
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38483999
202312290
10.1073/pnas.2312290121
research-articleResearch ArticlemicrobioMicrobiology423
Biological Sciences
Microbiology
Distinct early role of PTEN regulation during HCMV infection of monocytes
Chesnokova Liudmila S. a b 1
Mosher Bailey S. a b 1
Fulkerson Heather L. a c
Nam Hyung W. d
Shakya Akhalesh K. a https://orcid.org/0000-0002-1739-8858

Yurochko Andrew D. andrew.yurochko@lsuhs.edu
a b c e f g 2 https://orcid.org/0000-0002-6452-5813

aDepartment of Microbiology and Immunology, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA 71103
bCenter for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA 71103
cCenter for Cardiovascular Diseases and Sciences, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA 71103
dDepartment of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA 71103
eFeist-Weller Cancer Center, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA 71103, Shreveport, LA 71103
fCenter for Excellence in Arthritis and Rheumatology, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA 71103
gCenter of Excellence for Emerging Viral Threats, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA 71103
2To whom correspondence may be addressed. Email: andrew.yurochko@lsuhs.edu.
Edited by Thomas Shenk, Princeton University, Princeton, NJ; received September 19, 2023; accepted December 1, 2023

1L.S.C. and B.S.M. contributed equally to this work.

14 3 2024
19 3 2024
14 9 2024
121 12 e231229012119 9 2023
01 12 2023
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

HCMV is a myelotropic virus with monocytes and macrophages serving as key cell types in viral dissemination and persistence following primary infection and reactivation from latency. Because of the clinical significance of HCMV infection of monocytes, an understanding of the mechanism for infection of these cells is needed to better define viral pathogenesis. Our results define a specific process by which pentamer binding drives molecular processes that enhances HCMV infection of clinically relevant monocytes, as well as suggests that wild-type pentamer-carrying virions that activate the β-integrin/c-Src signaling pathway exploit a distinct type of vesicle for internalization of these important cells.

Human cytomegalovirus (HCMV) infection of monocytes is essential for viral dissemination and persistence. We previously identified that HCMV entry/internalization and subsequent productive infection of this clinically relevant cell type is distinct when compared to other infected cells. We showed that internalization and productive infection required activation of epidermal growth factor receptor (EGFR) and integrin/c-Src, via binding of viral glycoprotein B to EGFR, and the pentamer complex to β1/β3 integrins. To understand how virus attachment drives entry, we compared infection of monocytes with viruses containing the pentamer vs. those without the pentamer and then used a phosphoproteomic screen to identify potential phosphorylated proteins that influence HCMV entry and trafficking. The screen revealed that the most prominent pentamer-biased phosphorylated protein was the lipid- and protein-phosphatase phosphatase and tensin homolog (PTEN). PTEN knockdown with siRNA or PTEN inhibition with a PTEN inhibitor decreased pentamer-mediated HCMV entry, without affecting trimer-mediated entry. Inhibition of PTEN activity affected lipid metabolism and interfered with the onset of the endocytic processes required for HCMV entry. PTEN inactivation was sufficient to rescue pentamer-null HCMV from lysosomal degradation. We next examined dephosphorylation of a PTEN substrate Rab7, a regulator of endosomal maturation. Inhibition of PTEN activity prevented dephosphorylation of Rab7. Phosphorylated Rab7, in turn, blocked early endosome to late endosome maturation and promoted nuclear localization of the virus and productive infection.

HCMV
monocytes
signaling
cellular trafficking
phosphoproteomics
HHS | National Institutes of Health (NIH) 100000002 AI056077 Akhalesh K. ShakyaAndrew D. Yurochko HHS | National Institutes of Health (NIH) 100000002 AI127335 Akhalesh K. ShakyaAndrew D. Yurochko HHS | National Institutes of Health (NIH) 100000002 AI15962 Akhalesh K. ShakyaAndrew D. Yurochko HHS | National Institutes of Health (NIH) 100000002 P20GM134974 Akhalesh K. ShakyaAndrew D. Yurochko HHS | National Institutes of Health (NIH) 100000002 P20GM121307 Akhalesh K. ShakyaAndrew D. Yurochko HHS | National Institutes of Health (NIH) 100000002 P20GM121288 Akhalesh K. ShakyaAndrew D. Yurochko HHS | National Institutes of Health (NIH) 100000002 P30GM110703 Akhalesh K. ShakyaAndrew D. Yurochko
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pmcHuman cytomegalovirus (HCMV) is a β-herpesvirus that infects approximately 60 to 90% of the population worldwide (1, 2) HCMV infections are usually asymptomatic and well maintained by the immune system (3). However, HCMV is responsible for significant morbidity and mortality in immunocompromised patients, including AIDS patients and organ transplant recipients (1, 2). HCMV can also cross the placenta and put an unborn child at risk for severe birth defects (4–8).

HCMV glycoprotein B (gB) and the glycoprotein H and L (gH/gL) complex(s) are essential for viral entry (4). HCMV gB is the fusion protein and binds to and activates signaling via the epidermal growth factor receptor (EGFR) (9–14). HCMV expresses three gH/gL complexes. The trimer is composed of gH/gL and glycoprotein O (gO) and is considered essential and sufficient for infection of fibroblasts (15). The trimer is indispensable for infection of other cell types since it is required for membrane fusion (16–18). The pentamer complex consists of the glycoproteins gH/gL/UL128/UL130/UL131 and is found on all clinical strains of HCMV, although it is frequently lost in laboratory-adapted strains (19). The pentamer is required for infection of monocytes (9), endothelial cells (20), and epithelial cells (21). The recently discovered gH/UL116 is not well characterized (22, 23).

HCMV demonstrates broad cellular and tissue tropism (24, 25). The virus can bind to various cellular receptors such as the epidermal growth factor receptor [EGFR (11, 13, 14)], the platelet-derived growth factor receptor [PDGFR (18, 26–28)], and integrin αvβ3 or αvβ1 (16, 29). Two new receptors have recently been discovered to be involved in HCMV-epithelial cell tropism, OR14I1 (30) and neuropilin-2 (31). HCMV infection and nuclear translocation in fibroblasts and epithelial and endothelial cells occur relatively quickly. In fibroblasts, HCMV entry is pH- and clathrin-independent, and data support that it occurs either by direct fusion (32, 33) or by micropinocytosis (34, 35). This entry/internalization is followed by rapid nuclear translocation within 30 m (12, 36). For epithelial and endothelial cells, HCMV entry requires acidification, and the data support entry by micropinocytosis (16, 37) or by direct fusion in acidified endosomes mediated by Neuropilin-2/CD147, for example (31, 38, 39). In epithelial cells, HCMV IE2 protein expression can be detected as early as 2 hours postinfection (hpi) (33) documenting the rapid nature of nuclear translocation. In endothelial cells, HCMV DNA is detected in nuclei by as early as 30 minutes postinfection (mpi) to 4 hpi (36, 40). In human monocyte-derived dendritic cells, HCMV infection is pH-independent (37, 41) and occurs likely by macropinocytosis (37, 41).

