Human cytomegalovirus IE2 86 and IE2 40 proteins differentially regulate UL84 protein expression posttranscriptionally in the absence of other viral gene products

Abstract

It has previously been demonstrated that, during human cytomegalovirus infection, the viral IE2 86 and IE2 40 proteins are both important for the expression of an early-late viral protein, UL84. Here, we show that expression of the UL84 protein is enhanced upon cotransfection with either IE2 86 or IE2 40, although IE2 40 appears to play a more important role. The UL84 protein levels are tightly linked to the amount of IE2 40 present, but this does not appear to be true for IE2 86. RNA remains constant for all corresponding proteins, indicating posttranscriptional regulation of UL84. The first 105 amino acids of UL84 are necessary and sufficient for this phenotype, and this region is also required for an interaction with IE2 86 and IE2 40. Treatment with proteasome inhibitors shows that UL84 exhibits some proteasome-dependent degradation, and UL84 is not protected against this degradation when coexpressed with IE2 86 or IE2 40. UL84 also exhibits an inhibitory effect on IE2 86 and IE2 40 protein levels in these cotransfection assays. Further, we show that the amino acid sequence of UL84 is important for the enhancement governed by IE2 40. These results indicate that IE2 86, IE2 40, and UL84 serve to regulate protein expression in a posttranscriptional fashion and that this regulation is independent of other viral proteins.

FIG. 1.

UL84 and IE2 protein, RNA, and DNA expression in transfected 293FT cells. (A) 293FT cells were transfected with either the IE2 86 (86), IE2 40 (40), or UL84 (84) plasmids alone or in combination. Forty-eight hours later, cells were harvested, and UL84, IE2 86, and IE2 40 protein levels were assayed by Western blotting by loading equal amounts of protein per lane. Actin serves as a loading control. (B) Transfected cells with the same constructs as for panel A were assayed for DNA levels by quantitative real-time PCR. IE2 DNAs were assayed using a primer and probe directed against the 3′ end of the IE2 86 gene, thus recognizing both forms of the IE2 constructs. UL84 DNA levels were also assayed alone or in combination with the IE2 constructs using primers and a probe that recognize the N terminus of UL84. Samples were tested in duplicate in each experiment, and the graphs shown are averages of 3 experiments. DNA for each sample was normalized to the cellular GAPDH promoter as a control for the amount of input DNA in each reaction. Graphs are represented as relative amplification (Amp), with the value of the first sample in each graph set to 1. (C) RNA expression for each of the samples was measured by quantitative real-time RT-PCR. Each of the samples was measured using the same primer and probes as for panel B and are normalized to the housekeeping G6PD gene. Graphs represent at least 2 experiments.

FIG. 2.

Titration of IE2 40 DNA affects UL84 protein expression. (A) Decreasing amounts of IE2 40 (40) DNA were transfected (0.8, 0.4, 0.2, and 0.05 μg), while a constant amount of UL84 (84) DNA was transfected into cells. The levels of transfected DNA were analyzed by quantitative real-time PCR and normalized to the GAPDH promoter as a loading control. Values are shown as relative amplification (Amp), with the lowest value set to 1. Both IE2 and UL84 DNAs were assessed. (B) IE2 40 and UL84 RNAs from the same samples as in panel A were measured by quantitative real-time RT-PCR and normalized to the cellular housekeeping G6PD gene. (C) UL84 and IE2 40 protein expression was analyzed from the same samples as in panels A and B by Western blotting (left). UL84 expression was also measured with an antibody that recognizes the C-terminal tag HA to confirm UL84 antibody results (left). UL84 expression was assessed after cotransfection with 20-fold-less IE2 86 plasmid [right; 84/86 (0.05)]. Actin serves as a loading control.

FIG. 3.

The N-terminal 105 aa of UL84 are important for the enhancement of protein expression governed by the IE2 proteins. (A) Deletions throughout the N terminus of UL84 were created by PCR mutagenesis and included the first 68 (Δ68), 105 (Δ105), 135 (Δ135), and 200 (Δ200) aa. The schematic is not to scale. (B) Protein expression of UL84 (wt and mutants), IE2 86, and IE2 40 was measured using Western blot analyses. Actin serves as a loading control. Mutants are represented by the corresponding amino acid deletions (68, 105, 135, and 200). (C) RNA for each sample was analyzed as for Fig. 1B. Only the Δ68 and Δ105 mutants are shown. Values are shown as relative amplification (Amp) and are relative to the 84/86 sample, with a value set to 1.

