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. 2018 Aug 16;92(17):e00162-18.
doi: 10.1128/JVI.00162-18. Print 2018 Sep 1.

The Human Cytomegalovirus Protein UL148A Downregulates the NK Cell-Activating Ligand MICA To Avoid NK Cell Attack

Liat Dassa  1 , Einat Seidel  1 , Esther Oiknine-Djian  2   3   4 , Rachel Yamin  1 , Dana G Wolf  2   3   4 , Vu Thuy Khanh Le-Trilling #  5 , Ofer Mandelboim #  6
Affiliations

The Human Cytomegalovirus Protein UL148A Downregulates the NK Cell-Activating Ligand MICA To Avoid NK Cell Attack

Liat Dassa et al. J Virol. .

Abstract

Natural killer (NK) cells are lymphocytes of the innate immune system capable of killing hazardous cells, including virally infected cells. NK cell-mediated killing is triggered by activating receptors. Prominent among these is the activating receptor NKG2D, which binds several stress-induced ligands, among them major histocompatibility complex (MHC) class I-related chain A (MICA). Most of the human population is persistently infected with human cytomegalovirus (HCMV), a virus which employs multiple immune evasion mechanisms, many of which target NK cell responses. HCMV infection is mostly asymptomatic, but in congenitally infected neonates and in immunosuppressed patients it can lead to serious complications and mortality. Here we discovered that an HCMV protein named UL148A whose role was hitherto unknown is required for evasion of NK cells. We demonstrate that UL148A-deficient HCMV strains are impaired in their ability to downregulate MICA expression. We further show that when expressed by itself, UL148A is not sufficient for MICA targeting, but rather acts in concert with an unknown viral factor. Using inhibitors of different cellular degradation pathways, we show that UL148A targets MICA for lysosomal degradation. Finally, we show that UL148A-mediated MICA downregulation hampers NK cell-mediated killing of HCMV-infected cells. Discovering the full repertoire of HCMV immune evasion mechanisms will lead to a better understanding of the ability of HCMV to persist in the host and may also promote the development of new vaccines and drugs against HCMV.IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen which is usually asymptomatic but that can cause serious complications and mortality in congenital infections and in immunosuppressed patients. One of the difficulties in developing novel vaccines and treatments for HCMV is its remarkable ability to evade our immune system. In particular, HCMV directs significant efforts to thwarting cells of the innate immune system known as natural killer (NK) cells. These cells are crucial for successful control of HCMV infection, and yet our understanding of the mechanisms which HCMV utilizes to elude NK cells is partial at best. In the present study, we discovered that a protein encoded by HCMV which had no known function is important for preventing NK cells from killing HCMV-infected cells. This knowledge can be used in the future for designing more-efficient HCMV vaccines and for formulating novel therapies targeting this virus.

Keywords: HCMV; NK cells; immune evasion.