Peripheral blood monocytes are the primary target of HCMV infection in vivo and act as an essential vehicle for viral dissemination (12–14, 29, 36, 42–44) throughout the various organ systems in the body following both primary infection and reactivation from a latent infection (45, 46). We have previously shown that the early steps that lead to productive infection of monocytes include gB attachment to and activation of EGFR, as well as pentamer complex attachment to β1/β3-integrins and activation of downstream integrin signaling (29, 45). These receptor engagements mediate activation of the c-Src/MAPK and PI3K/Akt signaling pathways required for correct viral trafficking to the nucleus (42, 43, 47–49). Analysis of HCMV entry into monocytes and postentry events revealed an intracellular trafficking and nuclear translocation of viral DNA pathway that is distinct from that observed in other cell types (36). In monocytes, internalized mature (fully enveloped) HCMV is found in EEA1–positive endosomes 10 to 45 mpi (13, 36, 50). The mature virus in these EEA1-positive endosomes is then transported to the trans-Golgi network (TGN) and then to Rab11+ recycling endosomes, where the virus de-envelops (starting around ~24 hpi), followed by trafficking of the capsid to the nuclear membrane, where viral DNA is translocated to the nucleus starting 3 days postinfection (dpi) (12, 13, 36). Functionally, HCMV gB binds to EGFR and promotes its dimerization and activation of its intrinsic kinase activity (12–14). We recently showed that the virus and EGFR are internalized together from the external membrane and that the gB remains bound to EGFR throughout the viral trafficking pathway until de-envelopment in Rab11+ vesicles (13). The pentameric complex also plays a critical role in monocyte infection (29, 36, 43) through binding to integrins and the subsequent activation of the c-Src/paxillin-signaling pathway (29, 43). Furthermore, pentamer-induced signaling is required for efficient viral trafficking from the early endosome to the TGN and prevents endocytosed HCMV from lysosomal degradation (12, 29, 43, 44, 51).

PTEN phosphatase activity opposes PI3K through downregulation of PI3K/Akt signaling (52, 53), RTK signaling (54), and integrin signaling (55, 56). PTEN is a membrane-residing protein (57, 58) found in the cytoplasm distributed along microtubules and bound directly to endosomes through the early endosomal lipid phosphatidylinositol 3-phosphate [PtdIns-(3)-P1] (59, 60). It is through this interaction with endosomes that allows PTEN to regulate endocytic trafficking (56, 60). PTEN belongs to a large class of dual-specificity phosphatases (lipid and protein). PTEN dephosphorylates phosphotyrosine residues (61) on important regulators of endocytic processes, such as the late endosomal protein Rab7 (62). Rab7 regulation is required for the Rab5/Rab7 switch (63) that controls endosomal maturation. PTEN also binds to all phosphatidylinositol phosphates phosphorylated at position 3 of the inositol ring but only hydrolyzes with high-efficiency phosphatidylinositol (3, 4, 5)-tris-phosphate [PtdIns-(3, 4, 5)-P3] (64–66). These lipid substrates play important roles in cell metabolism (63, 67). Regulation of PTEN phosphatase activity occurs through phosphorylation of the cluster of serines and threonines on the C-terminal tail domain (57, 68–70). PTEN phosphorylation is usually irreversible, and phosphorylated PTEN is eventually degraded (71, 72). A link between HCMV infection and an increase in PTEN phosphorylation at 24 hpi was reported in connection with HCMV-triggered activation of antiapoptotic signaling (73).

We report here in blood monocytes infected with HCMV that pentamer signaling resulted in rapid (15 mpi) PTEN phosphorylation at Ser380 and Ser370 that inhibited PTEN phosphatase activity (69, 70). We showed that the pentamer-triggered activation of the c-Src kinase likely directly phosphorylated PTEN (74, 75) and led to activation of enzymes such as glycogen synthase kinase-3α/β [GSK-3α/β (76, 77)] and AMP-activated Liver Kinase B1 [LKB1 (78)] that also phosphorylate PTEN on tail domains known to inhibit PTEN activity. When silencing PTEN expression with siRNA or blocking PTEN phosphatase activity, we observed that entry of pentamer-carrying HCMV was inhibited in monocytes, but not in epithelial cells, showing cell type specificity to these key entry pathways. Our experiments also demonstrated that inhibition of PTEN phosphatase activity postentry could rescue defective, trimer-only-carrying virus. We showed that virus carrying only the trimer (pentamer-null), which usually moves to late endosomes and is degraded within 24 hpi (36), was found in the EEA1-positive endocytic vesicles upon inhibition of PTEN activity postentry. We showed that after PTEN inhibition, Rab7 was not dephosphorylated. Because the Rab5/Rab7 switch is the limiting step in endosomal maturation (79), and inhibition of the dephosphorylation of Rab7 prevents endosomal maturation, we argue that we have the initial identification of an essential mechanism by which pentamer binding to integrins on monocytes promotes productive viral trafficking. We conclude that inhibition of PTEN activity by pentamer-induced binding to β1/β3 integrins and appropriate downstream signaling represents a unique mechanism for preventing endocytosed HCMV in monocytes from degradation and for the promotion of viral trafficking to TGN/Rab11+ vesicles and subsequently to the nucleus.

Materials and Methods

Cells.

Retinal pigmented epithelium cells (ARPE-19, provided by J. Kamil, LSUHS), human embryonic lung fibroblasts (HEL) (ATCC), human fetal foreskin fibroblasts (HFFF provided by J. Kamil, LSUHS), and human fetal foreskin fibroblasts stably expressing tet-repressor [HFFF-tet, (80) obtained from Richard J. Stanton, Cardiff University School of Medicine, UK], were grown in 10% fetal bovine serum (FBS) in Dulbecco’s modified Eagle medium (DMEM), Corning.

Virus.

HCMV strains used in this study are BACs containing the wild-type HCMV genome TB40Ewt-GFP and TB40EΔUL128-131-GFP lacking the gH/gL/UL128-131 complex (30, 81); BADwt containing a frameshift mutation in UL131A and BADrUL131 with a repaired and functional gH/gL/UL128-131 complex, both of which are AD169-derived BACs (21); and TR-5 and TR-5ΔUL128-131 lacking the gH/gL/UL128-131 complex (36). The HCMV strains Merlin and MERLIN Bac pAL1393 were also used and were gifts from R. J. Stanton, Cardiff University School of Medicine, UK (80). Additional details are presented in SI Appendix, Materials and Methods.

Antibodies and Immunofluorescence.

A variety of primary and secondary Abs were used in the study. The specific list and catalog numbers are provided in SI Appendix, Materials and Methods.

Inhibitors of Signaling.

A description of the inhibitors and final concentrations used in the study are provided in SI Appendix, Materials and Methods. Monocytes were preincubated with an inhibitor or a corresponding volume of DMSO for 30 m, then washed, and infected with HCMV. With the exception of PP2, which we previously utilized (13), we tested a range of inhibitor concentrations to find the optimal concentration.

Human Peripheral Blood Monocyte Isolation.

Monocytes were isolated from human peripheral blood as described (82, 83) and as detailed in SI Appendix, Materials and Methods. Our full study protocol (H99-064) was approved by our LSUHSC-S IRB. We followed our study protocol to collect informed consent from healthy donors. We use adults 18 y and older and do not require parental permission. We do not discriminate based on gender, race, or ethnicity. We use flyers or verbal recruitment, and all donors sign an approved consent form.

Monocyte Treatment.

Unless stated in the figure legend or SI Appendix, Materials and Methods, human peripheral blood monocytes were infected with HCMV at multiplicity of infection (MOI) 10 or treated with an equal volume of RPMI 1640 for mock infection. Monocytes were treated in a suspension of 1% HS-RPMI at 37 °C with 5% CO2. SF1670 is a specific PTEN phosphatase inhibitor (84–86). Unless specifically noted, cells were preincubated with SF1670 at the final concentration of 20 µM for 30 m prior to HCMV infection.

siRNA Transfection.

A total of 3 × 106 freshly isolated human monocytes were nucleofected with SignalSilence control or SignalSilence PTEN siRNA I (Cell Signaling Technology) using an Amaxa nucleofector with the Human Monocyte Nucleofector Kit (Lonza) and the “Human Monocyte” program (29, 46). After nucleofection, monocytes were incubated for 48 h, harvested, and used for experimentation.

Virus Attachment and Internalization Assays.

