The lymphocytic choriomeningitis virus RING protein Z associates with eukaryotic initiation factor 4E and selectively represses translation in a RING-dependent manner

Abstract

Only a few host cell proteins that associate with arenaviruses have been identified. To date, the arenavirus Z protein associates with the promyelocytic leukemia protein PML and the ribosomal P proteins. The majority of PML is present in nuclear bodies which are translocated to the cytoplasm by infection with the arenavirus, lymphocytic choriomeningitis virus (LCMV). The Z protein is a small zinc-binding RING protein with an unknown function which is required for the viral life cycle. Here, we demonstrate an association between Z and the host cell translation factor, eukaryotic initiation factor 4E (eIF-4E) in infected and transfected cells. Z's association with both ribosomal proteins and this translation factor led us to investigate whether Z could modulate host cell translation. In cell culture, Z selectively represses protein production in an eIF-4E-dependent manner. Specifically, we see reduction in cyclin D1 protein production with no effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells transfected with Z. Previous reports indicate that cyclin D1 is sensitive to eIF-4E levels, whereas GAPDH is not. Consistent with this, we observe preferential downregulation of cyclin D1 during infection and no effect on GAPDH. Further, no changes in RNA levels were observed for cyclin D1 or GAPDH transcripts. The interaction between eIF-4E and Z may provide a mechanism for slower growth observed in infected cells and a viral strategy for establishing chronic infection.

FIG. 1

Z and eIF-4E colocalize. NIH 3T3 cells infected for 90 h with LCMV are shown. Subsequently, cells were stained with affinity-purified Z polyclonal antibody in green (A) and eIF-4E MAb in red (B), and the overlay is shown in yellow (C). In panels D and E uninfected and infected (90 h, inf) cells, respectively, were stained with MAb eIF-4E. The objective is ×100. Panels A to C were further magnified 1.5 times. Panels D and E were magnified 1.8 times. Confocal micrographs represent single slices through the plane of cells. FITC and Texas red channels were recorded independently.

FIG. 2

Z and eIF-4E associate physically. (A and B) Z and ZRINGmut coimmunoprecipitate eIF-4E. NIH 3T3 cells were transfected with Z (A) or ZRINGmut (B). The resulting lysates were immunoprecipitated with Z antisera. Nuclear, nuclear fraction; cyto., cytoplasmic fraction; total, total cell lysate; S, supernatant after immunoprecipitation (IP). Western blots were probed as indicated. (C) Cell lysates were immunoprecipitated with mouse IgG as a negative control. Western blots were probed with eIF-4E or actin as indicated. N, nuclear fraction; cyto., cytoplasmic fraction; T, total; S, supernatant after immunoprecipitation. (D) The specificity of the commercially obtained eIF-4E antibody (MAb eIF-4E) was assessed. Western blots of control cells lysates (3T3) or cells transfected with eIF-4E (3T3/eIF-4E) were probed with MAb eIF-4E.

FIG. 3

(A) ³⁵S-Met incorporation in cells overexpressing the indicated constructs. Total protein production was measured by autoradiography, and band intensities were quantitated. Mean band intensities and SDs for each transfection taken from three independent experiments are shown. (B) Z affects cyclin D1 and E production. Cells were transfected and metabolically labeled. The same amount of total protein from cell lysates was immunoprecipitated (IP) with antibodies to cyclins D1, E, or GAPDH as indicated. Immunoprecipitated protein was monitored by autoradiography. Values were normalized to those of GAPDH in each experiment. Mean band intensities and standard errors are given below each band. Results for three experiments were quantitated.

FIG. 4

Z does not inhibit transcription. (A) Z overexpression does not reduce levels of cyclin D1 transcripts. Total cyclin D1 mRNA levels were determined for cells transfected with the indicated constructs by using Northern analysis. Cyclin D1 mRNA levels were normalized to 28S and 18S rRNA band intensities. Experiments were carried out in duplicate and varied as shown. (B) Z does not alter transcription. CMV immediate-early promoter fragment (Promega) was transcribed in the presence of 30 μM (each) Z, GST, bovine serum albumin, or H₂O. RNA production was monitored by incorporation of [³⁵S]rUTP and autoradiography. The second panel shows the effect of heating extract prior to the assay (H.E.). “Cold” refers to the use of unlabeled rUTP, and “No ex.” means no extract was used. (C) Z does not alter the distribution of cyclin D1 mRNA. RNA distribution in cellular fractions overexpressing the listed constructs was monitored. The percentage of nuclear cyclin D1 (solid bars) or GAPDH (open bars) RNA is given. Values of band intensities in each lane were normalized to band intensities of 28S and 18S rRNA to correct for differences in loading. RNA levels were normalized to the distribution of total RNA in the cell. Experiments were carried in duplicate and varied by the percentage given.

FIG. 5

Western blot analysis of infected HeLa cells. Times indicate hours p.i., and “V” refers to virions. Blots were probed as indicated.