Measles virus N protein inhibits host translation by binding to eIF3-p40

Abstract

The nonsegmented, negative-sense RNA genome of measles virus (MV) is encapsidated by the virus-encoded nucleocapsid protein (N). In this study, we searched for N-binding cellular proteins by using MV-N as bait and screening the human T-cell cDNA library by yeast two-hybrid assay and isolated the p40 subunit of eukaryotic initiation factor 3 (eIF3-p40) as a binding partner. The interaction between MV-N and eIF3-p40 in mammalian cells was confirmed by coimmunoprecipitation. Since eIF3-p40 is a translation initiation factor, we analyzed the potential inhibitory effect of MV-N on protein synthesis. Glutathione S-transferase (GST)-fused MV-N (GST-N) inhibited translation of reporter mRNAs in rabbit reticulocyte lysate translation system in a dose-dependent manner. Encephalomyocarditis virus internal ribosomal entry site-mediated translation, which requires canonical initiation factors to initiate translation, was also inhibited by GST-N. In contrast, a unique form of translation mediated by the intergenic region of Plautia stali intestine virus, which can assemble 80S ribosomes in the absence of canonical initiation factors, was scarcely affected by GST-N. In vivo expression of MV-N induced by the Cre/loxP switching system inhibited the synthesis of a transfected reporter protein, as well as overall protein synthesis. These results suggest that MV-N targets eIF3-p40 and may be involved in inhibiting MV-induced host translation.

FIG. 1.

Interaction between eIF3-p40 and MV-N. (A) Yeast strain AH109 was cotransfected with the following pairs of expression vectors and plated onto the selection medium without His, Leu, and Trp: (1) BD vector+AD-eIF3-p40, (2) BD-MV-N+AD-vector, (3) BD-MV-N+AD-eIF3-p40, (4) BD-lamin C+AD-eIF3-p40, (5) BD-MV-N+AD-SV40 large T antigen, and (6) BD-P53+AD-SV40 large T antigen. (B) Cos-7 cells were mock transfected (lane 1) or transfected with myc-tagged MV-N (lane 2) or myc-tagged MV-N and HA-tagged eIF3-p40 (lane 3). Immunoprecipitation was performed on total cell lysates using anti-myc antibody. Immunoprecipitates were detected by SDS-PAGE, followed by autoradiography. (C) COBL-a cells were infected with MV-HL at an MOI of 0.001. At 48 h postinfection, cells were cross-linked with formaldehyde, and the cell lysate was immunoprecipitated with anti-N monoclonal antibody. Immunoprecipitates were de-cross-linked by boiling and then detected by Western blotting with the indicated antibodies.

FIG. 2.

Inhibition of in vitro translation by GST-fused MV-N. (A) Increasing amounts of recombinant GST, GST-N, or GST-P were incubated with rabbit reticulocyte lysates. A reporter RNA encoding the luciferase gene was added and translated in the presence of [³⁵S]methionine. Translated products were separated by SDS-PAGE and detected by autoradiography. The amount of luciferase synthesized was quantified by densitometric analysis. The results shown represent the means of three experiments. (B) A reporter RNA was preincubated with increasing amount of GST-N and then added to rabbit reticulocyte lysates. Translated products were quantified in a similar manner as panel A.

FIG. 3.

Mapping the eIF3-p40-interacting domain of MV-N. (A) Parts of the N gene corresponding to amino acid residues 1 to 80, 81 to 192, 193 to 252, 253 to 336, 337 to 420, and 421 to 525 were deleted, and the resulting deletion clones were inserted into a BD vector for yeast two-hybrid analysis. These constructs were cotransformed with the AD-eIF3-p40 vector into yeast and plated onto selection medium lacking Leu and Trp (SD/−Leu/−Trp). The colonies obtained were spotted on SD/−His/−Leu/−Trp/3-AT plates and incubated at 30°C for 4 days. The apparent binding strength was assessed by the degree of growth and is scored as strong (+++), intermediate (++), weak (+), or absent (−). (B) Myc-tagged MV-N and its deletion clones, NΔ1 and NΔ2, were coexpressed with eIF3-p40 in Cos-7 cells and coimmunoprecipitated using anti-myc antibody. Asterisks indicate immunoprecipitated MV-N or its deletions. An arrowhead indicates the band of eIF3-p40 that was coprecipitated. (C) Parts of the NΔ2 deletion region corresponding to amino acid residues 81 to 120, 121 to 160, and 161 to 192 were further deleted, and the resulting deletion clones were coexpressed with eIF3-p40 in Cos-7 cells and coimmunoprecipitated. The asterisks indicate MV-N or its deletion clones precipitated using anti-myc antibody. Arrowhead indicates the band of eIF3-p40 coprecipitated.

FIG. 4.

MV-N lacking eIF3-p40-binding ability shows no inhibitory effect on translation. Increasing amounts of recombinant GST or GST-NΔ2c were incubated with rabbit reticulocyte lysates. A reporter RNA encoding the luciferase gene was added and translated in the presence of [³⁵S]methionine. Translated products were separated by SDS-PAGE and detected by autoradiography. The amount of luciferase synthesized was quantified by densitometric analysis. The results shown represent means of three experiments.

FIG. 5.

MV-N selectively inhibits cap-dependent and EMCV-IRES-mediated translation but not IGR-IRES-mediated translation. Increasing amounts of GST-N were incubated with rabbit reticulocyte lysate. m⁷G-luc RNA (A), EMCV-IRES-luc RNA (B), and IGR-IRES-luc RNA (C) were added and translated in the presence of [³⁵S]methionine. Translated products were separated by SDS-PAGE and detected by autoradiography. The amount of luciferase synthesized was quantified by densitometric analysis. The results shown represent the means of three experiments.

FIG. 6.

Inhibition of protein synthesis by MV-N in vivo. (A to D) 293-MVN cells or 293 cells were plated in 24-well plates and infected with a recombinant adenovirus, AxCANCre, expressing Cre recombinase at an MOI of 10, 20, or 50. (A) At 24 h postinfection, switching expression of MV-N in 293-MVN cells was detected by Western blotting. (B) At 24 h postinfection, a reporter plasmid encoding the luciferase gene was transfected and incubated for 24 h. Cells were harvested, an aliquot was lysed, and luciferase activity was measured by using a luminometer. Another aliquot was harvested, and the total RNA was analyzed for luciferase transcripts using one-step real-time RT-PCR. The luciferase activity and mRNA levels in 293-MVN cells are expressed relative to those in 293 cells. The results shown represent means of three experiments. (C) At 24 h postinfection, cells were labeled with [³⁵S]methionine for 24 h. An aliquot of the cells was lysed, and the level of radioactivity incorporated in all proteins was measured by using a scintillation counter. An aliquot of the cells was harvested, and the total RNA was analyzed for transcripts of the housekeeping gene, GAPDH, using one-step real-time RT-PCR. The levels of radioactivity and GAPDH mRNA in 293-MVN cells are expressed relative to those in 293 cells. The results shown represent means of three experiments. (D) At 48 h postinfection, cells were lysed and endogenous GAPDH was detected by Western blotting.