The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing.

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells without prior sensitization. One pivotal activating NK receptor is NKG2D, which binds a family of eight ligands, including the major histocompatibility complex (MHC) class I-related chain A (MICA). Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus causing morbidity and mortality in immunosuppressed patients and congenitally infected infants. HCMV encodes multiple antagonists of NK cell activation, including many mechanisms targeting MICA. However, only one of these mechanisms, the HCMV protein US9, counters the most prevalent MICA allele, MICA*008. Here, we discover that a hitherto uncharacterized HCMV protein, UL147A, specifically downregulates MICA*008. UL147A primarily induces MICA*008 maturation arrest, and additionally targets it to proteasomal degradation, acting additively with US9 during HCMV infection. Thus, UL147A hinders NKG2D-mediated elimination of HCMV-infected cells by NK cells. Mechanistic analyses disclose that the non-canonical GPI anchoring pathway of immature MICA*008 constitutes the determinant of UL147A specificity for this MICA allele. These findings advance our understanding of the complex and rapidly evolving HCMV immune evasion mechanisms, which may facilitate the development of antiviral drugs and vaccines

Dynamic Co-evolution of Host and Pathogen: HCMV Downregulates the Prevalent Allele MICA∗008 to Escape Elimination by NK Cells

Natural killer (NK) cells mediate innate immune responses against hazardous cells and are particularly important for the control of human cytomegalovirus (HCMV). NKG2D is a key NK activating receptor that recognizes a family of stress-induced ligands, including MICA, MICB, and ULBP1-6. Notably, most of these ligands are targeted by HCMV proteins and a miRNA to prevent the killing of infected cells by NK cells. A particular highly prevalent MICA allele, MICA∗008, is considered to be an HCMV-resistant “escape variant” that confers advantage to human NK cells in recognizing infected cells. However, here we show that HCMV uses its viral glycoprotein US9 to specifically target MICA∗008 and thus escapes NKG2D attack. The finding that HCMV evolved a protein dedicated to countering a single host allele illustrates the dynamic co-evolution of host and pathogen

MicroRNA Editing Facilitates Immune Elimination of HCMV Infected Cells

<div><p>The human cytomegalovirus (HCMV) is extremely prevalent in the human population. Infection by HCMV is life threatening in immune compromised individuals and in immune competent individuals it can cause severe birth defects, developmental retardation and is even associated with tumor development. While numerous mechanisms were developed by HCMV to interfere with immune cell activity, much less is known about cellular mechanisms that operate in response to HCMV infection. Here we demonstrate that in response to HCMV infection, the expression of the short form of the RNA editing enzyme ADAR1 (ADAR1-p110) is induced. We identified the specific promoter region responsible for this induction and we show that ADAR1-p110 can edit miR-376a. Accordingly, we demonstrate that the levels of the edited-miR-376a (miR-376a(e)) increase during HCMV infection. Importantly, we show that miR-376a(e) downregulates the immune modulating molecule HLA-E and that this consequently renders HCMV infected cells susceptible to elimination by NK cells.</p></div

HCMV infection of human decidua organ culture induces ADAR1 and reduces HLA-E expression.

<p>(a–d) ADAR1 levels were assessed in decidua organ culture 36 hr (a and b) and 7days (c and d) after HCMV infection (Blue-DAPI, green-HCMV-infected cells, red-ADAR1). (a and c) Quantification of ADAR1 fluorescence intensity 36 hr (a) and 7 d (c) after infection. (a) Relative average ADAR1 intensity±SEM of GFP^neg cells (uninfected cells): 0.80±0.05, n = 123, Relative average ADAR1 intensity±SEM of GFP^pos cells (HCMV infected cells): 1.13±0.03, n = 123, *<i>P</i><0.05, by student t-test. (c) Relative average ADAR1 intensity±SEM of GFP^neg cells (uninfected cells): 0.81±0.03, n = 143, Relative average ADAR1 intensity±SEM of GFP^pos cells (HCMV infected cells): 1.15±0.05, n = 109, *<i>P</i><0.05, by student t-test. Data are summation of two independent experiments. (b and d) Representative examples. (e–f) HLA-E levels were assessed in decidua organ culture 36 hr (e and f) and 7days (g and h) after HCMV infection (Blue-DAPI, green-HCMV-infected cells, red-HLA-E). (e and g) Quantification of HLA-E fluorescence intensity 36 hr (e) and 7 d (g) after infection. (e) Relative average HLA-E intensity±SEM of GFP^neg cells (uninfected cells): 0.82±0.03, n = 193, Relative average HLA-E intensity±SEM of GFP^pos cells (HCMV infected cells): 1.14±0.03, n = 241, *<i>P</i><0.05, by student t-test. (c) Relative average HLA-E intensity±SEM of GFP^neg cells (uninfected cells): 1.12±0.06, n = 70, Relative average HLA-E intensity±SEM of GFP^pos cells (HCMV infected cells): 0.85±0.04, n = 75, *<i>P</i><0.05, by student t-test. Data are summation of two independent experiments. (f and h) Representative example.</p

ADAR1-p110 is involved in the editing of miR-376a.