For the binding assay, monocytes were washed with cold RPMI and cold PBS. DNA was isolated using a QIAmp DNA Blood Mini Kit (Qiagen USA, Germantown, MD), and the number of virus genome copies bound per cell was determined by quantitative real-time PCR (qPCR, see below). For the internalization assay, virus binding to cells was performed as described above for the binding assay, followed by warming of the cells to 37 °C for 1 h. Cells were then placed on ice and treated with Proteinase K (Roche) for 45 m to remove virus remaining at the cell surface (87). Cells were washed and DNA isolated with a QIAmp DNA Blood Mini Kit (Qiagen) for determination of the number of virus genome copies remaining per cell (by qPCR).

qPCR and qRT-PCR.

qPCR to determine the number of virus genomes per cell was done as previously described (29). The number of bound or internalized viral particles was expressed as a number of viral genomes per cell [a ratio of UL123 copy number to C-reactive protein (CRP) gene copy number]. See SI Appendix, Materials and Methods for additional details.

Phosphoproteomic Screen (Full Moon BioSystem, Cat.# PEX100).

The manufacturer’s protocol was followed with the slight modifications, documented in ref. 88, with additional information in SI Appendix, Materials and Methods. Image quantification was performed by the Full Moon BioSystem Company.

Rab7 Immunoprecipitation (IP).

Monocyte lysate as defined in SI Appendix, Materials and Methods was used for IP with rabbit Ab to Rab7 (D95F2) XP® (Cell Signaling Technology) for 2 to 5 h. The protein complex was bound with Protein A Sepharose for 1 h. After washes in a RIPA buffer containing HALT inhibitor cocktail, the sample was diluted in reducing Laemmli buffer (Bio-Rad Laboratories, Hercules, CA).

SDS-PAGE and Western Blot Analysis.

The proteins were separated in a discontinuous gradient gel (89). Proteins were transferred to nitrocellulose or PVDF membranes (Bio-Rad), blocked, and probed with the appropriate Abs as noted in SI Appendix, Materials and Methods. Fluorescence was visualized using an Odyssey infrared imager (LiCor Biosciences) and quantified with LiCor software (SI Appendix, Materials and Methods).

Lipid Extraction and Profiling Using Liquid Chromatography and Mass Spectrometry (LC–MS).

Phosphatidylinositol phosphates were extracted from monocytes based on published protocols (90, 91). Analysis of the lipid profile was carried out using Waters Acquity Ultra Performance Liquid Chromatography (UPLC) coupled with a XEVO TQ-MS ESI mass spectrometer. [PtdIns–(3)–P1], [PtdIns–(4, 5)-P2], and [PtdIns-(3, 4, 5)-P3] (Cayman Chemical Company) were used as standards (SI Appendix, Materials and Methods).

Immunofluorescence Microscopy.

High-resolution images were acquired using a Nikon N-SIM E Super-Resolution Microscope system using a 100× objective. Each image appearing in the text is a representative single slice from a z-stack. The distances between gB and the closest appropriate organelle were quantified. Data were combined from three biological replicates. Each data point represents an individual viral particle to the organelle measurement, and each condition includes at least 50 data points. See SI Appendix, Materials and Methods for more details.

Flow Cytometric Analysis.

Cells were fixed incold methanol for 10 m with subsequent washing in cold 0.5% BSA and 0.01% NaN3 in PBS. Cells were stained with Abs to EGFR conjugated with Alexa Fluor 488 (Sigma-Aldrich, Inc.; #16-246); to CD29 (Integrin β1) conjugated with allophycocyanin APC 633/650 (BD Biosciences, #559883); and to CD61 (Integrin β3) conjugated with FITC 494/520 (BD Biosciences, #555753) for 60 m on ice. Cells were washed with cold 0.5% BSA and 0.01% NaN3 in PBS and resuspended in the same buffer and analyzed with an Agilent Novocyte Quanteon using NovoExpress 1.4.1. Software (Santa Clara, CA).

Statistics.

GraphPad software online calculator was used to perform t tests to determine the statistical significance between two groups of data; Social Science Statistics one-way ANOVA calculator was used to perform comparison of several groups of data.

Results

HCMV Infection with and without the Pentamer Shows Cell Type–Specific Differences.

We showed that efficient HCMV internalization into monocytes requires the pentamer complex engagement of integrins (29, 36, 43, 92). We next compared various strains of HCMV with and without the pentamer to evaluate attachment, entry, and rapid changes in protein phosphorylation to identify the critical components that integrate attachment and the signaling required for productive entry of monocytes. The viral strains we analyzed included clinical-like and lab-adapted viruses that express the pentamer [strains TB40wt (30), TR-5wt (36), Merlin 5-mer (80), and BADrUL131 (21)] and laboratory-adapted or mutated strains that do not express the pentamer [BADwt (21); TB40ΔUL131-128 (30); TR-5ΔUL131-128 (36); and Merlin Bac pAL1393 (Merlin 3-mer (80)]. In addition to monocytes, we also tested infection of epithelial ARPE19 cells to compare cell type–specific changes. Virions of all selected virus strains were added to cells at an MOI = 1. Viral binding was measured by qPCR and expressed as the number of viral genomes per cell (ratio of UL123 copy number to CRP gene copy number) (Fig. 1 A and B). HCMV attachment to epithelial cells was generally uniform (Fig. 1A) and did not depend on whether the pentamer complex was expressed since no significant differences were noted when comparing paired viruses with and without the pentamer. However, for monocytes, the trimer-only strains demonstrated a significantly higher affinity of binding to their receptors compared to the analogous pentamer-expressing strains (Fig. 1B). To correlate this initial cell type–specific difference in binding affinities between viruses and their mutant counterparts, we next evaluated HCMV entry/internalization (14) (Fig. 1 C and D). We observed that, as expected, the pentamer was required for efficient viral entry/internalization in both cell types. The temperature-switching process involved in synchronous infection for entry assays did not affect virus internalization (SI Appendix, Fig. S1). These data (Fig. 1A vs. 1B) suggest that there are biological differences in pentamer binding between monocytes and epithelial cells.

Fig. 1. HCMV attachment/internalization into monocytes and epithelial cells is different. Pentamer-carrying viruses (TB40 wt, TR-5, BADrUL131, and Merlin 5-mer) and trimer-only carrying viruses [TB40Δ, TR-5Δ, BADwt, Merlin Bac pAL1393, and tet UL128L tet+ operator upstream of UL128 3-mer (Merlin 3-mer)] were used to perform viral binding assays and entry assays for epithelial ARPE-19 cells [(A) and (B)] and for primary human monocytes [(C) and (D)]. DNA was isolated and the ratio of viral genomes per cell (UL123/CRP) was determined by qPCR. Error bars indicate the SD of three experiments with monocytes from different blood donors or three independent experiments with ARPE-19 cells. A single asterisk indicates P < 0.05 and two asterisks indicate P < 0.01.

Pentamer-Carrying HCMV Rapidly Activates the Signaling Network in Monocytes.

To assess the role that phosphorylation (93, 94) played in the earliest events of the pentamer-mediated entry process, we performed a phosphoproteomic screen using an ELISA-based phosphoproteomic array (88). Freshly isolated monocytes were mock-infected or infected with either BADwt or BADrUL131 for 15 m and prepared as described (88). Compared to mock-infected cells, infection with the pentamer-carrying BADrUL131 showed increased phosphorylation of multiple residues (Fig. 2; complete array values displayed in SI Appendix, Table S2) involved in signaling, whereas BADwt, which lacks the pentamer, demonstrated a general inhibition of signaling (Fig. 2). Fig. 2 only shows key signaling molecules likely to be important for viral infection. In agreement with previously reported data, we found that the pentamer signaling induced rapid c-Src and Akt phosphorylation (29, 43, 44, 47), paxillin and STAT1 phosphorylation (43, 44). EGFR signaling, which is induced following HCMV gB binding (13, 14), showed increased phosphorylation after infection with both viruses. The most prominent pentamer-biased phosphorylated protein was a member of the PI3K/Akt signaling pathway, a dual phosphatase PTEN at Ser380 (8.5-fold induction); the trimer-only virus showed the opposite effect on PTEN phosphorylation. A similar trend was also observed with PTEN at Ser370. Phosphorylation at Ser380, as well as at Ser370, Ser382, and Ser385 of the PTEN tail domain, is known to inhibit PTEN phosphatase activity and block its substrate-binding and catalytic sites (70).

