Protein arginine methylation during lytic adenovirus infection

Abstract

Arginine methylation of proteins affects major processes in the cell, including transcriptional regulation, mRNA metabolism, signal transduction and protein sorting. Arginine methylation of Ad (adenovirus) E1B 55-kDa-associated protein E1B-AP5 was recently described by us [Kzhyshkowska, Schutt, Liss, Kremmer, Stauber, Wolf and Dobner (2001) Biochem. J. 358, 305-314]. In this first example of protein arginine methylation analysis in Ad-infected cells, we investigated methylation of the E1B-AP5 and the viral L4-100 kDa protein. We demonstrate that E1B-AP5 methylation is enhanced during the course of infection in a cell-type-specific manner. We also show that L4-100 kDa is efficiently methylated in Ad-infected cells. L4-100 kDa formed complex with methyltransferase in vivo during productive infection, and can be methylated by HRMT1L2 (human protein arginine methyltransferase 1) in vitro. Comparative analysis of E1B-AP5 and L4-100 kDa protein methylation in Ad-infected HeLa, MCF-7 and H1299 cells revealed that the profile of protein arginine methylation correlates with the efficiency of Ad proteins production. Our results suggest that protein arginine methylation is an important host-cell function required for efficient Ad replication.

Figure 1. Experimental scheme for analysing protein arginine methylation in productive Ad-infected cells

At various time points after infection (22–10 h) and control, non-infected (0 h) cells were transferred to a methionine-deficient medium in the presence or absence of protein synthesis inhibitors (+NTM or −NTM) as described in the Experimental section. To monitor protein methylation, the methyl-group donor L-[methyl-³H]methionine was added to the NTM-containing medium. Under these conditions, incorporation of ³H into proteins can only occur through post-translational methylation. Inhibition of protein synthesis was confirmed by parallel cell labelling with [³⁵S]methionine in the presence of NTM. In contrast, total protein synthesis was monitored by labelling with [³⁵S]methionine in the absence of NTM. Samples were subsequently analysed by SDS/PAGE of total cell lysates or immunoprecipitates.

Figure 2. Protein methylation in H5wt300-infected HeLa, MCF-7 and H1299 cells

At the indicated time points after infection (hpi), cells were incubated with MEM-lacking methionine and labelled either with L-[methyl-³H]methionine in the presence of +NTM (A, C) or with [³⁵S]methionine but without −NTM (B). Inhibition of protein synthesis was confirmed by parallel cell labelling with [³⁵S]methionine +NTM (lanes C in A–C) and cells were harvested 22 h post-infection. (A) Methylation of E1B-AP5 during the time course of infection. E1B-AP5 was immunoprecipitated from total cell extracts using anti-E1B-AP5 mAb 4A11. Samples were analysed by SDS/PAGE followed by autoradiography. The signal was analysed after 7 days of exposure. (B) Total protein synthesis during the time course of infection. Proteins (20 μg of samples) from each time point were separated by SDS/PAGE followed by autoradiography (6 h exposure). (C) Total protein methylation during the time course of infection. Proteins (20 μg of samples) from each time point were separated by SDS/PAGE followed by autoradiography (3 days exposure). Arrows indicate E1B-AP5 120 kDa (A) and methylated L4-100 kDa proteins (C).

Figure 3. Protein steady-state level in HeLa, MCF-7 and H1299 cells infected with H5wt300

Aliquots of lysates (20 μg of samples) used in Figure 2 were separated by PAGE and analysed by Western blotting using the following antibodies: (A) (B6-8) mouse hybridoma supernatant recognizing Ad5 E2A 72 kDa to control the infection efficiency; (B) rat hybridoma supernatant 4A11 anti-E1B-AP5; (C) rat hybridoma supernatant L4-6B10 anti-L4-100 kDa.

Figure 4. Ad5 L4-100 kDa is methylated in H5wt300-infected cells

Cells were labelled with L-[methyl-³H]methionine or [³⁵S]methionine in the presence (+NTM) or absence (−NTM) of NTM as indicated. Total cell extracts were prepared from non-infected cells (0 h) and 16 h post-infection and subjected to IP using anti-L4-100 kDa mAb 2100K-1 (A) or anti-hexon mAb (B). The immunocomplexes were separated by SDS/PAGE and proteins were visualized by autoradiography.

Figure 5. Ad5 L4-100 kDa is methylated by hPRMT1/HRMT1L2 in vitro

HeLa cells, grown in the presence of the methylation inhibitor AdOx, were infected with H5wt300 at a multiplicity of 20 plaque-forming units/cell. Total cell extracts were prepared 16 h post-infection and subjected to IP using anti-L4-100 kDa mAb 2100K-1 (IP α-L4-100 kDa). To inactivate co-precipitated methyltransferase activity half of the precipitate was incubated at 70 °C for 6 min (lanes 3 and 4). GST-hPRMT1 (0.2 μg) was added to the reactions (lanes 2 and 4) and samples were analysed by SDS/PAGE followed by autoradiography. Lanes 5–8 show control reactions where two different fragments of E1B-AP5 fused to GST were used as substrates for GST-hPRMT1 (lanes 6 and 8). GST-AP5-RGG (lanes 7 and 8) contains the RGG domain of E1B-AP5 previously shown to be methylated by GST-hPRMT1 in vitro [24].

Figure 6. Ad5 L4-100 kDa protein synthesis (A) and methylation (B) during the time course of infection

H5wt300-infected HeLa and MCF-7 cells were labelled with L-[methyl-³H]methionine or [³⁵S]methionine in the presence (+NTM) or absence (−NTM) of NTM. At the indicated time points after infection (hpi), total cell extracts were prepared and subjected to IP using anti-L4-100 kDa mAb 2100K-1. The immunocomplexes were separated by SDS/PAGE and L4-100 kDa was visualized by autoradiography [6 h exposure for (A) and 2 days exposure for (B)].