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. 2017 Feb 14;91(5):e01206-16.
doi: 10.1128/JVI.01206-16. Print 2017 Mar 1.

Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34+ Human Progenitor Cells

Jung Heon Kim  1 , Donna Collins-McMillen  1 , Jason C Buehler  2 , Felicia D Goodrum  2   3 , Andrew D Yurochko  4   5   6
Affiliations

Human Cytomegalovirus Requires Epidermal Growth Factor Receptor Signaling To Enter and Initiate the Early Steps in the Establishment of Latency in CD34+ Human Progenitor Cells

Jung Heon Kim et al. J Virol. .

Abstract

The establishment of human cytomegalovirus (HCMV) latency and persistence relies on the successful infection of hematopoietic cells, which serve as sites of viral persistence and contribute to viral spread. Here, using blocking antibodies and pharmacological inhibitors, we document that HCMV activation of the epidermal growth factor receptor (EGFR) and downstream phosphatidylinositol 3-kinase (PI3K) mediates viral entry into CD34+ human progenitor cells (HPCs), resulting in distinct cellular trafficking and nuclear translocation of the virus compared to that in other immune cells, such as we have documented in monocytes. We argue that the EGFR allows HCMV to regulate the cellular functions of these replication-restricted cells via its signaling activity following viral binding. In addition to regulating HCMV entry/trafficking, EGFR signaling may also shape the early steps required for the successful establishment of viral latency in CD34+ cells, as pharmacological inhibition of EGFR increases the transcription of lytic IE1/IE2 mRNA while curbing the expression of latency-associated UL138 mRNA. EGFR signaling following infection of CD34+ HPCs may also contribute to changes in hematopoietic potential, as treatment with the EGFR kinase (EGFRK) inhibitor AG1478 alters the expression of the cellular hematopoietic cytokine interleukin 12 (IL-12) in HCMV-infected cells but not in mock-infected cells. These findings, along with our previous work with monocytes, suggest that EGFR likely serves as an important determinant of HCMV tropism for select subsets of hematopoietic cells. Moreover, our new data suggest that EGFR is a key receptor for efficient viral entry and that the ensuing signaling regulates important early events required for successful infection of CD34+ HPCs by HCMV.IMPORTANCE HCMV establishes lifelong persistence within the majority of the human population without causing overt pathogenesis in healthy individuals. Despite this, reactivation of HCMV from its latent reservoir in the bone marrow causes significant morbidity and mortality in immunologically compromised individuals, such as bone marrow and solid organ transplant patients. Lifelong persistent infection has also been linked with the development of various cardiovascular diseases in otherwise healthy individuals. Current HCMV therapeutics target lytic replication, but not the latent viral reservoir; thus, an understanding of the molecular basis for viral latency and persistence is paramount to controlling or eliminating HCMV infection. Here, we show that the viral signalosome activated by HCMV binding to its entry receptor, EGFR, in CD34+ HPCs initiates early events necessary for successful latent infection of this cell type. EGFR and associated signaling players may therefore represent promising targets for mitigating HCMV persistence.

Keywords: CD34+ HPC; EGFR; HCMV; latency; virus entry.