Fig. 2. Infection of monocytes with HCMV carrying the pentamer rapidly activates signaling pathways. Monocytes were infected with HCMV (BADrUL131 and BADwt) for 15 m at an MOI = 5, and then, phosphoprotein profiling using Full Moon BioSystems’ ELISA-based antibody array was performed. The black bars represent the ratio of the phosphorylation in monocytes infected with BADrUL131 vs. the phosphorylation seen in mock-infected cells. Error bars indicate the SD of three distinct experiments with monocytes from different blood donors [plus two technical replicates for each donor (six replicates)]. The gray bar represents the ratio of the phosphorylation in monocytes infected with BADwt vs. the phosphorylation seen in mock-infected cells. Error bars indicate the SD of two experiments with monocytes from different blood donors (four replicates). The values represent the fold change increase or decrease in phosphorylation (complete data are shown in SI Appendix, Table S2).

PTEN Phosphatase Activity Is Essential for HCMV Entry.

PTEN is a signaling phosphatase that regulates cellular membranes, endosomal maturation, and endocytosis (60, 95–97). The versatile role of PTEN in cell biology is shown in SI Appendix, Fig. S2. 1) As a tumor suppressor and metabolic regulator, PTEN opposes PI3K activity by dephosphorylating the signaling messenger PtdIns-(3, 4, 5)-P3. 2) PTEN is also involved in regulation of early endosome formation and 3) in dephosphorylating of the Rab7 that 4) controls endosomal maturation. PTEN colocalizes with the small GTPases Rab5, the hallmark protein of early endosomes (98), and with Rab7, the essential protein of late endosomes (99). PTEN dephosphorylates Rab7, prior to the Rab5-Rab7 switch, as a mechanism supporting endosomal maturation (99, 100). PTEN is found in the cytoplasm directly bound to the early endosome signature lipid PtdIns–[3]–P1 (59).

Phosphorylation of PTEN at Ser380 was the top hit in our phosphoproteomic screen. PTEN expression was silenced with siRNA (101) for 48 h to ensure maximal knockdown [experimentally determined half-life is reported to be 7.5 h (102)]. There was a substantial reduction of PTEN expression in response to cognate siRNA (almost sixfold) compared to control siRNA (Fig. 3A). We observed that PTEN knockdown did not affect entry/internalization of the trimer-only carrying strain BADwt into monocytes, but significantly decreased BADrUL131 entry/internalization into monocytes (Fig. 3B). We also observed this effect in clinical strains (TR-5/TR-5 delta) (SI Appendix, Fig. S3). In contrast, PTEN knockdown did not affect HCMV binding to monocytes (Fig. 3C) of either strain, suggesting that knockdown did not affect HCMV receptor accessibility. Loss of PTEN did not affect HCMV entry to epithelial cells (Fig. 3D), suggesting a cell type–specific role for PTEN during entry. The observed increase in phosphorylation of PTEN Ser380 and Ser370 suggested that HCMV signaling blocks PTEN phosphatase activity. To address if PTEN plays a role as a scaffold protein or if the observed change in virus entry was a result of a loss of PTEN enzymatic activity, we next treated cells with a PTEN-specific inhibitor, SF1670, which blocks lipid and protein phosphatase activity (84, 103). We observed that SF1670 inhibited HCMV entry/internalization into monocytes in a dose-dependent manner (Fig. 4A). Monocytes were preincubated with the drug in a range of concentrations from 1 µM to 40 µM, which corresponds (0.5 to 20) IC50. We found that SF1670 inhibited BADrUL131 entry, without affecting BADwt virus entry into monocytes. The data from the entry assay in Fig. 4A were used to calculate the inhibition parameters and the experimental IC50 for HCMV entry. The IC50 determined for inhibition of phosphatase activity (1.97 ± 0.24) µM was close to the reported IC50 of 2 µM (Fig. 4B) (84, 104). This similarity suggests a correlation between PTEN enzymatic activity and the pentamer-triggered viral entry. Treatment of cells with SF1670 (black bars, at the concentration 20 µM for 30 m at 37 °C) did not affect either HCMV entry into epithelial cells (Fig. 4C) or its attachment to monocytes (Fig. 4D). We reproduced these experiments with the clinical HCMV strains TR-5 (wt virus)/TR-5Δ (virus lacking the pentamer) and TB40/E and TB40/E delta (SI Appendix, Figs. S4 and S5). These results did not depend on viral gB content (SI Appendix, Fig. S6). Treatment with 20 µM SF1670 prior to infection effectively decreased pentamer-carrying HCMV entry across strains, without affecting pentamer-null virus entry.

Fig. 3. PTEN knockdown blocks entry of HCMV expressing the pentamer into monocytes but not into epithelial cells. (A–C) Monocytes were nucleofected with control or sense siRNA (29, 46), and 48 h posttransfection the samples were tested for PTEN and actin expression by western blot analysis without infection (A), for virus entry (B), or for virus attachment (C) following infection with BADwt or BADrUL131 (MOI = 5). The gray bars represent cells nucleofected with a negative control scrambled siRNA and the black bars represent cells nucleofected with sense siRNA to PTEN prior to infection. DNA was isolated and the ratio of viral genomes per cell (UL123/CRP) was determined by qPCR. Error bars indicate the SD of three experiments with monocytes from different donors. (D) ARPE-19 epithelial cells were nucleofected with the control and specific siRNA and infected 48 h later. Error bars indicate the SD of two independent experiments (four replicates). Two asterisks indicate P < 0.01.

Fig. 4. Inhibition of PTEN phosphatase activity blocks efficient entry of HCMV into monocytes. (A) Monocytes were treated with SF1670 prior to infection. Cells were preincubated with SF1670 at the indicated concentrations for 30 m at 37 °C. DNA was isolated, and the ratio of viral genomes per cell (UL123/CRP) was determined with qPCR. Entry of BADwt is shown in diamond bars and entry of BADrUL131 as solid bars. The solid lines represent significant differences in the values (one-way ANOVA) and the dashed line represents no statistical differences between values (one-way ANOVA). A single asterisk indicates P < 0.05, and two asterisks indicate P < 0.01. (B) Numeric data from the entry assay were described in a curve [curve fitting was conducted using the program KaleidaGraph (Synergy Software, Reading, PA)]. Parameters of inhibition are written in the inlet. (C) HCMV (MOI = 5) entry into ARPE-19 cells was examined following either pretreatment with SF1670 (20 µm, black bars) or DMSO as a control (gray bars). Error bars indicate the SD of four experiments and NS represents no statistical significance between samples. (D) HCMV infection of monocytes was examined following pretreatment with SF1670 (20 µm, black bars) or DMSO as a control (gray bars). Error bars indicate the SD of three experiments with monocytes from different donors and NS represents no statistical significance between samples.

SF1670 has been shown to reversibly inhibit the signaling membrane tyrosine phosphatase, CD45 (105, 106). Results of our phosphoproteomic screen revealed that monocyte infection with both pentamer-carrying BADrUL131 and trimer-only-carrying BADwt produced a similar effect on CD45 phosphorylation (SI Appendix, Fig. S7A). When we used siRNA to knock down CD45 expression, we did not see differences in entry between viruses with and without the pentamer (SI Appendix, Fig. S7B).

