Disruption of Adherens Junctions Liberates Nectin-1 To Serve as Receptor for Herpes Simplex Virus and Pseudorabies Virus Entry

Disruption of Adherens Junctions Liberates Nectin-1 To Serve as Receptor for Herpes Simplex Virus and Pseudorabies Virus Entry

Des études contrôlées sur des facteurs potentiellement interférents ont démontré que les caractéristiques du test ne sont pas modifiées par des anticoagulants (citrate de sodium, EDTA, héparine), par une hémolyse (jusqu’à 1000 mg/dL d’hémoglobine), une lipémie (jusqu’à 3000 mg/dL de triglycérides), une bilirubinémie (jusqu’à 20 mg/dL de bilirubine) ou les cycles de congélation/décongélation des échantillons. We now show that cationic oligomers of β-amino acids (“β-peptides”) inhibit HSV-1 infection. The incubation period following genital acquisition of HSV-1 or -2 is approximately four days (range, 2-12 days). In this study, we applied an experimental feedback system control (FSC) method and rapidly identified optimal drug combinations that inhibit herpes simplex virus-1 infection, by only testing less than 0.1% of the total possible drug combinations. While typically HSV-1 causes oral lesions and HSV-2 genital ones, both are capable of infecting either region (1). At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. Once infection is established and an immune response is made, autoinoculation is extremely rare.

Herpes simplex virus (HSV) infections are extremely widespread in the human population. Herpes simplex virus type 1 (HSV-1) is a linear double-stranded DNA virus that, as a major human pathogen, mainly infects epithelial and neuronal cells, causing a variety of potentially fatal diseases1. The inhibitory effect of various concentrations of Aloe vera was observed one hour after the Vero cell was infected with HSV-1. Cytotoxicity assay, determining the 50% tissue culture infectious dose (TCID50), and incubation of HSV-1 with licorice root extract prior to viral infection were performed. The most reliable method by which to deliver oncolytic HSV-1 to solid tumors is direct inoculation. The reconstruction showed further that, compared to mature HSV-1 capsids, closed spherical capsids are more open structures in which the capsid floor layer is less pronounced. The subsequent interaction of virion gD with one of its receptors triggers the penetration of virus, which occurs through fusion of the viral envelope with the cell membrane and requires four virion glycoproteins, gB, gD, gH, and gL.

Three classes of cell surface molecules can serve as gD receptors for the entry of HSV-1 or HSV-2 (30). Lors de la présence d’une infection à la naissance, entre 40% et 60% des nouveau-nés peuvent être affectés. Following attachment, gD interacts with any of three cellular receptors: herpes virus entry mediator, nectin-1, and 3-O-sulfated heparan sulfate, leading to a conformational change in gD (12, 13, 21, 22, 34, 39, 54, 62). Human nectin-1 and nectin-2 can serve as entry receptors for PRV as well as HSV. Within these pathways and protein complexes, single targets have been found that upon drug manipulation can disrupt viral replication. Polyethylene sheets, from McMaster Carr (Atlanta, GA), were cut into 2.5×2.5-cm slides. coupled an exothermic reaction with an engineered phase change material to enable incubation of loop-mediated amplification (LAMP) reactions at the point of care [11], [12].

The nectins are Ca2+-independent cell adhesion molecules that can engage in homophilic or heterophilic interactions. HSV gD receptors are cell-surface molecules derived from three structurally unrelated families. Viral infection generally results in dramatic changes in cellular mRNA expression including the pattern of cellular miRNA expression23, which represents a disastrous event in the life of host cells. Most of the membrane-bound isoforms can bind through their cytoplasmic tails to the PDZ domain in l-afadin. The nectins and l-afadin colocalize with E-cadherin and catenins in adherens junctions of epithelial cells (31, 32). Vero cell monolayers were infected with the virus serially diluted 10–fold for a standard plaque assay to determine the virus titer. If nectin-1 or nectin-2 is localized to adherens junctions in epithelial cells, are these gD receptors accessible for binding to virus?