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Figures

FIG 1
FIG 1
HCMV strain VarS fails to downregulate the NKG2D ligand MICA. (A) Scheme of HCMV genome organization. UL, unique long; US, unique short; TRL, terminal repeat long; IRS, internal repeat short; TRS, terminal repeat short; IRL, internal repeat long; RL, repeat long. (B to F) FLS1 HFFs (MICA*004/*009:01-*049) were either mock infected or infected with the indicated HCMV strains. Cells were harvested at 72 h postinfection (hpi), and the surface expression of the following NKG2D ligands was assayed by flow cytometry. (B) MICA. (C) MICB. (D) ULBP1. (E) ULBP2/5/6. (F) ULBP3. Black, blue, and red histograms represent mock-infected, BAC2-infected, and VarS-infected HFFs, respectively. Gray-filled histograms (BG [background]) represent secondary antibody staining of the mock-infected cells, which produced similar results for all cells.
FIG 2
FIG 2
Deletion of UL148A hampers the reduction in MICA surface expression during HCMV infection. (A) Schematic representation of AD169-ULb′ ORFs. UL140 to UL144 are depicted in gray, since they are deleted in part or completely in the VarL variant. Red lines indicate gene blocks deleted in the BAC2 mutants. (B to I) FLS3 HFFs overexpressing MICA*004-HA were mock infected, BAC2 infected, or infected with the indicated BAC2 deletion mutants. Cells were harvested at 72 hpi, and MICA surface expression was assessed by flow cytometry. Black, blue, and red histograms represent mock-infected, BAC2-infected, and BAC2 deletion mutant-infected HFFs, respectively. Gray-filled histograms represent secondary antibody-only staining of mock-infected cells, which produced similar results for all cells. Histograms represent combined results of independent experiments.
FIG 3
FIG 3
UL148A downregulates full-length MICA alleles but not the truncated allele MICA*008. The indicated HFFs were mock infected, BAC2 infected, or infected with one of the following BAC2 deletion mutants: BAC2 ΔUL148A or BAC2 ΔUL148B. Cells were harvested, and MICA surface expression was assessed by flow cytometry. (A to C) FLS3 HFFs overexpressing MICA*004-HA, harvested 48 hpi. (D to F) FLS3 HFFs overexpressing MICA*008-HA, harvested 48 hpi. (G to I) FLS1 HFFs (MICA*004/*009:01-*049), harvested 24 hpi. Black histograms represent MICA staining of mock-infected cells, red histograms represent MICA staining of cells infected with the indicated virus, and gray-filled histograms represent isotype control staining of mock-infected cells, which produced similar results for all cells.
FIG 4
FIG 4
UL148A requires a viral interaction partner external to the ULb′ region. (A to C) FLS1 HFFs (MICA*004/*009:01-*049) transduced with an EV (A), with UL148A (B), or with C-His UL148A (C) were either mock infected (black histograms) or VarS infected (red histograms). Cells were harvested 24 hpi, and MICA surface expression was assessed by flow cytometry. Gray-filled histograms represent isotype control staining of mock-infected cells, which produced similar results for all cells. (D and E) FLS1 HFFs (MICA*004/*009:01-*049) transduced with an EV or with C-His UL148A were either mock infected or infected with VarS or BAC2. (D) Cells were lysed 24 hpi, and Western blotting was performed using anti-MICA antibody for detection of MICA, anti-HIS antibody for detection of UL148A, and anti-vinculin antibody as a loading control. (E) Quantification of two independent experiments. MICA protein levels were quantified relative to the loading control (vinculin). RQ, relative quantification. Error bars represent SEM. Student's t test was performed to evaluate significance. *, P < 0.05. (F) FLS1 HFFs (MICA*004/*009:01-*049) transduced with an EV or with C-His UL148A were either mock infected or infected with mutant BAC2 ΔUL148A or BAC2. (F) Cells were lysed 24 hpi, and Western blotting was performed using anti-MICA antibody for detection of MICA, anti-HIS antibody for detection of UL148A, and anti-vinculin antibody as a loading control.
FIG 5
FIG 5
UL148A's downregulation of MICA is functional, and UL148A targets MICA to lysosomal degradation. (A) FLS3 MICA*004-HA cells were mock infected or infected with BAC2 or the BAC2 ΔUL148A mutant. NK cells were incubated 48 hpi with a blocking anti-NKG2D MAb or with no antibody and then incubated with the aforementioned HFFs. Data represent results from two independent experiments. Error bars represent SEM. Student's t test was performed to evaluate significance. *, P < 0.05; **, P < 0.005; N.S., nonsignificant. (B to E) FLS3 MICA*004-HA cells were mock infected (M) or infected with BAC2 (B) or with BAC2 ΔUL148A (Δ) and were incubated for 12 h with a vehicle-only treatment (Vehicle), lysosome inhibitors (in panel B, leupeptin [Leu], NH4Cl, or concanamycin A [ConA]), or proteasome inhibitors (in panel C, epoxomicin [Epx] or MG132). Cells were lysed 48 hpi, and Western blotting was performed using anti-MICA antibody for detection and anti-vinculin antibody as a loading control. (D and E) Quantification of two independent lysosome (D) or proteasome (E) inhibition experiments. MICA protein levels were quantified relative to the loading control (Vinculin). RQ, relative quantification. Error bars represent SEM. Student's t test was performed to evaluate significance. *, P < 0.05. The ConA panel is derived from a separate gel with a vehicle-only control.
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