Inhibition of PTEN Depletes a Membrane Pool of the Early Endosome Signature Lipid PtdIns–(3)–P1.

Phosphatidylinositol phosphates play multiple roles in cell physiology (65, 107, 108). PTEN dephosphorylates all phosphatidylinositol phosphates phosphorylated in position 3 of the inositol ring. Its lipid substrates are molecules of high significance for cell signaling and trafficking: PtdIns-[3]-P1 is a structural lipid of early endosomes (63, 67); PtdIns-(3, 4)-P2 is a signaling lipid mostly localized in the plasma membrane (67); and PtdIns-(3, 4, 5)-P3 is a signaling molecule, which activates the PI3K/Akt pathway (54). In cells, PTEN is found bound to PtdIns-(3)-P1 of early endosomes tethered to microtubules (59). We next examined the extracted lipid composition of monocytes with and without treatment with SF1670 (Fig. 5A) or infected monocytes with either BADwt or BADrUL131 (Fig. 5B) using ultra-performance liquid chromatography–mass spectrometry. We observed that the phosphatidylinositol phosphate composition of monocyte membranes changes with SF1670 treatment or infection. Compared to the untreated mock-infected cells, inhibition of PTEN phosphatase activity in uninfected cells increased the relative amount of PtdIns-(3, 4, 5)-P3, a short-lived signaling molecule and a major PTEN lipid substrate (Fig. 5A). The increase was not significant, but an expected trend was seen. Perhaps a longer incubation with SF1670 (only 30-m inhibition was used to match our experimental entry setup) would show a greater accumulation of PtdIns-(3, 4, 5)-P3. The effect of BADrUL131 infection on PtdIns-(3, 4, 5)-P3 levels was even stronger than the effect of SF1670 alone (Fig. 5B). PtdIns-(3, 4, 5)-P3 accumulation following infection can be explained by activation of its synthesis by PI3K and reduction of dephosphorylation due to inhibition of PTEN phosphatase activity by the pentamer. In agreement with our phosphoproteomic data, infection with trimer-only carrying virus (BADwt) did not affect PtdIns-(3, 4, 5)-P3 levels (Fig. 5B).

Fig. 5. Examination of phospholipids in monocytes following inhibition of PTEN or infection with wt or mutant virus. Phosphatidylinositol phosphates were extracted and analyzed using liquid chromatography and mass spectrometry. The intensity of a peak corresponding to each phosphatidylinositol phosphate was normalized to the intensity of mock-infected cells. PtdIns-(3,4,5)-P3, PtdIns-(4,5)-P2, and PtdIns-(3)-P1 and are depicted as black, gray, and diamond bars, respectively. (A) Effect of inhibition of PTEN activity on major phosphatidylinositol phosphates in uninfected monocytes following inhibition of PTEN (SF1670, 20 µM) activity. Statistical differences between control cells and SF1670-treated cells were not significant for PtdIns-(3,4,5)-P3 and PtdIns-(4,5)-P2 (dotted and dashed lines) but were significant for PtdIns-(3)-P1 (solid line; single asterisk indicates P < 0.05). (B) Major phosphatidylinositol phosphates were examined in short-term infected [BADrUL131 and BADwt (MOI = 5)] monocytes (at 15 mpi). Error bars indicate the SD of three experiments with monocytes from different donors. A single asterisk indicates P < 0.05; two asterisks indicate P < 0.01; three asterisks indicate P < 0.001; and, NS indicates no significance.

PtdIns-(4, 5)-P2 is the product of PtdIns-(3, 4, 5)-P3 when dephosphorylated by PTEN (67). It is known that PtdIns-(3, 4)-P2 is abundant in cells and is usually found at higher levels than PtdIns-(3, 4, 5)-P3 or PtdIns-(3)-P1 (109). The increase in the PtdIns-(3, 4, 5)-P3 level after monocyte infection with BADr-UL131, which as we showed above blocks PTEN phosphatase activity, was accompanied by a consistent decrease in PtdIns-(4, 5)-P2 in all donors, although it did not reach the level of statistical significance at 15 mpi. Infection with BADwt alone (Fig. 5B) showed a statistically significant decrease of PtdIns-(4, 5)-P2, which could be explained by the global signaling declination observed after BADwt infection. We were especially interested in the effect of SF1670 and infection on PtdIns-(3)-P1, the essential lipid of early endosomes. The results shown in Fig. 5 A and B indicate that SF1670 alone and infection with HCMV led to strong depletion of accessible PtdIns-(3)-P1, which would affect endocytic vesicle formation and involvement of this lipid in dynamic metabolic processes.

Inhibition of PTEN activity decreased the amount of PtdIns-(3)-P1 in uninfected cells (Fig. 5A), which would explain the inhibition of entry of the pentamer-carrying virus reported in Figs. 3 and 4 when cells were pretreated with the PTEN inhibitor. The data indicating that the lack of PtdIns-(3)-P1 only decreased pentamer-carrying HCMV entry, but not trimer-only HCMV entry, into monocytes were very exciting to us. First, it suggested that there are specific lipid components and specific vesicles [regulated in this case by PTEN or at least requiring a certain level of PtdIns-(3)-P1] that are required following pentamer engagement of integrins for the macropinocytosis event that promotes internalization of HCMV into monocytes. These lipid components and possible vesicle structure are not used for the inefficient entry of virus lacking the pentamer. We do note a decrease in levels of PtdIns-(3)-P1 after BADwt infection (Fig. 5B), which we believe is explained at least in part by the global negative signaling outcome when monocytes are infected with this defective virus. The data also hinted that because pentamer signaling following infection [at 15 mpi (Fig. 5B)] also decreased PtdIns-(3)-P1 that there was a role for PTEN inhibition postentry in the inhibition of maturation of early endosomes to late endosomes.

Inhibition of PTEN Phosphatase Activity Rescues the Pentamer-Minus Virus from Degradation in Late Endosome and Restores Viral Trafficking.

Internalized mature virus is rapidly found in EEA1-positive endosomes, then in the TGN, and finally in Rab11+ recycling endosomes as a mature viral particle (13, 36). De-envelopment only occurs in Rab11+ endosomes starting ~1 dpi, prior to the nuclear translocation of viral DNA, which begins at ~3 dpi (13, 36). In the absence of the pentamer, HCMV is promptly transported into late endosomesand degraded (36). We hypothesized that pentamer-driven inhibition of PTEN activity is the key driver of the unique viral trafficking pattern observed in monocytes and that targeted inhibition would restore this trafficking in viruses lacking the pentamer. Therefore, we used SF1670 to block PTEN activity in monocytes 1) prior to infection (Fig. 6) and then 2) postentry (Fig. 7, which we theorized would functionally mimic downstream pentamer activity) to see whether we could restore productive trafficking in viruses lacking the pentamer. We infected monocytes with either BADwt or BADrUL131, fixed, permeabilized, and then stained the cells with Abs to the HCMV envelope (gB), early endosomes (EEA1), late endosomes CI-M6PR (M6PR), and recycling endosomes (Rab11). We measured and quantified the distance between the viral particles and the closest target organelle with a N-SIM E Super-Resolution Microscope system (13). Shorter distances indicate colocalization (13). At 30 mpi, most of the pentamer-carrying viral particles are colocalized (≤0.2 µm) with EEA1+ endosomes, while in contrast, trimer-only-carrying viral particles are generally not colocalized (>0.2 µm) with EEA1+ endosomes (Fig. 6A). In monocytes with PTEN activity inhibited, the trimer-only BADwt viral particles became significantly more associated with EEA1+ vesicles, similar to that seen with viral particles containing the pentamer, suggesting that PTEN inhibition rescues pentamer-lacking virus from degradation.

