The host ubiquitin-dependent segregase VCP/p97 is required for the onset of human cytomegalovirus replication

Abstract

The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97, results in loss of IE2 expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. RNAseq analysis showed increased levels of IE1 splicing, with a corresponding decrease in IE2 splicing following VCP knockdown. Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression, including UL112/113. Furthermore, Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus. Finally, we show that NMS-873, a small molecule inhibitor of VCP, is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection.

Fig 1. Schematic representation of differential splicing of IE1 and IE2.

IE1 and IE2 are derived from the same primary transcript, driven by the major immediate early promoter. Differential splicing and polyadenylation of the terminal exon dictates expression of IE1 or IE2. Filled boxes indicate coding exons whereas the open box represents a non-coding exon.

Fig 2. Knockdown of the cellular gene VCP results in a complete block in HCMV replication.

Human primary fibroblast cells were transfected with 160 siRNA pools against membrane organization genes then infected with a GFP expressing HCMV virus. (A) Relative GFP fluorescence levels compared to cells transfected with a negative control siRNA for all 160 siRNA pools. Assays were performed in duplicate with three biological repeats with standard deviations shown. (B) GFP levels ranked by Z-score. (C) VCP deconvoluted siRNAs were tested for VCP knockdown by western blot and virus replication by relative GFP fluorescence to negative control siRNA transfected cells. GFP levels were measured 144 HPI and assays performed in duplicate. (D) HCMV replication was measured by plaque analysis following transfection with VCP siRNA or negative control siRNA. Cells were infected at an MOI of three and performed in duplicate with two biological repeats.

Fig 3. Knockdown of VCP results in a specific loss of the major immediate early protein IE2.

(A) Fluorescence microscopy demonstrates that all cells are infected with HCMV at 48 HPI following transfection with VCP siRNA, indicating no major effect on virus entry and translocation to the nucleus. (B) Western blot analysis for viral proteins of each major kinetic class shows a clear loss of IE2 expression and downstream early and late viral proteins.

Fig 4. Knockdown of VCP results in substantial reduction in IE2 transcript.

(A) Following transfection with VCP siRNA or negative control siRNA, cells were treated with proteasome or protease inhibitors, then infected with HCMV. Viral protein levels were measured by western blot analysis. Inhibition of protein degradation did not rescue IE2 expression in VCP knockdown cells. (B) Fibroblast cells were transfected with VCP or negative control siRNA and infected with HCMV. Total RNA was harvested at the indicated time points and IE1 and IE2 transcript levels determined by Northern blot analysis. 28S rRNA stained with ethidium bromide is shown as loading control.

Fig 5. Knockdown of VCP does not reduce IE2 transcript stability.

(A) Fibroblast cells were transfected with siRNA against VCP or negative control siRNA and infected with HCMV 48 hours post transfection. Forty-eight HPI cells were treated with actinomycin D to inhibit transcription and total RNA harvested at the indicated time points. Stability of IE1 and IE2 transcript was determined by Northern blot analysis. Bands were quantified using Image J software [54]. Quantification of transcript levels shown in (A) were plotted and transcript half-life determined using equations derived from linear trend lines. Knockdown of VCP results in an increase in transcript stability of 1.8 fold for IE1 (5.8 for VCP knockdown cells versus 3.2 for control cells) (B) and 1.5 fold for IE2 (5.4 for VCP knockdown versus 3.6 for control cells) (C).

Fig 6. Knockdown of VCP alters splicing of MIE transcripts.

RNA-seq analysis was performed to measure relative splicing levels of the MIE region following VCP knockdown. (A) The proportion of total reads mapping to each of the five exons was calculated, with the absolute difference in these values between VCP knockdown and corresponding negative control shown (numbers within exons). Knockdown of VCP has no effect on the proportion of reads originating from exons 1, 2 or 3, but is associated with a greater proportion of reads derived from exon 4 (IE1) and decreased numbers from exon 5 (IE2). (B) Log₂ ratios of the proportions of reads mapping to exon 3 whose matching pair maps to either exons 2, 4 or 5 are shown. The proportion of read pairs spanning the shared splice junction between exon two and three is unaffected by VCP knockdown, whereas exon 3 to 4 frequency increases and 3 to 5 decreases.

Fig 7. Expression of a subset of viral genes remains high following VCP knockdown, despite loss of IE2 expression.

Read counts were aligned to the TB40E genome sequence and normalised for total reads and CDS length. Difference in transcription at each time point was determined by calculating the Log₂ ratio in normalised read counts between control infected cells and VCP knockdown infected cells.

Fig 8. Altered MIE splicing is not due to changes in cell cycle following VCP knockdown.

Fibroblast cells were transfected with negative control siRNA or VCP siRNA and cells were fixed and stained with propidium iodide 4 days post transfection. Untransfected cells were treated with 0.5 μg/ml nocodazole as a positive control of G2/M arrest. DNA content was then measured by FACS analysis and defined as G1, S or G2 using FlowJo software.

Fig 9. VCP colocalises with viral replication compartments in the nucleus.

The cellular localisation of VCP was determined by confocal microscopy during HCMV infection. Cells were infected at high MOI and fixed at 24, 48 and 72 HPI and co-stained for VCP and IE2. Nuclei are stained with DAPI.

Fig 10. VCP inhibitor NMS-873 is a potent HCMV antiviral.

(A) Treatment with NMS-873 causes the same phenotype as knockdown of VCP. Cells were pretreated with 1μM NMS-873 then infected at high MOI with HCMV. Total RNA and protein were harvested at the indicated times and IE1 and IE2 protein and RNA levels measured by Western and Northern blot analysis. (B) Cells were treated with the indicated concentrations of NMS-873 or Ganciclovir then infected 24 hours post treatment. Cell and supernatant was harvested at the indicated time points and virus levels determined by plaque analysis. (C) Cells were treated with NMS-873 24 hours before infection, at the same time as infection, or 24 HPI. Supernatant was collected seven days post infection and virus titers determined by plaque assay. (D) Cell viability was determined five days post treatment at the indicated concentrations of NMS-873 using two independent commercial kits, Cell Titer-Glo and Cell Titer-Blue. (B-D) Data represents two biological repeats.