Proteasome subunits relocalize during human cytomegalovirus infection, and proteasome activity is necessary for efficient viral gene transcription

Abstract

We have continued studies to further understand the role of the ubiquitin-proteasome system (UPS) in human cytomegalovirus (HCMV) infection. With specific inhibitors of the proteasome, we show that ongoing proteasome activity is necessary for facilitating the various stages of the infection. Immediate-early protein 2 expression is modestly reduced with addition of proteasome inhibitors at the onset of infection; however, both early and late gene expression are significantly delayed, even if the inhibitor is removed at 12 h postinfection. Adding the inhibitor at later times during the infection blocks the further accumulation of viral early and late gene products, the severity of which is dependent on when the proteasome is inhibited. This can be attributed primarily to a block in viral RNA transcription, although DNA synthesis is also partially inhibited. Proteasome activity and expression increase as the infection progresses, and this coincides with the relocalization of active proteasomes to the periphery of the viral DNA replication center, where there is active RNA transcription. Interestingly, one 19S subunit, Rpn2, is specifically recruited into the viral DNA replication center. The relocalization of the subunits requires viral DNA replication, but their maintenance around or within the replication center is not dependent on continued viral DNA synthesis or the proteolytic activity of the proteasome. These studies highlight the importance of the UPS at all stages of the HCMV infection and support further studies into this pathway as a potential antiviral target.

FIG. 1.

HCMV protein expression is delayed by proteasome inhibitor treatment. HFFs infected with HCMV at an MOI of 2 were treated with MG132 (2.5 μM) from 0- to 12 h p.i. with cells harvested at the times indicated (A) or lactacystin (Lact; 10 μM) from 24 to 36, 36 to 48, or 48 to 60 h p.i. with cells harvested at the end of treatment (B) and processed for Western blot analyses with antibodies to HCMV proteins IE2, IE1, UL44, UL57, UL99, UL83, UL85, and UL86. Actin is shown as a loading control.

FIG. 2.

Proteasome inhibition prevents further accumulation of viral transcripts. HFFs infected with HCMV at an MOI of 2 or mock infected were treated with lactacystin (10 μM) at the times indicated and harvested at the end of treatment. Total RNA was isolated and processed for Northern blot analyses with probes to the 3′ coterminal regions of UL92 to UL99, UL44, UL83, and UL82. GAPDH mRNA and 28S rRNA are shown as loading controls.

FIG. 3.

RNAP II expression is increased during HCMV infection but not affected upon proteasome inhibition. HFFs infected with HCMV at an MOI of 2 or mock infected were treated with lactacystin (10 μM) at the times indicated and harvested at the end of treatment for Western blot analyses with antibodies to RNAP IIo/a (ARNA3), pSer5-RNAP IIo (H14), pSer2-RNAP IIo (H5), p53, wee1, and actin.

FIG. 4.

DNA synthesis is downregulated upon proteasome inhibitor treatment. Viral DNA accumulation was measured by qPCR with primers and probe to the UL77 gene using total DNA isolated from HCMV-infected HFFs (MOI of 2) treated with DMSO or MG132 (2.5 μM) for 24 h at the postinfection times indicated. Values were normalized to that for the GAPDH promoter and are expressed as the fold inhibition of DNA accumulation in MG132-treated samples compared to DMSO control samples during the 24-h period. Mean values from two experiments are shown.

FIG. 5.

Proteasome activity and subunit expression increases during HCMV infection. (A) Mock-infected (M) or virus-infected (V) HFFs were harvested at the times indicated. Cell lysates were processed for an in-gel peptidase assay by using a fluorigenic peptide to measure proteasome activity. Western blot assays for 19S Rpn2 and 20S α6 were done in parallel on separate gels to identify the 26S and 20S proteasome fractions. (B) Western blot analyses of proteasome subunit expression in mock- or virus-infected HFFs over an infection time course. Representative subunits from each subcomplex are shown. Actin was used as a loading control.

FIG. 6.