<p>(a) The rate of miR-376a(e) in HFF cells either mock treated or HCMV infected was determined by next generation small RNA deep sequencing. Data are combined of two independent experiments. The rate in mock treated cells: 0.5943, in HCMV infected cells: 0.7912, *P-value: 4.38E-13. The rate of miR-376a(e) was calculated as the proportion of miR-376a(e) out of all the miR-376a species after allowing two mismatches. Hence no specific normalization was required. (b and c) WB analysis (b) and quantification (c) of ADAR1-p110 expression in mock treated or HCMV infected HFF cells transduced either with shADAR1 vector or with a control vector. (b) Data are representative of three independent experiments. Numbers indicate fold change in intensity compared to the expression of ADAR1-p110 observed after mock infection (M) in the control vector transduced cells. (c) Quantification of all experiments performed in (b). Shown are the relative average intensities ± S.D., *<i>P</i><0.001 by Student's t-test. (d) The rate of miR-376a(e) in HFF cells transduced either with a control vector (Ctrl) or shADAR1, and HCMV infected was determined by next generation small RNA deep sequencing. Data are combined of two independent experiments. The rate in control cells: 0.2806, in shADAR1 cells: 0.0.1461, *P-value: 1.6E-4. The rate of miR-376a(e) was calculated as the proportion of miR-376a(e) out of all the miR-376a species after allowing two mismatches. Hence no specific normalization was required. (e and f) WB (e) and quantification (f) of ADAR1-p110 expression in ADAR1-p110-transduced HFF cells. Data are representative of three independent experiments. Numbers indicate fold change in intensity of ADAR1-p110 compared to HFF cells transduced with an empty vector. (f) Shown are the relative average intensities of ADAR1-p110±S.D., *<i>P</i><0.002 by Student's t-test. (g) The rate of miR-376a(e) in HFF cells transduced either with a control vector or ADAR1-p110 was determined by next generation small RNA deep sequencing. Data are combined of two independent experiments. The rate in control cells: 0.3517, in ADAR1-p110 cells: 0.7653, *P-value: 3.97E-50. The rate of miR-376a(e) was calculated as the proportion of miR-376a(e) out of all the miR-376a species after allowing two mismatches. Hence no specific normalization was required.</p

ADAR1-p110 is induced during HCMV infection.

<p>(a–f) WB analysis of ADAR1 expression during various viral infections. (a) Time course analysis of ADAR1-p150 and ADAR1-p110 expression during infection of HFF cells with the AD169 strain of HCMV. Quantification of ADAR1-p110 was performed relative to expression at time 0 h (the fold increased numbers are indicated). Data are representative of four independent experiments. (b) Analysis of ADAR1-p110 expression during infection of HFF cells with the indicted HCMV strains. The analysis was performed 48 hrs after infection. Quantification of ADAR1-p110 was performed relative to expression in uninfected HFF cells (the fold increased numbers are indicated below the blot). Data are representative of three independent experiments. CI – clinical isolate (c) Analysis of ADAR1-p110 expression at different time points following infection of ARPE-19 cells with the TB40/E strain of HCMV. Quantification of ADAR1-p110 was performed relative to expression at time 0 h (the fold increased numbers are indicated below the blot). Data are representative of four independent experiments. (d) Analysis of ADAR1 expression during infection of HFF cells with HSV1 and HSV2, 24 hrs after infection. Quantification of ADAR1-p110 was performed relative to expression in uninfected HFF cells (the fold increased numbers are indicated below the blot). Data are representative of three independent experiments. (e and f) Analysis of ADAR1 expression during infection of A549 cells with Adenovirus (AdV, e), Influenza (f) and human metapneumovirus (hMPV, f), 48 hrs and 96 hrs after infection, respectively. Quantification of ADAR1-p110 was performed relative to expression in uninfected A549 cells (the fold increased numbers are indicated below the blot). Data are representative of three independent experiments. (a–f) Alpha-tubulin served as a loading control.</p

The regulation of HLA-E by miR-376a(e) during infection affects NK cell cytotoxicity.

