Nuclear export of the human cytomegalovirus tegument protein pp65 requires cyclin-dependent kinase activity and the Crm1 exporter

Abstract

We have previously shown that treatment of human cytomegalovirus-infected cells with the cyclin-dependent kinase (cdk) inhibitor roscovitine has significant effects on several stages of the virus life cycle depending on the time of addition (V. Sanchez, A. K. McElroy, J. Yen, S. Tamrakar, C. L. Clark, R. A. Schwartz, and D. H. Spector, J. Virol. 78:11219-11232, 2004; V. Sanchez and D. Spector, J. Virol. 80:5886-5896, 2006). In this report, we add to these findings by demonstrating alterations in the phosphorylation and localization of pp65 (UL83) in cells treated with roscovitine. We observed that inhibition of cdk activity causes the retention of pp65 within the nucleus at late times postinfection. At the same time, we observed a change in the phosphorylation pattern of the protein. Interestingly, mutation of potential cdk phosphorylation sites did not affect the ability of pp65 to localize to the nucleus or to relocalize to the cytoplasm late in infection. However, we found that the cytoplasmic accumulation of pp65 late in infection was sensitive to the Crm1 inhibitor leptomycin B.

FIG. 1.

Treatment of HCMV-infected fibroblasts with roscovitine results in nuclear retention of pp65 at late times during infection. Cells were treated with 20 μM roscovitine from 24 or 48 h p.i. and were fixed at 96 h p.i. For experiments testing the reversibility of the drug, roscovitine was added at 24 h p.i. and removed at 48 h p.i. as described in Materials and Methods. Cells were stained with monoclonal antibody (MAB) 28-19 (A) or 65-8 (B) against pp65. The nuclei were counterstained with Hoechst stain (blue), and the anti-pp65 antibody was detected with a fluorescein isothiocyanate-conjugated secondary antibody (green). Original magnification, ×400.

FIG. 2.

The phosphorylation pattern of pp65 changes upon treatment of cells with roscovitine from 24 h p.i. Infected cells were labeled with ³²P_i beginning at 66 h p.i. in the presence or absence of the drug. The pp65 from labeled lysates was immunoprecipitated with monoclonal antibody 28-19. The protein was isolated from SDS-polyacrylamide gels and subjected to digestion and separation as described in Materials and Methods. (A) Peptides separated at a high pH; (B) peptides separated at a low pH. Arrows indicate peptides whose intensities decreased, and stars indicate peptides whose intensities increased, in cells treated with roscovitine.

FIG. 3.

Mutation of putative cdk sites in EGFP-pp65 does not alter relocalization to the cytoplasm late in infection. (A) Sequences of putative cdk sites in pp65. aa, amino acid. (B) Human fibroblasts were transfected with plasmids encoding wild-type (wt) or mutant (T66A or T555A) pp65 proteins fused to EGFP. Cells were either infected with HCMV Towne at a high MOI or mock infected. Cells were fixed at 90 h p.i. Arrowheads indicate cells displaying cytoplasmic pp65 localization. Original magnification, ×400.

FIG. 4.

Mutation of putative cdk sites in pp65 does not alter relocalization to the cytoplasm in cells infected with the pp65-null virus RV. Human fibroblasts were transfected with plasmids encoding wild-type (WT) or mutant (T66A or T555A) pp65 proteins. Cells were either infected with RV at a high MOI or mock infected. Cells were fixed at 96 h p.i. and stained with monoclonal antibody 65-8 directed against pp65. The nuclei were counterstained with Hoechst stain (blue), and the anti-pp65 antibody was detected with a fluorescein isothiocyanate-conjugated secondary antibody. Original magnification, ×400.

FIG. 5.

The cytoplasmic accumulation of pp65 late in infection is reversed by LMB. HCMV-infected cells were treated with LMB or methanol (MeOH) in the presence of cycloheximide (CHX) beginning at 72 h p.i. Cells were fixed at 73 or 75 h p.i. and stained with monoclonal antibody 65-8. The nuclei were counterstained with Hoechst stain (blue), and the anti-pp65 antibody was detected with a fluorescein isothiocyanate-conjugated secondary antibody. Original magnification, ×400.

FIG. 6.

Late addition of roscovitine to infected cells causes the nuclear accumulation of pp65. Roscovitine (20 μM) was added at 72 h p.i., a time when pp65 is predominantly localized in the cytoplasm. Cells were fixed at 96 h p.i., and coverslips were stained with monoclonal antibody 28-19. The nuclei were counterstained with Hoechst stain (blue), and the anti-pp65 antibody was detected with a fluorescein isothiocyanate-conjugated secondary antibody. Original magnification, ×400.