Nucleocytoplasmic shuttling of human cytomegalovirus UL84 is essential for virus growth

Abstract

Human cytomegalovirus (HCMV) UL84 is a multifunctional protein that is the proposed initiator for lytic viral DNA synthesis. Recently it was shown that UL84 displays nucleocytoplasmic shuttling. The role of shuttling in lytic DNA replication and virus growth is unknown. We now show that expression of the nonshuttling UL84 mutant failed to complement oriLyt-dependent DNA replication in the transient assay under conditions where core replication and ancillary proteins were expressed under the control of their native promoters. However, constitutive expression of the core replication proteins, along with the nonshuttling UL84 mutant, resulted in efficient oriLyt amplification, suggesting that shuttling may contribute to the activity of one of the auxiliary replication proteins. A recombinant HCMV bacterial artificial chromosome plasmid (BACmid) expressing the nonshuttling UL84 mutant (NS84 BAC) was defective for production of infectious virus. Quantitative PCR showed that NS84 BAC had decreased accumulation of viral DNA in both cellular and supernatant samples. Analysis of the accumulation of select viral mRNAs showed no difference in total cellular mRNA accumulation for IE2, IRS1, TRS1, UL102, UL105, and UL75 in cells transfected with the NS84 BAC. However, examination of cytoplasmic RNA and subcellular localization of IRS1 revealed a decrease in IRS1 mRNA accumulation and displaced protein localization, strongly suggesting that UL84 facilitated the localization of IRS1 mRNA to the cytoplasm. RNA pulldown assays showed that UL84 interacted with IRS1 mRNA. These results indicate that nucleocytoplasmic shuttling is essential for virus growth and strongly suggest that UL84 is responsible for localization of at least one virus-encoded transcript, IRS1 mRNA.

FIG. 1.

Conditional requirement for nucleocytoplasmic shuttling of UL84 for oriLyt-dependent DNA replication. HFF cells were cotransfected with plasmids encoding all of the required core replication proteins under the control of the SV40 promoter or their native promoters plus an IE2 expression plasmid, oriLyt, and either a plasmid that expresses native UL84 or the nonshuttling mutant. Lanes: 1, SV40 promoter expression of replication proteins plus nonshuttling UL84 with IE2 and oriLyt; 2, SV40 promoter expression of replication proteins plus IE2 with oriLyt; 3, native promoter expression of replication proteins plus wt UL84 with IRS1, UL112/113, IE2, and oriLyt; 4, native promoter expression of replication proteins plus nonshuttling UL84 with IRS1, UL112/113, IE2, and oriLyt; 5, native promoter expression of replication proteins plus IRS1 with UL112/113, IE2, and oriLyt; 6, native promoter expression of replication proteins plus wt UL84 with UL112/113, IE2, and oriLyt. The arrow indicates replicated oriLyt.

FIG. 2.

Generation of HCMV nonshuttling UL84 recombinant BAC. (A) Schematic of HCMV genome showing the relative location of UL84 and the amino acid changes made to generate the HCMV NS84 BAC. (B) HindIII restriction enzyme cleavage of BAC DNA. Lanes: 1, wild-type HCMV BAC; 2 to 4 miniprep of three BAC colonies after recombination. The arrow shows the location of the wild-type band that is missing in the recombinant BAC DNA clones due to the insertion of the galK-kan cassette. (C) Southern blot analysis using the galK probe. The arrow indicates the location of the inserted galK-kan cassette in the recombinant BAC DNA. MW, molecular weight.

FIG. 3.

HCMV NS84 BAC is defective for virus growth. HFF cells were transfected with NS84 BAC, revertant BAC, or wt BAC, and cells were allowed to incubate for up to 27 days posttransfection. Cells were visualized using a fluorescent microscope at 3, 4, 5, and 7 days posttransfection. For the NS84 BAC, images are also shown for 13 and 27 days posttransfection.

FIG. 4.

Nonshuttling UL84 BAC is defective for DNA replication. (A) Evaluation of supernatant viral DNA accumulation from wt HCMV BAC and nonshuttling UL84 BAC-transfected cells. HFF cells were transfected with BAC DNA, supernatant virus was isolated, and viral DNA was extracted at 3, 4, 5, and 8 days posttransfection and assayed for viral DNA accumulation by real-time PCR. The data are presented as fold increase in viral DNA accumulation over the amount found at day 3 posttransfection. Error bars are standard deviations for three separate experiments. (B) Evaluation of intracellular viral DNA accumulation from wt HCMV BAC-transfected and NS84 BAC-transfected cells. The data are presented as fold increase in viral DNA accumulation over the amount of day 2 DNA.

FIG. 5.

Total cellular mRNA accumulation levels are similar in NS84 BAC- and wt HCMV BAC-infected cells for representative virus-encoded transcripts. HFF cells were transfected with either wt BAC or NS84 BAC DNA, and total cellular RNA was harvested at various days posttransfection and analyzed by qPCR. The data are presented as fold increase over mRNA accumulation measured on day 2 posttransfection. Error bars are the standard deviations from the averages of three separate experiments. Input DNA was measured at 20 h p.t. and found to be approximately equal for the wt BAC and NS84 BAC.

FIG. 6.

IE2 and IRS1 display an aberrant subcellular localization pattern in NS84 BAC-transfected cells. HFF cells were transfected with wt or NS84 BAC DNA, and cells were visualized for the presence of UL84, IE2, UL44, UL83, and IRS1 by immunofluorescence using specific antibodies at 10 days posttransfection. Images are at a magnification of ×40 using Alexa Fluor 555-conjugated secondary antibodies. The presence of the indicated protein is shown in merged differential interference contrast (DIC) and bright-field images. DAPI (4′,6′-diamidino-2-phenylindole)-stained cells are also shown to the right of DIC images.

FIG. 7.

Decreased accumulation of cytoplasmic IRS1 mRNA in NS84 BAC-transfected cells. HFF cells were transfected with wt or NS84 BAC DNA, and cytoplasmic RNA was harvested at 2 or 3 days posttransfection and analyzed by qPCR. Transfected BAC DNA was normalized, and the results shown are from three separate experiments (error bars). Total cellular RNA was evaluated using the same sample collected for cytoplasmic RNA evaluation. Data are reported as fold increase over the level at 1 day posttransfection.

FIG. 8.

UL84 interacts with IRS1 mRNA in infected cells. An RNA-protein pulldown assay was performed using HCMV-infected cells. Protein-associated RNA was used to generate cDNA, which was subsequently amplified using primers specific for various HCMV-encoded transcripts. For nested PCR, first-round PCR products were used as a template for a second round of PCR using primers that corresponded to sequences within the first-round PCR product. Control immunoprecipitations were done using anti-K-bZIP antibody (KSHV). The arrow indicates the presence of a positive PCR signal.