Human cytomegalovirus UL84 insertion mutant defective for viral DNA synthesis and growth

Abstract

Human cytomegalovirus (HCMV) UL84 is required for oriLyt-dependent DNA replication, and evidence from transient transfection assays suggests that UL84 directly participates in DNA synthesis. In addition, because of its apparent interaction with IE2, UL84 is implicated as a possible regulatory protein. To address the role of UL84 in the context of the viral genome, we generated a recombinant HCMV bacterial artificial chromosome (BAC) construct that did not express the UL84 gene product. This construct, BAC-IN84/Ep, displayed a null phenotype in that it failed to produce infectious virus after transfection into human fibroblast cells, whereas a revertant virus readily produced viral plaques and, subsequently, infectious virus. Real-time quantitative PCR showed that BAC-IN84/Ep was defective for DNA synthesis in that no increase in the accumulation of viral DNA was observed in transfected cells. We were unable to complement BAC-IN84/Ep in trans; however, oriLyt-dependent DNA replication was observed by the cotransfection of UL84 and BAC-IN84/Ep. An analysis of viral mRNA by real-time PCR indicated that, even in the absence of DNA synthesis, all representative kinetic classes of genes were expressed in cells transfected with BAC-IN84/Ep. The detection of UL44 and IE2 by immunofluorescence in BAC-IN84/Ep-transfected cells showed that these proteins failed to partition into replication compartments, indicating that UL84 expression is essential for the formation of these proteins into replication centers within the context of the viral genome. These results show that UL84 provides an essential DNA replication function and influences the subcellular localization of other viral proteins.

FIG. 1.

Generation of BAC-IN84/Ep. (A) Schematic of wt HCMV BAC pHB5 with the genomic region encoding UL84, the mutant BAC-IN84/Ep, showing the location of the insertion of an EGFP-puromycin expression cassette, and the revertant BAC_84R. The predicted sizes from cleavages with SalI are shown. Also shown are the positions of hybridization probes used for Southern blots. (B) Southern blot of wt HCMV BAC pHB5 and mutant BAC-IN84/Ep. (Left) BAC clones cleaved with SalI and hybridized with probe 1; (Right) BAC clones cleaved with SalI and hybridized with probe 2. (C) Generation of UL84 revertant BAC (BAC_84R). The Southern blot shows the mutant BAC-IN84/Ep and revertant BAC_84R cleaved with SalI and hybridized with probe 2.

FIG. 2.

BAC-IN84/Ep does not produce viral plaques or infectious virus. (A) Electroporation of HFFs with BAC-IN84/Ep shows the appearance of green cells at 96 hpt. The transfection efficiency was monitored by measuring EGFP expression by fluorescence microscopy. (B) HFFs were electroporated with either BAC-IN84/Ep, wt HCMV BAC, or revertant BAC (BAC-84R). The cells were incubated for 8 days and then visualized by light microscopy. wt HCMV BAC- and BAC-84R-transfected cells showed typical viral plaques, whereas BAC-IN84/Ep-transfected cells failed to show any plaque formation. Viral plaques are shown within circled regions. (C) Single-step growth curves of reconstituted wt HCMV BAC virus (RVHB5) and revertant BAC virus (RV84R). Supernatant virus was harvested from cells electroporated with revertant BAC or wt HCMV BAC DNA. The resultant virus, RVHB5 (wt HCMV BAC) or RV84R (UL84 revertant BAC), was harvested and used to infect fresh HFFs. The supernatants from infected cells were collected on various days postinfection, and viral titers were determined by standard plaque assays. Error bars are the standard deviations from the average titers of three separate experiments.

FIG. 3.

BAC-IN84/Ep is defective for DNA replication. (A) Evaluation of viral DNA accumulation from wt HCMV BAC- or BAC-IN84/Ep-transfected cells. HFFs were electroporated, and total cellular DNAs were extracted on various days posttransfection and assayed for viral DNA accumulation by real-time PCR. The data are presented as fold increases in viral DNA accumulation over the amount of input DNA (day 1). Error bars are standard deviations for three separate experiments. (B) BAC-IN84/Ep can complement oriLyt-dependent DNA replication in the presence of UL84. The wt HCMV BAC pHB5 or mutant BAC-IN84/Ep was transfected into HFFs by electroporation along with HCMV oriLyt and with or without a plasmid encoding UL84. DNAs were extracted at 8 days posttransfection, cleaved with EcoRI and DpnI, transferred to a nylon membrane, and hybridized with pGEM-7Zf(−) (the parental vector of HCMV oriLyt). Lanes: 1, pHB5 cotransfected with oriLyt; 2, pHB5 cotransfected with pGEM-7Zf(−); 3, BAC-IN84/Ep cotransfected with oriLyt (no UL84); 4, BAC-IN84/Ep cotransfected with oriLyt and a plasmid encoding UL84 (ZP13). The arrow indicates the replicated, DpnI-resistant oriLyt plasmid.

FIG. 4.

Immediate-early gene expression patterns for BAC-IN84/Ep- and wt HCMV BAC-transfected cells. HFFs were electroporated with either BAC-IN84/Ep or wt HCMV BAC DNA, and the total cellular RNA was harvested on various days posttransfection and analyzed by real-time PCR. Real-time PCR primers and probes specific for IE2 or TRS1 were used to evaluate relative mRNA levels. The data are presented as fold increases over the mRNA levels measured on day 1 postelectroporation for the wt HCMV BAC. Error bars are the standard deviations from the averages of three separate experiments.

FIG. 5.

Early and late mRNAs are expressed in BAC-IN84/Ep-transfected cells. HFFs were electroporated with either BAC-IN84/Ep or wt HCMV BAC DNA, and the total cellular RNA was harvested on various days posttransfection and analyzed for early (UL44, UL105, and UL84), early-late (UL83), and late (UL75) gene expression by real-time PCR. The data are presented as fold increases over the mRNA levels measured on day 1 postelectroporation for the wt HCMV BAC. Also shown is a quantitative analysis of cyclophilin mRNA as a control to ensure that equal amounts of cDNA were added to the reaction mixtures. Error bars are the standard deviations from the averages of three separate experiments.

FIG. 6.

Replication protein UL44 and immediate-early protein IE2 fail to partition into replication compartments in BAC-IN84/Ep-transfected cells. An immunofluorescence assay was performed on HFFs transfected with the wt pHB5 BAC or BAC-IN84/Ep at 96 hpt. Transfected cells were incubated with an anti-IE2 or anti-UL44 antibody and an Alexa fluor 555-conjugated secondary antibody, as shown in red. Hoechst stain was used to identify the nuclei of transfected cells, which are shown in blue. (A) Incubation with anti-IE2-specific antibody in either pHB5 (top)- or BAC-IN84/Ep (bottom)-transfected cells. (B) Incubation with anti-UL44-specific antibody in either pHB5 (top)- or BAC-IN84/Ep (bottom)-transfected cells.