Fig. 6. Inhibition of PTEN phosphatase activity prior to infection rescues virus without the pentamer allowing trafficking to early endosomes (A and B) and through the TGN to recycling endosomes (C). Monocytes were treated with SF1670 (20 µM, for 30 m at 37 °C) and then infected with either BADwt or BADrUL131 at an MOI = 10, fixed, permeabilized, and then stained with specific primary Abs to detect EEA1 (early endosomal marker), CI-M6PR (M6PR, late endosomal marker), Rab11 (recycling endosome marker), or HCMV gB (viral envelope marker). To detect the colocalization of gB and EEA1, the cells were infected for 30 mpi; to detect the colocalization of gB and M6PR, the cells were infected for 30 mpi; and, to detect the colocalization of gB and Rab11, the cells were incubated for 24 hpi. High-resolution images were acquired using a Nikon N-SIM E Super-Resolution Microscope. The distances between gB and the closest appropriate organelle were quantified. Data are combined from three biological replicates. Each data point represents an individual viral particle to the organelle measurement, and each condition includes at least 50 data points. A single asterisk indicates P < 0.05; two asterisks indicate P < 0.01; three asterisks indicate P < 0.001; and NS indicates no significance.

Fig. 7. Inhibition of PTEN phosphatase activity after HCMV internalization rescues virus without the pentamer. Monocytes were infected with either BADwt or BADrUL131 at an MOI = 10 and 10 mpi treated with the inhibitor of PTEN phosphatase activity SF1670 (20 µM). To detect the colocalization of gB and EEA1 cells were infected for 30 mpi (A). To detect the colocalization between gB and M6PR cells were infected for 30 mpi (B). Infected monocytes then were washed, fixed, permeabilized, and stained with specific primary antibodies to detect EEA1 (early endosomal marker), M6PR (late endosomal marker), or HCMV gB (viral envelope marker). High-resolution images were acquired using a Nikon N-SIM E Super-Resolution Microscope. The distances between gB and organelle were quantified. Data are combined from three biological replicates. Each data point represents an individual viral particle to the organelle measurement and each condition includes at least 50 data points. A single asterisk indicates P < 0.05; two asterisks indicate P < 0.01; three asterisks indicate P < 0.001; and NS indicates no significance.

When looking at viral particles in late endosomes at the same point (30 mpi), we noted several findings. First, that most of the particles regardless of virus or treatment do end up in late endosomes (Fig. 6B). We previously noted a bottleneck prior to movement of the normal virus (those with the pentamer complex) to the TGN and that only a lower number of viral particles made it to the TGN from the total input and those found in EEA1+ endosomes. Second, that the number of viral particles that do move through the TGN seemed constant as the virus moved to Rab11+ vesicles (13). These data are consistent with data from another study from the lab that looked at translocated DNA in the nucleus of infected monocytes (29). We now present evidence as to why that bottleneck occurs—most of the virus ends up in late endosomes in monocytes. This finding also probably explains why a fibroblast MOI of 10 is roughly equivalent to a monocyte MOI of 1 to 2.

In Fig. 6B we note that SF1670 treatment had no effect on the localization of pentamer-containing virus, but does show that virus without the pentamer (plus SF1670 treatment) is less likely to be colocalized with the late endosomes, consistent with the data from the parallel-timed experiment shown in Fig. 6A. At 24 hpi (Fig. 6C), we observed colocalization between the virus particles carrying pentamer and recycling endosomes, while viruses that lack the pentamer do not colocalize with recycling endosomes. Viruses not found in recycling endosomes at 24 hpi are degraded (29, 36). Pretreatment of cells with the PTEN inhibitor SF1670 rescued pentamer-lacking virus allowing for colocalization of the viral particles with recycling endosomes (Fig. 6C). We note that SF1670 treatment affects pentamer containing viral infection at later times of infection in a negative manner, while rescuing the pentamer-null virus. We suggest that normal virus with the pentamer does inhibit PTEN activity, but likely not to zero activity, and perhaps under these normal conditions with pentamer signaling, a low level of PTEN activity is required. We use the presence of the virus in recycling endosomes as a marker of productive infection (13, 36). Similar results were observed in PTEN knockdown cells (SI Appendix, Fig. S8). Additionally, we pretreated monocytes with SF1670 and monitored amount of viral genome remaining in the cells at 3 dpi, a time point in which we have previously published that the majority of viral genomes are within the nucleus (13, 36) (SI Appendix, Fig. S9). Treatment with SF1670 led to an approximate 24-fold increase in BADwt DNA, suggesting that pentamer-associated inhibition of PTEN is an important factor for efficient HCMV nuclear translocation and subsequent infection. Because the addition of SF1670 did not fully restore BADWT DNA levels to that of its pentamer-carrying counterpart (BADrUL131), excitingly, the data also suggest that other pentamer-associated signaling events necessary for nuclear translocation exist. The results shown in Fig. 6 imply that inhibition of PTEN rescues trafficking of defective, trimer-only-carrying HCMV. We are aware that lack of PTEN activity previral infection inhibits efficient viral entry, however, the low number of particles that did enter, trafficked correctly when PTEN was inhibited. Nevertheless, we next wanted to inhibit PTEN activity postentry to confirm that PTEN inhibition could rescue defective virus trafficking. PTEN activity was inhibited 10 mpi after the virions were internalized and found in EEA1+ endosomes (Fig. 7). We observed the same trend that SF1670 treatment rescued the viral trafficking pathway process in viruses lacking the pentamer (Fig. 7 A and B). Representative immunofluorescence images from Figs. 6 and 7 and SI Appendix, Fig. S8 are shown in SI Appendix, Fig. S10. From these data, we argue that we have uncovered a molecular step regulating entry/internalization to this clinically relevant cell type.

Monocyte Treatment with the PTEN Phosphatase Inhibitor Does Not Rescue HCMV-Induced Signaling.

EGFR and integrin signaling cascades are independently essential for translocation of the viral particle to the nucleus and for activation of the PI3K/Akt signaling axis (110). PTEN, as a signaling phosphatase, inhibits PI3K/Akt signaling and as well as some RTK and integrin signaling pathways (53, 55, 111). In addition, PTEN can positively regulate some pathways such as p53 (52, 97, 112). We next wanted to test the impact of inhibition of PTEN activity on downstream signaling pathways important in viral trafficking [Akt (SI Appendix, Fig. S11A), c-Src (SI Appendix, Fig. S11B), and ERK (SI Appendix, Fig. S11C)]. We inhibited PTEN with SF1670 and infected cells and analyzed the expression of various signaling proteins (pan-c-Src vs. P-Src, pan-Erk 1/2 vs. P-Erk 1/2, and pan-Akt vs. P-Akt). The results shown in SI Appendix, Fig. S11 A–C demonstrate that although inhibition of PTEN expectedly increased signaling through c-Src, Erk 1/2 and Akt in control cells, there was no increase in these signaling pathways when cells were pretreated with SF1670 and infected with the defective virus (SI Appendix, Fig. S11 A–C; representative blots show in SI Appendix, Fig. S12). The results suggest that rather than restoration of virus-induced signaling PTEN inhibition seems to instead rescue viral particles from degradation via management of endosomal maturation.

c-Src and Other Kinases Phosphorylate PTEN.

