Gain of function experiment shows increase in infection with RLM5 in K562 cells following expression of αvβ6-integrin.

<p>(A, B) wt-K562 cells were uninfected (A) or infected with RLM5 (B). (C–F) K562 cells expressing αvβ6-integrin (K562_αvβ6) cells were infected with RLM5. (D–F) Inhibition of RLM5 infection in K562_αvβ6 cells exposed to MAb to αvβ6-integrin (D), or to αv-integrin (E) or to PAb to HVEM (F) for 1 h prior to infection with RLM5 in the same medium, and incubated with MAb-containing medium until harvest at 6 h after infection. The extent of infection was determined as percentage of cells expressing GFP. The figures reported in each panel represent percentage of cells in the lower right quadrant and represent the mean and standard deviation of four independent experiments.</p

Kinetic constants (±SD) for gH/gL binding to soluble truncated integrins.

<p>Kinetic constants (±SD) for gH/gL binding to soluble truncated integrins.</p

Inhibition of HSV-1 infection by silencing of β6- or β8-integrin.

<p>(A) Extent of R8102 infection in silenced cells was quantified as detailed legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003806#ppat-1003806-g003" target="_blank">Fig. 3</a>. 100% infection is the value obtained with siRNActrl cells. Values represent the average of quadruplicates. Bars show SD. (B–D) Red or green columns represent extent of β6 or of β8-integrin expression in 293 T cells, either mock-silenced (full columns), or silenced (hatched columns). Striped black columns represent extent infection with RLM5 as detected by FACS. (E) Inhibition of R8102 infection in 293T cells silenced with siRNActrl (black diamond), or with siRNA to β6- (red triangle), β8- (green circle), or β6-plus β8- (blue square) integrins. Cells were infected at increasing MOI (pfu/cell), and harvested at 16 h after infection, as detailed in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003806#ppat-1003806-g003" target="_blank">Fig. 3</a>. each point represents the average of triplicates ± SD.</p

Schematic representation of the intervention of integrins in the process of HSV entry and in the cascade of activation of the fusion glycoproteins.

<p>Following HSV attachment to cells via heparan sulphate glycosaminoglycans (1), the interaction of gD with one of its receptors activates gD (2); the activation is propagated to gH/gL; the details of this interaction are still under investigation. Integrins intervene (3): they promote endocytosis, and, most likely, induce or contribute to conformational changes, hence to activation of gH/gL. The gH/gL activation is propagated to gB. Endosomal acidification (4). Fusion execution by gB (5). Color code of the glycoproteins as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003806#ppat.1003806-Atanasiu2" target="_blank">[69]</a>. gH/gL and gB are shown in the resolved post-fusion structures.</p

Inhibition of HSV-1 infection by function blocking MAbs to αvβ6- or αvβ8–integrin.

<p>293T (A), HeLa (B), SW480 (C), HaCaT (D), SK-N-SH (E) cells were exposed to increasing amounts of MAb to nectin1 (black diamond), to αvβ6-integrin (red triangle), to αvβ8- integrin (green circle) for 1 h, infected with R8102 (3 pfu/cell) in the same medium, and overlaid with MAb-containing mediun until harvest at 6–8 h after infection. The extent of infection was quantified from Lac-Z gene engineered in the viral genome under the immediate-early α27-promoter. Extent of β-Gal activity, measured as conversion of ONPG substrate, reflects the amount of infection. Each point represents the average of triplicates. ONPG conversion was measured by O.D. reading at 405 nm <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003806#ppat.1003806-Cocchi1" target="_blank">[5]</a>. 100% infection is the value obtained with murin IgG used as a control. Bars show SD.</p

αvβ6 and αvβ8 -integrins do not substitute for nectin 1, yet they increase infection of R8102 in J cells expressing nectin 1 at low level.

<p>(A) The receptor-negative J cells were transfected with nectin1 alone, αvβ6-integrin alone, or αvβ8-integrin alone. (B) J cells were transfected with low amount (75 ng DNA/24 well) of nectin1 alone, or with the same amount of nectin1 plus αvβ6-integrin (300 ng DNA/24 well), or plus αvβ8–integrin (300 ng DNA/24 well). 48 h after transfection, cells were infected with R8102, at increasing MOI (2.5–30 pfu/cell) and harvested at 16–18 h after infection. Experimental details as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003806#ppat-1003806-g003" target="_blank">Fig. 3</a>. Extent of β-Gal activity, measured as ONPG conversion was measured by O.D. reading at 405 nm. Each point represents the average of triplicates. Bars show SD.</p

Steps in HSV infection inhibited by function-blocking MAbs to αvβ6- and αvβ8–integrins, or by integrin silencing.