Proteasome subunits relocalize around the viral replication center during HCMV infection. Mock- or virus-infected HFFs were fixed onto coverslips at 48 h p.i., processed for IFA, and imaged by deconvolution microscopy of 0.2-μm sections at a magnification of 1,000× under oil immersion conditions. Midsectional planes of representative cells are shown. (A) Mock-infected HFFs were fixed at 48 h p.i. and costained for 19S Rpt6 (red) and 20S α6 (green), with Hoescht stain in blue. (B) HCMV-infected HFFs (MOI of 5) were fixed at 48 h p.i. and costained with antibodies to 19S Rpn12 (green) and UL57 (red), 19S Rpn10 (green) and UL57 (red), 19S Rpt6 (red) and UL57 (green), or 20S α6 (green) and 11S REGγ (red).

FIG. 7.

Relocation of proteasome subunits to the replication center periphery occurs after the onset of viral DNA replication. (A) Mock- or HCMV-infected HFFs (MOI of 2) were treated with DMSO or PAA at 0 to 48 h p.i. and fixed at 48 h p.i. for IFA with antibodies to 19S Rpt6, 20S α6, and UL57. (B) DMSO or PAA was added at 36 to 48 h p.i. to inhibit viral DNA synthesis during this 12-h period. Cells were fixed at 36 h p.i. prior to treatment and at 48 h p.i. and processed for IFA with antibodies to 19S Rpt6, 20S α6, and UL57. Cells were imaged by deconvolution microscopy with 0.2-μm sections at a magnification of 1,000× under oil immersion conditions. Midsectional planes of representative cells are shown merged with Hoescht stain (blue). Magnified images of the prereplication foci (as indicated by the white arrow) are shown adjacent to subpanels 7 and 15 in panel A.

FIG. 8.

The perireplication center region is transcriptionally active. Mock- or virus-infected HFFs (MOI of 2) were treated as indicated and processed by IFA with Hoescht stain (in blue). Midsectional planes of representative cells imaged by deconvolution microscopy with 0.2-μm sections at a magnification of 1,000× under oil immersion conditions are shown. (A) Cells were pulsed with BrU (30 min) or BrdU (60 min) at 36 h p.i. and then fixed for IFA. Incorporated BrU or BrdU is shown in green and UL57 is shown in red. (B) Cells were pulsed with BrU at 36 h p.i. for 30 min and stained for 19S Rpt6 (red) and incorporated BrU (green). (C and D) Cells were fixed at 48 h p.i. and stained for 19S Rpt6 (red) and H3K4 (green) (C) or UL57 (red) and pSer2-RNAP II by the H5 antibody (green) (D). (E) Cells were treated with MG132 from 36 to 48 h p.i. and then fixed. Cells stained for 19S Rpt6 (red) and UL57 (green) are shown.

FIG. 9.

Enhanced proteolytic activity occurs in the perireplication center region as HCMV infection progresses. HCMV- or mock-infected HFFs were microinjected with DQ-ova (green) at 40 h p.i. Cells were fixed 30 min postinjection and stained for 19S Rpt6, 20S α6, or UL57 (shown in red). Midsectional planes of representative cells imaged by deconvolution microscopy with 0.2-μm sections at a magnification of 600× under oil immersion conditions are shown.

FIG. 10.

Proteasome subunit 19S Rpn2 relocalizes to the viral replication center after the onset of viral DNA replication. Cells were fixed at the times indicated and stained for 19S Rpn2 (green), UL57 (red), and Hoescht (blue). Midsectional planes of representative cells imaged by deconvolution microscopy with 0.2-μm sections are shown. (A) HFFs infected with HCMV (MOI of 0.01) were fixed daily. Representative cells at different stages of infection from day 4 are shown. Examples of prereplication center foci (white arrows), midsize replication centers (pink arrows), and a large coalesced replication center (orange arrows) are indicated. Cells were imaged at a magnification of 400× under oil immersion conditions. (B) Mock- or virus-infected HFFs (MOI of 2) were treated with DMSO or PAA at the onset of infection and fixed at 48 h p.i. Cells were imaged at a magnification of 1,000× under oil immersion conditions. (C) Mock- or virus-infected HFFs (MOI of 2) were treated with DMSO, PAA, or MG132 from 36 to 48 h p.i. Cells were fixed prior to treatment at 36 h p.i. and after treatment at 48 h p.i. Images were taken at a magnification of 1,000× under oil immersion conditions.