<p>(a) FACS analysis of MICB levels on HFF cells transduced either with an anti-miR-376a sponge (black histogram), or an anti-miR-376a(e) sponge (grey histogram). Filled empty histogram represents background staining. Data are a representative of four independent experiments. (b) FACS analysis of HLA-E levels on HFF cells transduced either with a control sponge (empty grey histogram), an anti-miR-376a(e) sponge (black histogram, left), or an anti-miR-376a sponge (black histogram, right). Filled empty histogram represents background staining. Data are a representative of five independent experiments. (c) The percentage of NKG2A⁺ or NKG2C⁺ or CD94⁺ NK cells obtained from HCMV^neg (grey bars) and HCMV^pos (black bars) donors as detected by FACS. NK cells were identified by gating on CD56⁺CD3⁻ cells. (d and e) Killing of HCMV infected HFF cells transduced either with a control sponge or with a specific anti-miR-376a(e)-sponge (d) or with a control miRNA or miR-376a(e) (e). Killing was assessed 96 hrs post infection and was performed with bulk NK cells derived from four HCMV^neg and four HCMV^pos donors. The NK cells derived from the various donors were preincubated either with a CD94 blocking mAb (+block, dark grey bars), or with an isotype control (black bars) and then incubated with the ³⁵S methaionine-labeled target cells. Data shown are the relative killing by each specific donor, and the relative average killing by all HCMV^neg or HCMV^pos donors combined (x-axis). The killing of the control-sponge (Ctrl, d) or control-miRNA (Ctrl, e) expressing HFF cells was set as 1 (marked by a dashed line). Shown are relative average killing ± S.D. *<i>P</i><0.05, by Student's t-test. Of note, all single experiments also showed statistically significant effects (<i>P</i><0.05, by Students' t-test) based on relative mean±S.D. of quadruplets.</p

CEACAM1-Mediated Inhibition of Virus Production

Cells in our body can induce hundreds of antiviral genes following virus sensing, many of which remain largely uncharacterized. CEACAM1 has been previously shown to be induced by various innate systems; however, the reason for such tight integration to innate sensing systems was not apparent. Here, we show that CEACAM1 is induced following detection of HCMV and influenza viruses by their respective DNA and RNA innate sensors, IFI16 and RIG-I. This induction is mediated by IRF3, which bound to an ISRE element present in the human, but not mouse, CEACAM1 promoter. Furthermore, we demonstrate that, upon induction, CEACAM1 suppresses both HCMV and influenza viruses in an SHP2-dependent process and achieves this broad antiviral efficacy by suppressing mTOR-mediated protein biosynthesis. Finally, we show that CEACAM1 also inhibits viral spread in ex vivo human decidua organ culture

ADAR1-p110 is induced via a specific promoter.

<p>(a) Schematic description of the alternative promoters of the ADAR1 gene (black arrows) and their alternative splicing (dotted lines). Exons 1B, 1C and 2 (white boxes), drive the expression of the ADAR1-p110, while exon 1A (black box) drives the expression of ADAR1-p150. The genomic locations of the DNA fragments, which were cloned upstream to a Firefly luciferase, are indicated at the bottom. (b) Dual luciferase assay was performed on HFF cells that were transfected with the reporter vectors containing different genomic regions of the ADAR1 promoters as indicated, 4 hrs after transfection the cells were either mock treated (Mock) or treated with IFN-α (1000 u/ml), IFN-β (1000 u/ml), or infected with HCMV (at MOI 1) for 48 hours. The Firefly/Renilla ratio of each treatment was normalized to the ratio in mock HFF cells. Data are representative of four independent experiments, shown are mean ± S.D. of triplicates. *<i>P</i><0.01, **<i>P</i><5E-5 by two-tailed Student's t-test. (c) ARPE-19 cells were transfected with the indicated reporters (x-axis). Four hours after transfection the cells were infected with the TB40/E strain of HCMV. 48 hrs after infection the Firefly/Renilla ratio was assessed and calculated relatively to the activity in the uninfected cells. Data are representative of four independent experiments; shown are mean ± S.D. of triplicates. *<i>P</i><0.001, by Student's t-test. (d) HFF cells were transfected with the 1B reporter, and then infected with HCMV in the presence or absence of the inhibitor GCV. Firefly/Renilla activity ratio was assessed 48 hrs after infection, and was calculated relatively to the activity in the uninfected calls. Data are representative of three independent experiments; shown are mean ± S.D. of triplicates. ns-not significant. (e) HFF cells were transfected with the 1B reporter. Firefly/Renilla activity ratio was assessed at the indicated time points in mock treated cells (Mock), in cells infected with HCMV, or in cells infected with an UV-inactivated virus (UV-inact). The Firefly/Renilla ratio was calculated relatively to the ratio in mock HFF cells. Data are representative of three independent experiments; shown are mean ± S.D. of triplicates. *<i>P</i><0.002, **<i>P</i><0.0003, by Student's t-test.</p