RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

<p>Human Cytomegalovirus (HCMV), an ubiquitous β-herpesvirus, is a significant pathogen that causes medically severe diseases in immunocompromised individuals and in congenitally infected neonates. RhoB belongs to the family of Rho GTPases, which regulates diverse cellular processes. Rho proteins are implicated in the entry and egress from the host cell of mainly α- and γ-herpesviruses, whereas β-herpesviruses are the least studied in this regard. Here, we studied the role of RhoB GTPase during HCMV lytic infection. Microscopy analysis, both in fixed and live infected cells showed that RhoB was translocated to the assembly complex/compartment (AC) of HCMV, a cytoplasmic zone in infected cells where many viral structural proteins are known to accumulate and assembly of new virions takes place. Furthermore, RhoB was localized at the AC even when the expression of the late HCMV AC proteins was inhibited. At the very late stages of infection, cellular projections were formed containing RhoB and HCMV virions, potentially contributing to the successful viral spread. Interestingly, the knockdown of RhoB in HCMV-infected cells resulted in a significant reduction of the virus titer and could also affect the accumulation of AC viral proteins at this subcellular compartment. RhoB knockdown also affected actin fibers' structure. Actin reorganization was observed at late stages of infection originating from the viral AC and surrounding the cellular projections, implying a potential interplay between RhoB and actin during HCMV assembly and egress. In conclusion, our results demonstrate for the first time that RhoB is a constituent of the viral AC and is required for HCMV productive infection.</p

The Downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 Axis and by Human Cytomegalovirus (HCMV) Associated Factors Allows the Activation of the HCMV Major IE Promoter and the Transition to Productive Infection

<div><p>Earlier studies had suggested that epigenetic mechanisms play an important role in the control of human cytomegalovirus (HCMV) infection. Here we show that productive HCMV infection is indeed under the control of histone H3K27 trimethylation. The histone H3K27 methyltransferase EZH2, and its regulators JARID2 and NDY1/KDM2B repress GFI1, a transcriptional repressor of the major immediate-early promoter (MIEP) of HCMV. Knocking down EZH2, NDY1/KDM2B or JARID2 relieves the repression and results in the upregulation of GFI1. During infection, the incoming HCMV rapidly downregulates the GFI1 mRNA and protein in both wild-type cells and in cells in which EZH2, NDY1/KDM2B or JARID2 were knocked down. However, since the pre-infection levels of GFI1 in the latter cells are significantly higher, the virus fails to downregulate it to levels permissive for MIEP activation and viral infection. Following the EZH2-NDY1/KDM2B-JARID2-independent downregulation of GFI1 in the early stages of infection, the virus also initiates an EZH2-NDY1/ΚDM2Β-JARID2-dependent program that represses GFI1 throughout the infection cycle. The EZH2 knockdown also delays histone H3K27 trimethylation in the immediate early region of HCMV, which is accompanied by a drop in H3K4 trimethylation that may contribute to the shEZH2-mediated repression of the major immediate early HCMV promoter. These data show that HCMV uses multiple mechanisms to allow the activation of the HCMV MIEP and to prevent cellular mechanisms from blocking the HCMV replication program.</p></div

GFI1 mRNA and protein are degraded rapidly during HCMV infection and their degradation is followed by upregulation of EZH2, JARID2 and NDY1/KDM2B and downregulation of JMJD3.

<p><b>A</b>. The GFI1 mRNA levels in lysates of HFFs harvested at the indicated time points after HCMV infection were monitored by real time RT-PCR. <b>B</b>. The GFI1 protein levels in the same cells were monitored by western blotting. <b>C</b>. HCMV infection promotes the degradation of GFI1 at the RNA level. HFFs were mock-infected or infected with HCMV after treatment with Actinomycin D (5 µg/ml). GFI1 mRNA levels in cell lysates harvested at the indicated time points after exposure to the virus were measured by real time RT-PCR. <b>D</b>. Proteasomal inhibition blocks the rapid downregulation of GFI1 in HCMV-infected cells. HFFs were mock-infected or infected with HCMV after pretreatment with MG132 (10 µM). GFI1 protein levels in cell lysates harvested at the indicated time points after exposure to the virus were measured by western blotting. <b>E</b>. GFI1 protein levels in HFFs transduced with the indicated constructs and harvested before, and 2 hours after HCMV infection, were measured by western blotting. <b>F</b>. The expression of EZH2, NDY1/KDM2B, JARID2 and JMJD3 was measured by real time RT-PCR in lysates of HFFs, harvested at the indicated time points after HCMV infection.</p

Infection by HCMV depends on the downregulation of GFI1, a repressor of immediate-early gene transcription.

<p>Model summarizing the data on the interaction between the virus and the host. (Panel A) This panel describes the infection of wild type HFFs. The incoming virus rapidly degrades GFI1 to allow the activation of the MIEP of HCMV, and viral infection. In addition, virus infection alters the expression of NDY1/KDM2B, EZH2, JARID2 and JMJD3. The solid lines from these molecules to GFI1 indicate that they actively repress GFI1 both before and after infection, although due to HCMV-induced changes in their expression, the repression is enhanced after infection. (Panel B, Left) The repression of GFI1 in uninfected cells was blocked by the knockdown of NDY1/KDM2B, EZH2 or JARID2 and by the overexpression of JMJD3, resulting in significant up-regulation of GFI1 (dotted lines). (Panel B, Right) describes the infection of HFFs in the left side of panel B. The virus continues to degrade GFI1. However, the degradation of GFI1 by the virus is insufficient to downregulate it to levels that allow the activation of the MIEP and viral infection.</p

Primers set used for ChIP analysis.