Data from Fig. 5 show that HCMV containing the pentamer results in a distinct pattern of phospholipids following infection, which when coupled to the rapid phosphorylation of PTEN (Fig. 2), suggests that the pentamer via receptor-ligand signaling, mediates these processes during infection. Phosphorylation of various domains on PTEN is known to inhibit PTEN phosphatase activity (69). These PTEN residues can be phosphorylated by several kinases including c-Src (74, 75), CK2 (113, 114), GSK-3α and/or β [GSK (76, 77), and possibly LKB1 (78)]. We next asked whether specific inhibition of the 3 kinases where pharmacological inhibitors exist [c-Src (PP2 for c-Src (115), CK2 (TBB for CK2 (116), and GSKα and β (AR-A014418 for both GSK isoforms (117, 118)] affected levels of PTEN phosphorylation postinfection. At the time of these studies, inhibitors to LKB1 were not available for purchase. We observed significant loss of PTEN phosphorylation following inhibition of each of these three pathways (Fig. 8A; representative blot shown in SI Appendix, Fig. S12). Since we consider HCMV-pentamer-triggered integrin activation of c-Src signaling to be an essential step in HCMV trafficking, a phosphoproteomic screen was also performed for monocytes treated with the c-Src inhibitor PP2 (10, 13, 29, 36, 43). We observed (Fig. 8B; raw values displayed in SI Appendix, Table S3) that inhibition of c-Src activity resulted in inhibition of PTEN phosphorylation and the likely loss of activation GSK3α/β (as measured by decreased phosphorylation of several residues on both GSK isoforms). We saw no change in CK2, likely because this is a constitutively activated kinase (119).

Fig. 8. Examination of kinases that phosphorylate PTEN. (A) Western blot analysis of cells pretreated with inhibitors to CK2, c-Src, and GSK3α/β kinase activity prior to infection. Monocytes were infected with HCMV [BADrUL131 (MOI = 5)] for 15 m. Band densities were quantified by LiCor Image Studio software and normalized to mock-infected cell target protein level. The level of P-PTEN was expressed as a percent of the pan protein. Error bars indicate the SD of three experiments with monocytes from different blood donors. A single asterisk indicates P < 0.05 and two asterisks indicate P < 0.01. (B) Phosphoproteomic data showing kinases relevant to PTEN phosphorylation in infected monocytes at 15 mpi (BADrUL131 at an MOI = 5) pretreated with or without a selective c-Src inhibitor PP2 (12, 13, 43, 46). Fold change compares phosphorylation patterns between cells treated with and without PP2 (note three donors with two technical replicates serve as the data for no PP2 vs. two technical replicates from a single donor for with PP2).

Pentamer-Driven Inhibition of PTEN Prevents the Rab7 Dephosphorylation Required for Early Endosome Maturation to Late Endosomes.

Our IF experiments revealed that targeted inhibition of PTEN phosphatase activity rescued defective, trimer-only virus allowing for the restoration of viral trafficking previously shown to be dependent on pentamer-induced signaling (29). This rescue experiment uncovered a convergence of pentamer-binding, c-Src (and other kinase) signaling, PTEN phosphorylation and correct viral trafficking. But how might viral-induced PTEN inactivation promote successful trafficking and nuclear translocation? It has been shown that Rab7 is a protein substrate of the PTEN tyrosine phosphatase (62, 99). PTEN dephosphorylates Rab7 at Ser72 and Tyr183 prior to its recruitment to early endosomes (79). This step is required for endosomal maturation (99, 120). Therefore, we next examined the effect of HCMV infection on Rab7 phosphorylation status. Monocytes were treated with SF1670 or treated with a diluent control, infected with BADwt or BADrUL131 and cell lysate harvested for immunoprecipitation and immunostaining analysis with Abs to pan-Rab7 and for P-Tyr (to detect Tyr183) (Fig. 9). We observed that BADrUL131 signaling significantly increased Rab7 phosphorylation compared to mock-infected or BADwt-infected cells. Inhibition of PTEN phosphatase activity with SF1670 also blocked Rab7 dephosphorylation in mock-infected cells. In cells infected with BADwt, SF1670 treatment significantly increased the amount of phosphorylated Rab7 in cells (Fig. 9; representative blot shown in SI Appendix, Fig. S12). Because we can mimic the lack of pentamer signaling through PTEN inactivation either prior to entry or postentry, we suggest that the data strongly support our hypothesis on how pentamer-based signaling through PTEN inactivation and maintenance of Rab7 phosphorylation drives trafficking of the viral particle in monocytes.

Fig. 9. Inhibition of PTEN phosphatase activity in monocytes prevents Rab7 dephosphorylation. Monocytes were treated with SF1670 (20 µM for 30 m at 37 °C) prior to infection (black bars) or with an equal volume of DMSO (gray bars). Cells were then infected with BADwt or BADrUL131 (MOI = 5) for 30 m, washed, and harvested. Rab7 was immunoprecipitated and analyzed by western blot analysis. The level of P-Rab7 was expressed as a percent of pan-Rab7 in arbitrary units (a.u.). Error bars indicate the SD of four experiments with monocytes from different blood donors. A single asterisk indicates P < 0.05; two asterisks indicate P < 0.01; three asterisks indicate P < 0.001; and, NS indicates no significance.

Inhibition of PTEN Activity Does Not Affect Accessibility of HCMV Signaling Receptors.

It is possible that inhibition of PTEN prior to infection could affect the availability of viral receptors on the cell surface. Thus, we wanted to determine whether inhibition of PTEN activity affected accessibility of HCMV signaling receptors EGFR and beta integrins. Monocytes were treated with the PTEN phosphatase inhibitor (SF1670) and cells were subsequently stained with Abs to EGFR, β1-and β3-integrins. Receptor expression was then analyzed by flow cytometry. Results presented in SI Appendix, Fig. S13 indicate that cells express similar amount of EGFR (SI Appendix, Fig. S13A) and β1/β3 integrins (SI Appendix, Fig. S13 B–D) when treated with SF1670 compared to untreated cells.

Discussion

Peripheral blood monocytes are essential for HCMV dissemination throughout the body and the establishment of lifelong persistence (14, 42, 92, 121–124). To better understand how viral attachment drives entry, we compared infection of primary human monocytes infected with HCMV strains containing the pentamer vs. those without the pentamer and then performed a phosphoproteomic screen. We found that the most prominent pentamer-biased phosphorylated protein early after infection of monocytes was the lipid- and protein-phosphatase PTEN. Treatment of monocytes with siRNA targeting PTEN decreased pentamer-mediated viral entry. Inhibition of PTEN phosphatase activity showed the same effect. This effect was only seen when we examined viral entry into monocytes, suggesting these clinically relevant cells show differences in the nature of the vesicle required for viral internalization. Likely for monocytes, this vesicle requires the presence of PtdIns-(3)-P1 or at least high levels of this PtdIns, a scenario not required for viral internalization in epithelial cells. In addition, this effect was only seen when viruses contain the pentamer, further emphasizing that pentamer attachment and signaling drives a unique entry pattern in monocytes. This pentamer-biased signaling drives c-Src (and GSK) activation, which seems needed for the PTEN phosphorylation seen following infection. Constitutively active (or at least not c-Src driven) CK2 also seems to influence the status of PTEN phosphorylation. We found that regulation of PTEN also plays a role in HCMV infection postentry. We observed that pentamer-triggered inhibition of PTEN phosphatase during attachment is required to support the trafficking of virus-carrying EEA1-positive vesicles. Additionally, we show that loss of PTEN phosphatase activity prevents dephosphorylation of the late endosomal protein Rab7, inhibiting early-to-late endosomal maturation, thereby allowing viral particles to traffic from the early endosome to the TGN and then to recycling endosomes, while avoiding late endosomes and degradation of the viral particle. Lastly, our results demonstrate that we can rescue defective trafficking of pentamer-null/mutant viruses just via PTEN pharmacological inhibition.