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Figures

FIG 1
FIG 1
EGFR is expressed on the surfaces of CD34+ HPCs and is required for HCMV entry. (A) Uninfected CD34+ HPCs were stained with anti-EGFR antibodies and Alexa Fluor 594-conjugated secondary antibodies to detect EGFR expression on the cell surface (red). DAPI was used to counterstain the nucleus (blue). The contrast was adjusted in the differential interference contrast (DIC) image, and the cell membrane is outlined white in the DIC and merged images. Both adjustments were made so that the images could be more easily viewed. (B and C) Viral entry assays were performed in CD34+ HPCs. Cells were pretreated with AG1478 (AG) (EGFRK inhibitor), LY294002 (LY) (PI3K inhibitor), anti-EGFR blocking antibodies (α-EGFR), or DMSO/IgG antibody controls before HCMV infection. (B) Cells were infected with HCMV (TB40-UL32-HCMV/E) and stained with anti-EGFR antibodies and Alexa Fluor 594-conjugated secondary antibodies to detect EGFR (red). UL32-GFP was stained with an anti-GFP Alexa Flour 488-labeled antibody (green). DAPI was used to counterstain the nucleus (blue). Confocal microscopy was used to visualize HCMV bound to the cell surface or internalized into target cells. Representative slices from a Z stack are shown to compare the cell surface with intracellular space. (C) Quantification comparing the percentages of HCMV virions on the surfaces of or internalized into target cells. An average of 5 cells per experimental arm from two independent donors were used for counting. The means and standard errors of the mean (SEM) (error bars) of the results of two independent experiments performed.
FIG 2
FIG 2
EGFR/PI3K signaling is required for HCMV entry into CD34+ HPCs. (A and B) Viral entry assays were performed in CD34+ HPCs, as previously described in monocytes and fibroblasts (39, 42, 43). Cells were pretreated with AG1478 (EGFRK inhibitor), LY294002 (PI3K inhibitor), or DMSO solvent control (A) or anti-EGFR blocking antibodies (α-EGFR) or IgG control antibodies (B) before HCMV infection. The cells were infected with HCMV (low-passage-number Towne/E), and following proteinase K treatment, total DNA was isolated to detect viral entry by qPCR amplification of viral DNA corresponding to the UL123 gene region of the genome. The experiment was repeated with two different donors. For all the graphs, the levels of internalized HCMV DNA were normalized to the 4°C binding-only controls and to 18S rRNA as an internal control. Nontemplate controls (NTC) are also shown.
FIG 3
FIG 3
HCMV trafficking in CD34+ HPCs is enhanced by EGFR signaling activated during viral entry. (A) CD34+ HPCs were infected with HCMV (TB40-UL32-HCMV/E) and treated with DMSO or AG1478 at 30 mpi. The cells were cytospun onto slides at 1 hpi and 4 hpi and then stained and visualized by confocal microscopy. The cells were stained with anti-gB antibody (Ab) and Alexa Fluor 594-conjugated secondary Ab to detect gB (red). UL32-GFP was stained with an anti-GFP Alexa Fluor 488-labeled antibody (green). DAPI was used to counterstain the nucleus (blue). (B) Cells were infected with HCMV (low-passage-number Towne/E) and stained with specific Alexa Fluor 488-labeled HCMV DNA probe (green) according to the FISH protocol. DAPI was used to counterstain the nucleus (blue). (C) Stained cells (an average of 50 for each experimental group) were analyzed and quantitated to calculate the percentage of total viral DNAs detected in the cytoplasm or nucleus. The means and SEM (error bars) of the results of two independent experiments performed are shown.
FIG 4
FIG 4
HCMV-induced EGFR signaling favors latent over lytic viral gene expression in CD34+ HPCs. (A to C) CD34+ HPCs were mock infected or HCMV infected for 4 h and then treated with control DMSO or AG1478 (EGFRK inhibitor). RNA was isolated for RT-qPCR at 24 hpi, and expression of lytic IE1 mRNA (A) and lytic IE2 mRNA (B) versus that of latency-associated UL138 mRNA (C) was examined for two independent donors. The expression levels were normalized to 18S rRNA as an internal control. (A and C) Reverse transcriptase negative (RT−) and nontemplate (NTC) controls are also shown.
FIG 5
FIG 5
HCMV-induced EGFR signaling enhances IE1 expression in primary monocytes. Isolated monocytes were plated in 6-well dishes and mock infected or HCMV infected for 3 weeks. Beginning at 7, 10, or 14 dpi, control DMSO or AG1473 (EGFRK inhibitor) was added daily for the remainder of the infection period. No changes in cell survival were seen when they were added at these time points. After 3 weeks, RNA was isolated, and RT-qPCR was performed to determine expression of the IE1 viral transcript. The graph is representative of three replicates using different blood donors; the means and SEM (error bars) of the results of three independent experiments performed are shown. The expression levels of IE1 were normalized to that of 18S rRNA as an internal control; RT− and nontemplate controls are shown.
FIG 6
FIG 6
HMCV infection alters the transcription of cellular hematopoietic factors, in part via EGFR signaling. (A and B) CD34+ HPCs were mock infected or HCMV infected for 4 h to allow viral entry and trafficking to the nucleus. (A, top row, and B) At 4 hpi, RNA was harvested from a single cohort of HCMV-infected cells to identify a baseline level of cellular transcripts in infected cells (4 h). DMSO or AG1478 (EGFRK inhibitor) was added to the other cohorts at 4 hpi, and RNA was isolated from the groups at 24 hpi (24 h and AG). (A, bottom) DMSO or AG1478 was added at 4 hpi, and RNA was isolated at 24 hpi. RT-qPCR was performed on all the samples to quantitate IL-12 (A) and TGF-β (B) transcripts. Expression levels for both IL-12 and TGF-β were normalized to 18S rRNA as an internal control; RT− and nontemplate controls are shown. (A, top row, and B) Fold changes in cellular transcripts examined in two different donors. (A, bottom) Averages from three independent experiments with different donors (the means and SEM [error bars] of the results of three independent experiments performed) are shown. (C and D) CD34+ HPCs were mock infected for 4 h before treatment with DMSO or AG1478. At 24 hpi, the supernatants were collected for quantification of IL-12 by ELISA (C) and TGF-β by multiplexed bead-based immunoassay (D). The graphs represent the average expression levels of IL-12 and TGF-β from three independent experiments with different donors. The means and SEM (error bars) of the results of three independent experiments performed are shown.
FIG 7
FIG 7
Model for early HCMV infection of CD34+ HPCs. (Left) Various treatments used in this study to investigate HCMV-induced EGFR signaling in CD34+ HPCs. Pretreatment with anti-EGFR blocking antibodies was used to block HCMV-EGFR engagement at the cell surface and any direct signaling downstream of that specific engagement. Pretreatment with the signaling inhibitors AG1478 (EGFRK inhibitor) and LY294002 (PI3K inhibitor) was used to attenuate HCMV-induced molecular signaling associated with the viral binding and entry events. AG1478 was also used at 30 mpi or at 4 hpi to attenuate EGFR signaling after viral entry and trafficking commenced. (Right) Summary of our findings from this investigation. HCMV enters CD34+ HPCs via engagement of EGFR on the cell surface. The entry event is dependent upon both EGFRK and downstream PI3K signaling. HCMV de-envelopment occurs within 1 and 4 hpi, with nuclear translocation of the viral DNA occurring between 4 and 8 hpi. Both de-envelopment and delivery of HCMV DNA to the nucleus are enhanced by EGFR signaling. Once in the nucleus, HCMV initiates the appropriate cell-type-specific viral transcription program and also alters the transcription of the cellular genes that favor infection of CD34+ HPCs. EGFR signaling plays a role in shaping the induced viral and cellular transcriptional profiles during the initial stages of infection.
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