Inhibition of cellular DNA synthesis by the human cytomegalovirus IE86 protein is necessary for efficient virus replication

Abstract

Human cytomegalovirus (HCMV) expresses several proteins that manipulate normal cellular functions, including cellular transcription, apoptosis, immune response, and cell cycle control. The IE2 gene, which is expressed from the HCMV major immediate-early (MIE) promoter, encodes the IE86 protein. IE86 is a multifunctional protein that is essential for viral replication. The functions of IE86 include transactivation of cellular and viral early genes, negative autoregulation of the MIE promoter, induction of cell cycle progression from G0/G1 to G1/S, and arresting cell cycle progression at the G1/S transition in p53-positive human foreskin fibroblast (HFF) cells. Mutations were introduced into the IE2 gene in the context of the viral genome using bacterial artificial chromosomes (BACs). From these HCMV BACs, a recombinant virus (RV) with a single amino acid substitution in the IE86 protein was isolated that replicates slower and to lower titers than wild-type HCMV. HFF cells infected with the Q548R RV undergo cellular DNA synthesis and do not arrest at any point in the cell cycle. The Q548R RV is able to negatively autoregulate the MIE promoter, transactivate viral early genes, activate cellular E2F-responsive genes, and produce infectious virus. This is the first report of a viable recombinant HCMV that is unable to inhibit cellular DNA synthesis in infected HFF cells.

FIG. 1.

Multiple sequence alignment of the 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, a star indicates a residue that is identical in all aligned sequences, while a dot indicates a residue that is conserved 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. Lui, 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 (27, 30). 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. (C) Exon 5 of IE2 was amplified from the recombinant BACs by PCR and digested with the indicated restriction enzyme. Successful recombination of the Q548A or Q548R mutation introduced a new KasI or EagI restriction site, respectively, compared to WT.

FIG. 3.

Replication of recombinant viruses containing IE86 mutations. (A) HFF cells were transfected, in triplicate, with Towne, Q548A, Q548R, or Rev Q548R BAC DNA. Medium was changed on transfected cells every 4 days, and Q548R BAC-transfected cells were split 1:2 at day 14 to promote replication. Total DNA was harvested at 1, 5, 9, 13, 21, and 28 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. (B) HFF cells were infected, in triplicate, with equal DNA input of Towne, Q548A, or Q548R RV. Cells and supernatant were harvested at 1, 5, and 10 days postinfection, and triplicate samples were pooled and stored. A plaque assay was performed, in triplicate, with serial dilutions of stored virus, and plaques were counted at 10 to 14 days postinfection for Towne and Q548A RV-infected cells or 14 to 21 days postinfection for Q548R RV-infected cells.

FIG. 4.

Q548R mutant IE86 protein is able to negatively autoregulate expression from the MIE promoter. (A) HFF cells were transfected, in triplicate, with WT, H446A/H452A, or Q548R 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. (B) HFF cells were transfected with WT, H446A/H452A, or Q548R recombinant BAC DNA. Total protein was harvested at 24 h posttransfection. Western blot analysis was performed using antibodies to detect viral MIE proteins and cellular β-tubulin. In both figures, the H446A/H452A BAC contains mutations to the putative zinc finger of IE86, which is known to be defective in autoregulation of the MIE promoter (data not shown).

FIG. 5.

Q548R mutant IE86 protein is able to transactivate viral early genes. (A) 293 cells were transfected, in triplicate, with β-Gal expression vector DNA and shuttle vector DNA containing WT, Q548A, Q548R, or Q548 Rev IE2 and the UL127 early viral promoter driving the CAT reporter. Total protein was harvested at 4 days posttransfection. A CAT assay was performed to determine the ability of the IE86 protein to transactivate the viral early promoter, and a β-Gal assay was performed to determine transfection efficiency. Results are reported as CAT activity (percent acetylation) per microgram of protein. (B) HFF cells were infected, in triplicate, with equal DNA input of Towne, Q548A, or Q548R RV at an MOI equivalent to approximately 0.001. Total DNA was harvested at 4 hpi; total RNA was harvested at 18 hpi 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 Towne.

FIG. 6.

Q548R mutant IE86 protein is unable to inhibit cellular DNA synthesis in infected HFF cells. (A) Serum-starved, contact-inhibited HFF cells were synchronized in G₀ for 48 h. Cells were infected for 6 h in low-serum medium with Towne, Q548A, or Q548R RV at an MOI of approximately 0.9. Cells were then split onto glass coverslips for release from contact inhibition and grown in high-serum medium, to promote cell cycle progression, plus BrdU, to detect DNA synthesis, for 42 h. Cells were fixed and permeabilized in methanol and acetone (1:1), stained with anti-IE86 and anti-BrdU antibodies, counterstained with DAPI, and imaged by fluorescent microscopy. (B) Ten fields, with at least 30 cells per field, from the above experiment were counted to determine the percentage of uninfected and infected BrdU-positive cells for each sample.

FIG. 7.

Q548R mutant IE86 protein is unable to arrest cell cycle progression in infected HFF cells. (A) Serum-starved, nonconfluent HFF cells were synchronized in G₀ for 48 h. Cells were infected for 6 h in low-serum medium with Towne, Q548A, or Q548R RV at an MOI of approximately 0.9. Cells were then grown in high-serum medium to promote cell cycle progression for 42 h. Cells were permeabilized in 70% methanol, stained with anti-IE86 antibody, counterstained with DAPI, and subjected to FACS analysis. IE86-positive infected cells were sorted and counted based on DNA content. (B) Cell cycle profiles from the above experiment were analyzed using ModFit software to determine the percentage of infected cells in each phase of the cell cycle for each sample.

FIG. 8.

Q548R mutant IE86 protein is able to upregulate E2F-responsive genes in infected HFF cells. Confluent, contact-inhibited HFF cells were infected, in triplicate, with equivalent CPE input of Towne, Q548A, or Q548R RV, equivalent to an MOI of approximately 0.3. Cytoplasmic RNA was harvested at 48 hpi and separated on an agarose-formaldehyde-MOPS gel. RNA was transferred to a Nytran membrane, and Northern blot analysis was performed using ³²P-labeled DNA probes for the E2F-responsive genes Cdk2, MCM3, RR1, and TS and the cellular gene actin.