in different tissues from infected cynomolgus monkeys. Sections were obtained from cynomolgus monkeys 11 days after wtMV (A to D) or wtMV(C−) (E to H) inoculation. The MV antigen (light brown) in spleens (A and E), lymph nodes (B and F), tonsils (C and G), and larynxes (D and H) was detected with a rabbit antiserum against the MV N protein. Nuclei were counterstained with hematoxylin.