FIG. 4.

The first 105 aa of UL84 are important for interaction with IE2 86 and IE2 40. wt UL84 and each of the N-terminal mutants, Δ68 (68) and Δ105 (105), were assayed for their interaction with both IE2 86 and IE2 40 using immunoprecipitation assays as described in Materials and Methods. Expression of each of the proteins was analyzed before immunoprecipitation (Pre) in order to assess the relative expression levels. Following immunoprecipitation, the complexes were eluted from the agarose beads and subjected to Western blot analysis. Results are shown in the immunoprecipitation (IP) lanes. Antibodies to UL84 (wt and mutants), IE2 86, and IE2 40 were used in the analysis. The Pre samples are 10% of the sample used for IP.

FIG. 5.

Proteasome inhibitor treatment affects the level of UL84 expression, but coexpression with the IE2 proteins does not protect UL84 from proteasomal degradation. Thirty-six hours p.t., cells were incubated with a proteasome inhibitor (MG132; 10 μM; +) or mock treated with DMSO (−) and then assayed for protein expression 12 h later. UL84 expression was assayed by Western blotting with or without IE2 86 or IE2 40. Actin serves as a loading control.

FIG. 6.

The first 105 aa of UL84 are sufficient for enhancement of expression governed by the IE2 proteins but not for susceptibility to proteasomal degradation. (A) The first 105 aa were added to the amino terminus of another viral protein, UL44 (Hyb). Coexpression of this protein with IE2 86 and IE2 40 was assessed, as was the expression of UL44. The expression of the Hyb protein was assayed using an antibody directed against UL84 and UL44. (B) Proteasome treatment was conducted as in Fig. 5, except that amount of Hyb protein was analyzed in combination with IE2 86 (86) and IE2 40 (40). Cells were either treated with the proteasome inhibitor MG132 (+) or mock treated with DMSO (−). UL84 and UL44 protein expression was assayed as a control. Actin serves as a loading control in both panels A and B. (C) RNA was analyzed for IE2 86 (86), IE2 40 (40), UL44 (44), and Hyb samples. All samples were normalized to G6PD. Values are shown as relative amplification (Amp), with the first sample in the graph having a value set to 1.

FIG. 7.

Hyb RNA is expressed to the same level as UL84 RNA but to a significantly lower level than UL44 RNA. RNA was prepared from the same samples as in Fig. 6A and analyzed by quantitative real-time RT-PCR. Primers and probes directed near the 5′ end of the UL84 gene were used to compare the amounts of RNA in the Hyb and UL84 samples (UL84 RNA). Primers and probes to UL44 were used to compare the expression levels of the Hyb RNA and UL44 RNA (UL44 RNA). Values are shown as relative amplification (Amp), with the lowest value set to 1.

FIG. 8.

The UL84 amino acid sequence is important for regulation governed by IE2 86 and IE2 40 and for the interaction of UL84 with the IE2 proteins. (A) Schematic representation of the mutations (crosses) made in the RNA to shift the amino acid sequence to that encoded by the second and third open reading frames (ORFs). The original AUG is depicted and is the site of the first mutation, and all subsequent mutations are shown. The resulting RNA has four mutations, while the resulting protein is the product of a new ORF for the first 175 aa and is the product of the original ORF for the remainder of the protein. The resulting amino acid sequence is shown, with the new amino acid sequence indicated in boldface italics. (B) The RNA for each of the samples was measured by quantitative real-time RT-PCR. Analyses were conducted as for previous figures, and each of the relative RNA values is shown. Values are shown as relative amplification (Amp), with the sample 84/86 value set to 1. (C) The frameshift mutant (FS) was assayed by Western blotting in combination with IE2 86 and IE2 40. Actin serves as a loading control. (D) Interactions between each of the proteins assessed in panels B and C were measured using immunoprecipitation assays. Antibody specific for IE2 86 and IE2 40 was coupled to agarose beads, and then transfected samples were subjected to immunoprecipitation with the IE2-coupled beads. The amount of protein present before the immunoprecipitation is shown (Pre), followed by the amount of protein that was pulled down in the immunoprecipitation (IP).