The autoregulatory and transactivating functions of the human cytomegalovirus IE86 protein use independent mechanisms for promoter binding

Abstract

The functions of the human cytomegalovirus (HCMV) IE86 protein are paradoxical, as it can both activate and repress viral gene expression through interaction with the promoter region. Although the mechanism for these functions is not clearly defined, it appears that a combination of direct DNA binding and protein-protein interactions is involved. Multiple sequence alignment of several HCMV IE86 homologs reveals that the amino acids (534)LPIYE(538) are conserved between all primate and nonprimate CMVs. In the context of a bacterial artificial chromosome (BAC), mutation of both P535 and Y537 to alanines (P535A/Y537A) results in a nonviable BAC. The defective HCMV BAC does not undergo DNA replication, although the P535A/Y537A mutant IE86 protein appears to be stably expressed. The P535A/Y537A mutant IE86 protein is able to negatively autoregulate transcription from the major immediate-early (MIE) promoter and was recruited to the MIE promoter in a chromatin immunoprecipitation (ChIP) assay. However, the P535A/Y537A mutant IE86 protein was unable to transactivate early viral genes and was not recruited to the early viral UL4 and UL112 promoters in a ChIP assay. From these data, we conclude that the transactivation and repressive functions of the HCMV IE86 protein can be separated and must occur through independent mechanisms.

FIG. 1.

Amino acids LPIYE/D are conserved in CMV IE86 protein homologs. Amino acid sequences for the proteins from human CMV, Towne strain, IE2 (AAR31449), chimpanzee CMV IE2 (NP612745), rhesus CMV IE2 (AAB00488), African green monkey (AGM) CMV IE2 (AAB16881), mouse CMV IE3 (AAA74505), and rat CMV IE2 (AAB92266) were aligned using MultAlin. Multiple sequence alignments are displayed using BoxShade. Identical residues appear shaded in black, while similar residues appear shaded in gray. In the consensus sequence, an asterisk indicates a residue that is identical in all aligned sequences, while a dot indicates a residue that is identical in at least half of the aligned sequences. The numbers appearing between the species and the amino acid sequence represent the amino acid position for that particular species. A hyphen designates a gap in the sequence that was inserted for optimal alignment.

FIG. 2.

Construction and confirmation of recombinant BACs. (A) Recombinant BACs were constructed by homologous recombination of a NheI-linearized DNA fragment with Towne BAC, kindly provided by F. Liu, University of California, Berkeley. UL121 and UL128 served as flanking regions to introduce targeted mutations into exon 5 of IE2. The UL127 locus was replaced by the CAT reporter, as described previously (34, 38). A kanamycin resistance (Kan^r) cassette was inserted between UL127 and UL128 for selection of recombinant BACs. (B) The integrity of recombinant BACs was verified by digesting BAC DNA with the HindIII restriction enzyme. The presence of Kan^r in IE86 mutants or the presence of gentamicin resistance (Gen^r) in IE86 revertants (Rev) is indicated. (C) Exon 5 of IE2 was amplified from the recombinant BACs by PCR and digested with the indicated restriction enzymes. Successful recombination of the P535A/Y537A mutation introduced a new MscI restriction site, while the H446A/H452A and L534A mutations introduced a new KasI restriction site, compared to WT.

FIG. 3.

Recombinant BACs containing the P535A/Y537A and H446A/H452A IE86 mutations fail to replicate. HFF cells were transfected, in triplicate, with a viral pp71 expression vector and Towne (WT IE86), L534A, P535A/Y537A, Revertant (Rev)-PY, or H446A/H452A BAC DNA. Medium was changed on transfected cells every 4 days. Total DNA was harvested at 1, 5, 9, and 13 days posttransfection. Real-time PCR with primer/probe sets to detect HCMV gB DNA and cellular 18S rRNA genes was performed. BAC DNA replication was measured based on the amount of HCMV gB DNA, normalized to cellular 18S rRNA genes, relative to Towne-transfected cells at 1 day posttransfection. Mock samples contained pp71 but not BAC DNA.

FIG. 4.

P535A/Y537A mutant IE86 protein is able to negatively autoregulate expression from the MIE promoter. (A) HFF cells were transfected, in triplicate, with a viral pp71 expression vector and WT, P535A/Y537A, or H446A/H452A recombinant BAC DNA. Total RNA was harvested at 48 h posttransfection and converted to cDNA by reverse transcription. Real-time PCR was performed with primer/probe sets to detect HCMV MIE cDNA and cellular 18S complementary rRNA genes. MIE RNA transcription was measured based on the amount of HCMV MIE cDNA, normalized to cellular 18S complementary rRNA genes, relative to WT. Mock samples contained pp71 but not BAC DNA. (B) HFF cells were transfected with a viral pp71 expression vector and WT, L534A, P535A/Y537A (PY), revertant-PY (Rev), or H446A/H452A (HH) recombinant BAC DNA. Total protein was harvested at 72 h posttransfection. Western blot analysis was performed by cutting the membrane to separate IE86 from IE72, using antibodies to detect viral MIE proteins and cellular β-tubulin. Mock samples contained pp71 but not BAC DNA.

FIG. 5.