<p>Primers set used for ChIP analysis.</p

NDY1/KDM2B, EZH2 and H3K27 tri-methylation are required for immediate-early gene transcription.

<p><b>A and B</b>. HFFs were lentivirally or retrovirally transduced with the indicated constructs and they were subsequently infected with HCMV (MOI 0.5). The cells were fixed 5 hours later and the percentage of IE1-expressing cells was measured by FACS analysis. The bars show the percentage of IE1-positive cells (mean ± SD). <b>C</b>. Comparison of IE1 expression in HCMV-infected HFFs, transduced with pLKO.1, pLKO.1-shEZH2 or pLKO.1-shNDY1/KDM2B, prior to the infection. Cells were infected with HCMV (MOI 0.5). Western blots of cell lysates harvested at the indicated time points, were probed with anti-IE1 or anti-actin (loading control) antibodies. <b>D</b>. HFFs were infected with HCMV (MOI 0.5 PFU/cell) before and after a 30 minute pretreatment with the EZH2 inhibitor DZNep. The Western blotting shows the expression of EZH2 and IE1 in untreated and DZNep-pretreated cells at 24 hours from the start of the infection. <b>E</b>. HFFs were infected with HCMV (MOI 0.5 PFU/cell) before and after a 30-minute pretreatment with the EZH2 inhibitor DZNep. The infected cells were monitored by light microscopy 5 days later. In addition, they were stained for IE1 and counterstained with DAPI at 5 hours post-infection, and they were analyzed by epifluoerescence microscopy. Bar = 100 µm. <b>F</b>. The progeny virus harvested from the DZNep-treated and untreated cells as in D, 5 days after infection, was titrated by standard viral plaque assays. The bars show the viral titers (mean ± SD).</p

NDY1/KDM2B, EZH2 and JARID2 are selectively required for the infection of human foreskin fibroblasts with HCMV whereas the H3K27me3 demethylase JMJD3 inhibits viral infection.

<p><b>A</b>. HFF cells were transduced with pLKO.1-based lentivral constructs of shNDY1/KDM2B, shEZH2, shPHF2, shRBP2, or with the empty vector. Western blots of cell lysates were probed with the indicated antibodies. HCMV harvested from these cells was titrated using a plaque assay. The bars show the viral titers (mean ± SD). <b>B</b>. HFF cells were transduced with pBabe-based retroviral constructs of NDY1/KDM2B, EZH2, PHF2 and RBP2, or with the empty vector. Western blots of cell lysates were probed with the indicated antibodies. HCMV harvested from these cells was titrated using a plaque assay. The bars show the viral titers (mean ± SD). <b>C</b>. Growth of HCMV over time, in non-transduced cells or in cells transduced either with the empty pLKO.1 vector, or with pLKO.1-based lentiviral constructs of shEZH2, shNDY1/KDM2B, shPHF2 and shRBP2. Cells were infected with HCMV at an MOI of 0.5 PFU/cell and culture supernatants were harvested at time 0 and every other day after the infection for up to 9 days. The collected supernatants were titrated by plaque assay in HFFs and the results are presented as PFU/ml over time. <b>D</b>. HFFs were transduced with pLKO.1-based lentivral constructs of shJMJD3, or shJARID2, or with the empty vector. Alternatively, HFFs were transduced with pBabe-based constructs of JMJD3 or JARID2, or with empty vector. Western blots of lysates of these cells were probed with the indicated antibodies. HCMV harvested from these cells was titrated using a plaque assay. The bars show the viral titers (mean ± SD). <b>E</b>. HFFs were transduced with pLKO.1 or a pLKO.1-based construct of shEZH2 that targets sequences within the EZH2 3′ UTR. Some of the shEZH2 cultures were also transduced with pBabe-puro-based constructs of EZH2 (the catalytically-inactive myc-tagged EZH2ΔSET in lane 3, and the myc-tagged wild-type EZH2 in lane 4 in the inset), both of which are not targeted by shEZH2. HCMV was harvested 5 days p.i. from the transduced and non-transduced cells and titrated by a plaque assay. The bars show the viral titers (mean ± SD).</p

Lentiviral and retroviral constructs used.

<p>Lentiviral and retroviral constructs used.</p

Primer sets used in Real-Time PCR.

<p>Primer sets used in Real-Time PCR.</p

The knockdown of GFI1 partially rescues the ability of HCMV to infect HFF cells in which EZH2 was knocked down.

<p><b>A</b>. HFFs were transduced with a TRIPZ-shEZH2 construct and with the lentiviral vector pLKO.1, or with a pLKO.1-based shGFI1construct, as indicated. Cells transduced with TRIPZ-shEZH2 and pLKO.1 (third lane), or TRIPZ-shEZH2 and pLKO.1-shGFI1 (last lane) were treated with Doxycycline, to induce shEZH2. A1. Western blotting showing the expression of EZH2 and GFI1 in HFFs transduced with the indicated constructs, in the presence or absence of Doxycycline prior to HCMV infection. A2. Western blotting, monitoring the expression of IE1 in the cells shown in F1, upon infection with HCMV. Lysates were harvested at 5 days from the start of HCMV infection. A3. Titration of the progeny virus obtained 7 days after HCMV infection of the cells shown in A1. The bars show the number of plaques per ml (mean ± SD).</p