<p>(A, B) The indicated cells were exposed to MAb 2077Z to αvβ6-integrin, MAb 37E1 to αvβ8–integrin, MAb L230 to αv–integrin, MAb R1.302 to nectin1, or control IgGs. In the “Prior-during-post” treatment cells were exposed to the antibodies from 1 h prior to infection till time of harvest, at 6–8 h after infection. In the “Prior-during” treatment cells were exposed to the antibodies from 1 h prior to infection and during virus absorption. In the “Post” treatment, cells were exposed to the antibodies from the end of virus absorption till harvesting. Cells were infected with R8102 (3 pfu/cell). Infection was quantified in triplicates as detailed in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003806#ppat-1003806-g003" target="_blank">Fig. 3</a>, and expressed as percentage relative to cells treated with control IgGs. (C–E) Inhibition of R8102 absorption to integrin-silenced cells. β6- or β8–integrins were silenced by siRNAs. Control cells received siRNA control. Cells silenced for 2–3 days were infected with R8102 for 120 min at 4°C. Aliquots of the viral inoculum were withdrawn in duplicates at the indicated times, and immediately titrated in Vero cells. Extent of virus absorption is expressed as percentage of the amount of virus present in the inoculum at 0 time. Bars show SD. (F) The binding of gB_t, and not that of gH_t/gL, to cells is inhibited by heparin. One-StrEP tagged gB_t, gH_t/gL or GFP_t (2 µM each), the latter as a negative control, were preincubated or not with heparin (5 µg/ml) for 1 h at 4°C, and then added to cells grown in 96-well plates, for 1 h at 4°C. Binding was detected by means of HRP-conjugated MAb to the One-StrEP tag and the <i>o</i>-phenylenediamine substrate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003806#ppat.1003806-Gianni5" target="_blank">[41]</a>. Each point represents the average of triplicates. The values obtained with GFP_t were considered as background values and subtracted. Bars represent SD. (G–I). Determination of heparin sulphate binding sites by means of gB_t to integrin silenced and control-silenced cells. (G–I). 293T (G), HeLa (H) and SW480 (I) cells, control-silenced or integrn-silenced, were exposed to gB_t, pretreated or not heparin. Binding was detect as descibed in panel F. Columns represent the binding of gB_t, after subtraction of the values obtained in the presence of heparin. Each column represents the average of triplicates. Bars denote SD.</p

The RGD motif in gH is required for HSV infection and cell-cell fusion mediated by αvβ6–integrin, but not by αvβ8–integrin.

<p>(A–C). J cells were transfected with low amount of nectin1 alone (black triangle and square) (A), or nectin1 plus αvβ6-integrin (red triangle and square) (B), or nectin1 plus αvβ8-integrin (green triangle and square) (C), as detailed in legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003806#ppat-1003806-g007" target="_blank">Fig. 7</a>. 48 h later, cells were infected with increasing amounts (5–20 pfu/cell) of SCgHZ virus, a ΔgH HSV, complemented with gH_wt (black, red and green triangle) or with gH_ADA (black, red and green square), and harvested 16–18 h later. Extent of infection was expressed as detailed in legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003806#ppat-1003806-g003" target="_blank">Fig. 3</a>. Eeach point represents the average of triplicates. 100% of infection is the value obtained in J cells transfected with nectin1 alone and infected with 20 pfu of SCgHZ virus complemented with gH_wt. Bars show SD. (D) Cell-to-cell fusion between effector J cells expressing gH_wt, or gH_ADA plus the trio of gD, gL and gB, and luciferase, and target J cells expressing nectin1 alone, or nectin1 plus αvβ6-integrin, or nectin1 plus αvβ8-integrin plus Renilla luciferase. Fusion was quantified by means of a T7 promoter-driven reporter luciferase gene transfected in effector cells, and expressed as percentage relative luciferase units (R.L.U.). 100% is the value obtained in cells expressing nectin1 alone and gH_wt plus the trio of gD, gL, gB. Each point represents the average of triplicates. Bars show SD.</p

The Epstein-Barr Virus Glycoprotein gp150 Forms an Immune-Evasive Glycan Shield at the Surface of Infected Cells

<div><p>Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150’s cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle.</p></div

Glycan shielding of surface Ag-presenting molecules by gp150 occurs in human B cells and is reversed during productive EBV infection when gp150 is deleted.

<p>A-B) Latent AkataΔgp150 cells were lentivirally transduced to co-express either HA-gp150 or HA-gp150ΔC and GFP (from EF1a and PGK promoters, respectively) and were puromycin-selected to obtain pure populations of gp150⁺ cells. B cells already grow in suspension and gp150-positive B cells were maintained in culture for several weeks, indicating that gp150 expression was not toxic to the cells. A) Flow cytometry was used to assess total (intracellular staining with anti-gp150 Ab on permeabilized cells) and surface (anti-HA Ab on non-permeabilized cells) levels of EBVgp150. Expression levels of gp150 were compared to lytically induced AKBM cells (20 hours anti-human IgG treatment, rat CD2GFP⁺ cells). B) Akata+gp150 and non-transduced control cells were left untreated or were treated with neuraminidase (1U/μl, 60 min, 37°C). Surface levels of Ag-presenting molecules and cellular CD10 as a control were compared to gp150^- non-transduced cells. One representative experiment of three is depicted. C) Viral reactivation was induced in Akata wt and Δgp150 B cells by overnight culture with anti-human IgG and EBV-producing cells were identified by staining for the late viral protein gp350. Surface levels of Ag-presenting molecules and CD10 on lytic (gp350⁺) and latent (gp350^-) cells are depicted in overlay histograms. One representative experiment of at least four is depicted. Statistical analysis was performed for B and C) as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005550#ppat.1005550.g006" target="_blank">Fig 6</a>. * p<0.05, ** p<0.01.</p