P535A/Y537A mutant IE86 protein is unable to transactivate early viral genes. (A) HFF cells were transfected, in triplicate, with β-Gal expression vector DNA and shuttle vector DNA containing WT, L534A, P535A/Y537A, revertant (Rev)-PY, or H446A/H452A IE2 and the modified UL127 early viral promoter driving the CAT reporter. Total protein was harvested at 72 h posttransfection. A CAT assay was performed to determine the ability of the IE86 protein to transactivate the early viral promoter, and a β-Gal assay was performed to determine transfection efficiency. Results are reported as CAT activity (percent acetylation per microgram protein, normalized to β-Gal). Mock samples contained β-Gal and the UL127-CAT reporter but not IE86. (B) HFF cells were transfected, in triplicate, with a viral pp71 expression vector and recombinant BACs encoding WT, P535A/Y537A, or H446A/H452A IE86. Total DNA was harvested at 24 h posttransfection; total RNA was harvested at 48 h (IE) and 72 h (early) posttransfection and converted to cDNA by reverse transcription. Real-time PCR was performed with primer/probe sets to detect HCMV gB DNA, HCMV MIE and TRS1 IE cDNA, HCMV UL44, UL54, and IRL7 early cDNA, and cellular 18S complementary rRNA genes. HCMV IE and early RNA transcription was measured based on the amount of HCMV cDNA, normalized to cellular 18S complementary rRNA genes and gB DNA input, relative to WT. Mock samples contained pp71 but not BAC DNA.

FIG. 6.

P535A/Y537A mutant IE86 protein represses and is recruited to the MIE promoter in 293 and HFF cells. (A) 293 cells were transfected, in triplicate, with a β-Gal expression vector, a reporter construct consisting of the CAT gene under the control of the MIE promoter, including the autoregulatory MIE crs, and shuttle vector DNA encoding WT, L534A, P535A/Y537A, revertant (Rev)-PY, or H446A/H452A IE86. Total protein was harvested at 48 h posttransfection. A CAT assay was performed to determine the ability of the IE86 protein to repress the MIE promoter, and a β-Gal assay was performed to determine transfection efficiency. Results are reported as MIE-CAT activity (percent acetylation per microgram protein, normalized to β-Gal) relative to WT. Mock samples contained β-Gal and the MIE-CAT reporter but not IE86. (B) Western blot of ChIP lysates to detect equal expression of viral proteins (IE72) and to demonstrate the ability of the IP antibody to interact with the mutant proteins (IE86). An antibody specific to exon 4 (p63-27) demonstrates equal expression of IE72 in WT, L534A, P535A/Y537A (PY), and Rev-PY mutants, while the H446A/H452A (HH) mutant expresses higher levels of IE72 since it is unable to autoregulate the MIE promoter. An antibody specific to exon 5 (polyclonal 6655), which was used for IP, is able to detect phosphorylated (IE86p) and unphosphorylated IE86 in all mutants, in addition to a nonspecific (ns) protein that also appears in untransfected cells (mock). (C) HFF (shown) and 293 cells (not shown) were transfected with a plasmid containing the entire MIE locus, including the MIE crs and the WT or mutant IE2 gene. Cells were cross-linked with formaldehyde at 48 h posttransfection and ly- sed, and DNA/protein complexes were isolated. A ChIP assay was performed using polyclonal antibodies to immunoprecipitate (IP) HCMV IE86 or cellular TBP, or normal rabbit serum as a negative IP control, and nested PCR was performed to amplify the MIE promoter containing the MIE crs and TATA box. Purified PCR products were separated on an agarose gel. A positive (+) PCR control was performed using the crs-containing plasmid directly for nested PCR, while a negative (−) PCR control was performed without DNA template. The efficiency of the IP was compared using 10% of the input DNA from transfected cells, isolated directly from the lysed cells and not immunoprecipitated, for nested PCR. The specificity of the IP was compared using WT IE86-transfected cells, but immunoprecipitated using normal rabbit serum, for nested PCR.

FIG. 7.

P535A/Y537A mutant IE86 protein fails to transactivate and is not recruited to early viral promoters in 293 and HFF cells. (A) 293 cells were transfected, in duplicate, with a reporter construct consisting of the CAT gene under the control of the early UL4 or UL112 promoters and shuttle vector DNA encoding WT, P535A/Y537A, or H446A/H452A IE86. Total protein was harvested at 48 h posttransfection, and a CAT assay was performed to determine the ability of the IE86 protein to transactivate the UL4 and UL112 promoters. Results are reported as CAT activity (percent acetylation per microgram protein) relative to mock. Mock samples contained the UL4-CAT or UL112-CAT reporter, but not IE86. The positive control (Pos) consisted of HFF cells infected with recombinant HCMV containing a UL127-CAT reporter. The negative control (Neg) consisted of mock-infected HFF cells. (B) HFF (shown) and 293 cells (not shown) were transfected with a plasmid containing the UL4 promoter, and (C) HFF (not shown) and 293 (shown) cells were transfected with a plasmid containing the UL112 promoter. The cells were also transfected with a plasmid containing the WT or mutant IE2 gene. Cells were cross-linked with formaldehyde at 48 h posttransfection and lysed, and DNA/protein complexes were isolated. A ChIP assay was performed using polyclonal antibodies to immunoprecipitate (IP) HCMV IE86 or cellular TBP, or normal rabbit serum as a negative IP control, and nested PCR was performed to amplify the UL4 (B) or UL112 (C) promoter. Purified PCR products were separated on an agarose gel. A positive (+) PCR control was performed using the promoter-containing plasmid directly for nested PCR, while a negative (−) PCR control was performed without DNA template. The efficiency of the IP was compared using 10% of the input DNA from transfected cells, isolated directly from the lysed cells and not immunoprecipitated, for nested PCR. The specificity of the IP was compared using WT IE86-transfected cells, but immunoprecipitated using normal rabbit serum, for nested PCR.