We examined here HCMV attachment and entry (14, 29, 43, 83, 125) in different permissive cell types and found that HCMV attachment to monocytes was distinct relative to other cell types examined. The trimer-only containing strains of HCMV demonstrated a higher affinity of binding only to monocytes, but not to epithelial cells, compared to the pentamer-carrying strains. We speculate this difference in monocytes is related to the nature of the integrins engaged and how trimer and pentamer viruses engage integrins (29). In a previous report (29), we noted that in trimer-only viruses, gH bound to both β1- and β3-integrins, while when the virus contained the pentamer, gH only bound to β1-integrins and it was the UL128-131 complex that bound to β3-integrins. We suggest that affinity for attachment is a physical signal that along with the associated outside-in signaling dictates entry. Results of our phosphoproteomic screen showed that the pentamer triggered a rapid increase in global phosphorylation in monocytes, while the trimer-only viruses inhibited global phosphorylation.

The phosphoproteomic screen revealed that the lipid- and protein-phosphatase PTEN was rapidly and strongly phosphorylated. Knockdown of PTEN expression or blocking PTEN activity affected pentamer-mediated viral entry in monocytes, but not in epithelial cells. In these experiments in the absence of PTEN activity, pentamer-carrying virus entry was strongly reduced. Entry of the trimer-only carrying virus remained virtually unaltered by PTEN activity loss. We hypothesized that inhibition of PTEN prior to infection or during infection alters endosome function or endocytic process. The data from our mass spectrometry results supported that HCMV enters (or is internalized) into monocytes by an endocytic process, rather than by direct fusion, since loss of PTEN activity correlated with the loss of PtdIns-(3)-P1 content, which we know in turn affects vesicle formation. Loss of PtdIns-(3)-P1 as a result of PTEN inhibition by phosphorylation prior to infection (mock + SF1670) or during infection (the pentamer signaling followed by PTEN phosphorylation) could be explained by dissociation of substrate-protecting PTEN/PtdIns-(3)-P1 complexes. As a PTEN lipid substrate, PtdIns-(3)-P1 fits the substrate binding pocket, which PTEN uses for attachment to different types of early endosomes (59, 70, 126). This binding protects PtdIns–(3)–P1 from other enzymes (127). There is no indication that PTEN hydrolyzes PtdIns–(3)–P1 in vivo or in vitro. Phosphorylation of PTEN changes the protein conformation and blocks its substrate-binding and catalytic centers (70). Dissociation of a substrate–PTEN complex makes PtdIns–(3)–P1 available for dephosphorylation with its specific phosphatase TPIP (128) or for additional phosphorylation at position 5 by type III kinases such as PIKfyve (65, 129). The loss of the PtdIns-(3)-P1 needed for formation of new macropinosomes and/or early endosomes would result in decreased potential for endocytosis. This would influence the initial viral entry if vesicle formation was altered prior to infection, as we noted in Figs. 4 and 5, and would, if altered postinfection, block late endosome maturation and promote trafficking through the TGN and into recycling endosomes (noted here and in ref. 99). Any loss of PTEN activity due to phosphorylation would be expected to be irreversible and to lead to PTEN proteasomal degradation (71, 72, 130). On the other hand, when mutant viral (BADwt) infection occurs, the virus in whatever initial vesicle it is found in, would undergo subsequent maturation to late endosomes, creating a different explanation for the loss of PtdIns–(3)–P1 observed in Fig. 6. During this process, the vesicle maturation with the mutant virus being targeted for degradation, any PtdIns-(3)-P1 in these vesicles would be enzymatically metabolized during this process of cargo degradation (65, 131, 132).

We observed that inhibition of PTEN is a pentamer-specific event that inhibits endosomal maturation from early to late endosomes and redirects trafficking of the virus toward TGN and Rab11+ recycling endosomes and ultimately, the nucleus. Cells can exploit this trafficking route to protect a cargo in the trafficked vesicles from degradation (133). We examined the limiting step of endosomal maturation, Rab7 dephosphorylation prior to the Rab5/Rab7 switch. PTEN is the enzyme, which catalyzes this reaction (62, 134). We observed that inhibition of PTEN phosphatase activity by phosphorylation due to pentamer-induced signaling or directly with an inhibitory drug significantly decreased Rab7 dephosphorylation. Our results suggest that the lack of pentamer signaling and the resulting chronic PTEN activation following infection of mutant viruses promote endosomal maturation toward late endosomes and viral particle degradation. Because we can overcome this block through PTEN inactivation either prior to entry or postentry, we suggest that the data strongly support that pentamer-induced signaling through PTEN activation and maintenance of Rab7 phosphorylation drives trafficking of the viral particle in monocytes in a retrograde-like manner distinct from that seen in epithelial cells. Based on the evidence presented herein, we propose the model depicted in Fig. 10, in which the pentamer-induced phosphorylation of the PTEN tail domain serves as a rapid regulator of the early steps of HCMV infection of monocytes. Together, we suggest that in clinically relevant monocytes, in which the mature viral particle is retained in vesicles until de-envelope occurs after ~1 dpi and prior to nuclear translocation at 3 dpi, the virus has evolved an elaborate strategy to navigate the infection of a difficult immune cell that is designed to digest and degrade infectious agents.Fig. 10. Summary. Our screen identified that pentamer-induced phosphorylation of the PTEN tail domain (on Ser370 and Ser380) was rapidly regulated early after infection of monocytes. When PTEN activity was inhibited prior to infection, viral entry into monocytes, but not into epithelial cells, was inhibited. Only pentamer-driven viral entry into monocytes was affected. Analysis of changes in PtdIns following loss of PTEN activity documented diminished levels of PtdIns-(3)-P1, which has been shown to be important in macropinosome/early endosome formation. The data provide strong evidence that pentamer-driven (see red box) viral entry/internalization into monocytes occurs through an endocytic process (see green bracket) and that early endosome formation and the entry process in monocytes is distinct from that observed in epithelial cells. Furthermore, following infection, we also see loss of PtdIns-(3)-P1, which along with retained phosphorylated Rab7 prevents early to late endosome formation and degradation of the viral cargo (see Red “X”). We propose that in the absence of PTEN phosphatase activity [due to pentamer/integrin/c-Src/GSK signaling, plus a possible role for CK2 signaling (red arrow)], the virus is directed toward the early endosome, the TGN and recycling endosomes (green arrow). Mechanistically, we uncovered a single specific pentamer required event that promotes productive infection of monocytes. When monocytes were infected with a pentamer null virus that ordinarily gets degraded in late endosomes, we found that we can overcome this negative pathway by inhibiting PTEN function. Molecularly, our data showed that pentamer-induced inhibition of PTEN function allows Rab7 to remain phosphorylated, which blocks early endosome to late endosome maturation and promotes the smooth transition of virus in early endosomes to the TGN and then to recycling endosomes where de-envelopment occurs as a precursor to nuclear translocation and productive infection.

Supplementary Material

Appendix 01 (PDF)

We thank all blood donors who contributed for this study. We also thank D.C. (LSUHS Core Facility) for flow cytometric help and V.Z., G. Raft, and S. Tap for inspiration and data analysis. Portions of the paper were developed from the thesis/dissertation of B.S.M. The work was funded by the NIH Grants AI056077, AI127335, AI15962, P20GM134974, P20GM121307, P20GM121288, and P30GM110703 (A.D.Y.).

Author contributions

L.S.C., B.S.M., H.L.F., and A.D.Y. designed research; L.S.C., B.S.M., H.L.F., H.W.N., and A.K.S. performed research; L.S.C., B.S.M., H.L.F., H.W.N., A.K.S., and A.D.Y. analyzed data; and L.S.C., B.S.M., H.W.N., and A.D.Y. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.

Supporting Information

This article is a PNAS Direct Submission.
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