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Critical role for arginine methylation in adenovirus-infected cells - PubMed

Critical role for arginine methylation in adenovirus-infected cells

Demetris C Iacovides et al. J Virol. 2007 Dec.

Abstract

During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection.

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Figures

FIG. 1.
FIG. 1.

The RGG domain is well conserved at the C terminus of many adenovirus serotypes. The C terminus of Ad5 100K contains several RG and RGR motifs and an RGG domain between amino acids 727 and 743 with three RGG tripeptides. 100KΔC was constructed by deleting amino acids 651 to 805. We also constructed three mutant clones with each of the three RGG motifs with the RGG domain mutated to alanine residues.

FIG. 2.
FIG. 2.

RGG mutants fail to localize to the nucleus during infection. U2OS cells were transfected with plasmids expressing myc-tagged wild-type 100K, a C terminus truncated 100K (100KΔC), or an RGG mutant with positions 741 to 743 changed to alanine residues. 100KΔC has a deletion of amino acids 680 to 805, which encompasses the arginine-glycine rich region with the RGG, RGR, and RG motifs. Untransfected and transfected cells were infected with dl309 (wild-type Ad5). Slides were fixed at 18 or 40 h p.i. and stained with α-100K (two leftmost panels) or α-myc antibodies as indicated.

FIG. 3.
FIG. 3.

100K is methylated and binds PRMT1 methylase through its RGG domain. (A and B) Cells were transfected with myc-100K, myc-100KΔC, or myc-100KRGG3; infected with dl309 when indicated; and harvested at 24 h p.i. Equal amounts of lysates were subjected to immunoprecipitation with α-myc (A) or α-methyl arginine (B) antibodies, and samples were resolved by using an SDS-Tris-glycine 4 to 20% gradient gel and detected by immunoblotting with specific antibodies as shown.

FIG. 4.
FIG. 4.

Methylated 100K is exclusively nuclear and RGG mutants are defective for nuclear accumulation. (A) U2OS cells were transfected with myc-100K or myc-100KΔC; infected with dl309; and harvested at 18 and 36 h p.i. in a hypotonic lysis buffer. Lysates were then fractionated into nuclear and cytoplasmic fractions, and equal amounts were resolved by using an SDS-Tris-glycine 4 to 20% gradient gel and detected by immunoblotting with specific antibodies as indicated. N, nuclear fraction; C, cytoplasmic fraction; α-methyl-R, α-methyl arginine antibody. (B) Ionic currents detected in MS mode for selected ions at their maximum elution times. The sequences of the ions surveyed are EAAAAAATHGR*GGILGQSGR (ion at m/z = 467.0+4, containing monomethylated Arg727), EAAAAAATHGR**GGILGQSGR (ion at m/z = 470.5+4, dimethylated Arg727), GGFGR*GGGGHDGR (ion at m/z = 400.9+3, monomethylated Arg741), GGFGR**GGGGHDGR (ion at m/z = 405.5+3, dimethylated Arg741), and EAAAAAATHGR**GGILGQSGR* (ion at m/z = 474.0+4, simultaneously containing dimethylated R727 and monomethylated R736). R*, methylated arginine; R**, dimethylated arginine. The intensities of the different ions corresponding to species mono- or dimethylated at the same residue have been grouped and presented (as averaged values) in the graph. ColumnS: R727, ions at m/z = 467.0+4 and 470.5+4; R741, ions 400.9+3 and 405.5+3; R736R727, ion 474.0+4. M, monomethylated; DiM, dimethylated. Normalized ion counts are an average of the maximum intensity for every ion normalized against the maximum intensities of the reference peaks, as described in Materials and Methods. (C) Methylation inhibitors prevent 100K nuclear accumulation. Cells were infected with dl309, treated with 3 mM MTA at 12 h p.i., fixed with 4% PFA at 36 h p.i., and stained with a monoclonal antibody against 100K.

FIG. 5.
FIG. 5.

(A) U2OS:100KFL and U2OS:100KΔC cells were grown on glass chambers, fixed with 4% PFA, stained with an antibody against the myc tag and a fluorescently labeled secondary antibody, and visualized by confocal microscopy. In U2OS:100KFL cells, 100K is exclusively nuclear. However, the lack of the RGG domain in U2OS:100KΔC cells results in the cytoplasmic accumulation of 100K. (B) U2OS:vector, U2OS:100KFL, and U2OS:100KΔC cells were plated in the presence or absence of methylation inhibitor, labeled with S35 for 2 h, and harvested, and total lysates were run on a 4 to 20% gel, transferred to a polyvinylidene difluoride membrane, and visualized on PhosphorImager. Total protein synthesis was measured by using the ImageQuant software, as previously described (31), and the absolute value for each cell line is shown here (the results are the average value from two independent experiments).

FIG. 6.
FIG. 6.

U2OS:vector, U2OS:100KFL, and U2OS:100KΔC cells were infected with wild type or with an 100K ts mutant adenovirus at the nonpermissive temperature (39.4°C). At 36 h p.i., cells were labeled with S35 and harvested, and lysates were run on a 4 to 20% Tris-glycine gel as described in Materials and Methods. Proteins were transferred onto a polyvinylidene difluoride membrane, and the total labeled protein was visualized by using PhosphorImager. The total protein synthesis was measured by using ImageQuant and is depicted on this graph as a percentage relative to the protein synthesis in uninfected cells. The results are the average values from two independent experiments.

FIG. 7.
FIG. 7.

(A) Expression of late viral protein production is severely defective in MTA-treated cells. U2OS cells were infected with dl309 and treated with various concentrations of MTA at 12 h p.i.; lysates were normalized and resolved by using an SDS-Tris-glycine 4 to 20% gradient gel at 36 h p.i. and detected by immunoblot analysis using monoclonal antibodies against 100k, hexon, and fiber. Methyl-100K was detected by using an α-methyl arginine antibody. (B) U2OS cells were infected with dl309 in the presence or absence of the methylation inhibitor (MTA, added 12 h p.i.) and visualized under a light microscope at 48 h p.i. As a control, uninfected U2OS cells treated with the inhibitor were also included.

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