Human cytomegalovirus UL84 protein contains two nuclear export signals and shuttles between the nucleus and the cytoplasm

Abstract

Previous studies defined pUL84 of human cytomegalovirus as an essential regulatory protein with nuclear localization that was proposed to act during initiation of viral-DNA synthesis. Recently, we demonstrated that a complex domain of 282 amino acids within pUL84 functions as a nonconventional nuclear localization signal. Sequence inspection of this domain revealed the presence of motifs with homology to leucine-rich nuclear export signals. Here, we report the identification of two functional, autonomous nuclear export signals and show that pUL84 acts as a CRM-1-dependent nucleocytoplasmic shuttling protein. This suggests an unexpected cytoplasmic role for this essential viral regulatory protein.

FIG. 1.

pUL69 contains two active leucine-rich NESs. (A) Schematic representation of the UL84 protein showing the NLS/importin alpha binding domain (20). Two putative leucine-rich NES motifs located within the nonconventional NLS/importin alpha binding domain of pUL84 are highlighted. (B) Alignment and comparison of the two putative pUL84 NES motifs with known leucine-rich NESs and with the consensus sequence for leucine-rich NESs. The indicated pUL84 motifs are compared with the NES of the protein kinase inhibitor α (PKI) (31) or the fragile X mental retardation protein (FMRP) (11). Additionally, the NES of the human immunodeficiency virus type 1 Rev protein (HIV1-REV) (8) and a derived consensus NES are listed. Conserved residues in the NESs are shown in boldface. The numbers refer to the positions of the amino acid sequences within each protein. (C) Schematic representation of the UL84 coding sequence showing the two putative NESs fused to the C terminus of GST to produce GST-UL84-NES1 and GST-UL84-NES2. (D) Procaryotic expression and purification of GST-UL84-NES1 and GST-UL84-NES2. Shown is a Coomassie blue-stained gel: extracts from E. coli cells grown without isopropyl-β-d-thiogalactopyranoside (IPTG) (lanes 1 and 4) and grown in the presence of IPTG (lanes 2 and 5) are shown; lanes 3 and 6, purified GST fusion proteins. (E) GST fusion proteins were microinjected into the nuclei (a to d) or the cytoplasm (e to h) of HeLa cells, together with rabbit IgG as a marker for the injection site. At 1 h after injection, the cells were fixed and immunostained for GST-UL84-NES1 or GST-UL84-NES2 and the coinjected IgG control.

FIG. 2.

Nucleocytoplasmic shuttling of pUL84 in infected and transfected cells. (A) Heterokaryons were generated by fusion of HCMV-infected primary HFF and murine NIH 3T3 cells. Prior to and following heterokaryon formation, de novo protein synthesis was inhibited using cycloheximide. At 3.5 h after fusion, the cells were fixed, and a double-immunofluorescence analysis was performed with a polyclonal antiserum directed against pUL84 (b) and a monoclonal antibody against the IE1 protein (c). Staining with Hoechst 33258 (a) was used to differentiate between human and murine nuclei within the heterokaryon. Murine nuclei display a characteristic punctate pattern, whereas human nuclei are diffusely stained with the reagent; murine nuclei are indicated by arrows. Panel d shows the phase-contrast image of the heterokaryons; the cytoplasmic edge is highlighted by a broken line. (B) HeLa cells were cotransfected with expression plasmids for pUL84 and one of the internal control plasmids, β-Gal-NLS/NES or β-Gal-NLS, as indicated (a to d, UL84 and β-Gal-NLS/NES; e to h, UL84 and β-Gal-NLS). The transfected cells were subsequently analyzed in heterokaryon assays as described in the legend to panel A. Double-immunofluorescence analysis with polyclonal anti-pUL84 serum and a monoclonal antibody against β-Gal was performed in order to detect the expressed proteins. (C) pUL84 shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. Expression constructs for pUL84 and pUL69 were cotransfected into HeLa cells, and the transfected cells were subjected to heterokaryon assays. Three hours prior to fusion and throughout the experiment, the cells were incubated in the absence (−) (a to d) or presence (+) (e to h) of LMB. The indicated proteins were detected by double-label immunofluorescence using a polyclonal pUL84 antiserum and a monoclonal antibody directed against pUL69 (19).

FIG. 3.

Subcellular localization of UL84 mutants carrying mutated nuclear export signals. (A) A series of pUL84 mutants carrying alanine replacement mutations either in NES1 or NES2 was generated, and the subcellular localization of the resulting mutants was analyzed via indirect immunofluorescence analysis. The mutants indicated on the left were transiently expressed in HeLa cells, which were subsequently fixed and immunostained (right) using an anti-pUL84 antiserum (αUL84); DAPI, DNA staining of the transfected HeLa cells. (B) Western blot analysis of expression levels of the indicated UL84 mutants in transfected HeLa cell cultures.

FIG. 4.

Nucleocytoplasmic shuttling activities of pUL84 mutants carrying alanine substitutions within NES1, NES2, or NES1 and NES2. (Left) Schematic representation of the respective pUL84 mutants and β-Gal-NLS/NES, which was used as an internal shuttling control in the interspecies heterokaryon analysis. (Right) Heterokaryon experiments (as described in the legend to Fig. 2) were performed to visualize the nuclear-export activities of the indicated proteins.