In this study, we used Madin-Darby canine kidney (MDCK) cells to examine the effects of junctional disruption on cell surface localization of nectin-1, binding to the cells of HSV-1 gD and PRV gD, and susceptibility of the cells to viral entry. MDCK cells expressing transfected human nectin-1 or an endogenous entry receptor were used because, in either case, the only receptors available for HSV and PRV entry were human nectin-1 and/or a molecule antigenically related to human nectin-1, probably dog nectin-1. Thus, use of the MDCK cells facilitated investigation of the relationship between cell surface distribution of a single type of entry receptor and susceptibility of the cells to HSV and PRV entry. Lactoferricin binds to heparan sulfate, thereby blocking viral attachment (38). MDCK cells stably expressing human Flag-nectin 1α (32), designated here MDCK-nectin-1 cells, were provided by Y. Each drug combination was represented as a vector in the software. Coated condoms were prepared similarly, except that DMPEI was dissolved in butanol (23).

Ten normal, healthy volunteers were recruited for this study in accordance with protocol 14-211E, approved specifically for this study by the Rice University Internal Review Board. The β-galactosidase reporter viruses have been described previously (2, 12, 33). Similarly, virus inhibition and RT-PCR assays confirmed inhibitory potential of neem extract at virus replication step for Dengue virus type-2 (Parida et al., 2002). To optimize the time points, a time course of HSV-1 infection was performed to determine the suitable MOI for HSV-1 infection in HeLa cells. Ca2+ switch protocol.Cells were grown in DME containing 2 mM Ca2+ with 10% FCS (NC medium). To deplete the cells of Ca2+ as described previously (4), the cells were washed with phosphate-buffered saline and transferred for 1 or 2 h to Ca2+-free DME with 1 mM EDTA and 10% FCS that had been depleted of Ca2+ by passage over Chelex-100 (Bio-Rad) (LC medium, ∼2 μM Ca2+). For the study of ultrasound exposure immediately after viral inoculation, SAS cell monolayers were inoculated with 1 × 104 PFU of HSV-1 RH2, with or without 5 × 107 microbubbles, and then exposed to ultrasound for 10 s at room temperature.

HSV-1 gD:Fc has the first 345 amino acids (first 320 amino acids after signal peptide cleavage) of HSV-1(KOS) gD fused through a linker of 6 amino acids (YRARIH) to 231 amino acids at the C terminus of rabbit IgG heavy chain. The PRV gD:Fc is similar except that the first 408 amino acids (first 391 amino acids after signal peptide cleavage) of PRV(Kaplan) gD is fused through a 3-amino-acid linker (RIH) to the rabbit sequence. MDCK cells grown in 96-well plates were incubated with serial dilutions of HSV-1 gD:Fc or PRV gD:Fc for 1 h at 37°C. Cells were washed, fixed with 2% formaldehyde and 0.2% glutaraldehyde, and sequentially incubated with biotinylated anti-rabbit IgG (Sigma), Amdex streptavidin-conjugated horseradish peroxidase (HRP; Amersham), and HRP substrate (BioFx Lab). (A) Generic structures of α- and β3-amino acids. Alternatively, cells grown on coverslips were incubated with gD:Fc, and binding was visualized with Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes). To minimize variance generated from different batches of cells, the trial group and crossover group were tested and compared using the same batch of cells for each iteration.

Plaques were counted to determine the viral titer of the solutions. These measurements were taken for five volunteers. Cells were washed, permeabilized, and incubated with β-galactosidase substrate, O-nitrophenyl-β-d-galactopyranoside (ONPG; Sigma). Beatrice Yue, University of Illinois at Chicago) were grown in DMEM media supplemented with 10% FBS and 5% calf serum (CS) (Tiwari et al., 2006). To characterize this promoter, different lengths of DNA fragments upstream of the RCL1 ATG codon were cloned into a pGL3-basic vector as the miR-101-2/RCL1 promoter (following plasmid names: p1060, p421 and p334) (). The stained cells were photographed with an Olympus microscope (model CK2), equipped with a 35-mm camera, controlled by the Olympus automatic exposure photographic system (PM-10AK3). Immunofluorescence and microscopy.Indirect immunofluorescence was carried out as described previously (32).

The values were divided by those of the control and the rate was calculated. The secondary antibodies were Alexa 488-conjugated goat anti-mouse IgG and Alexa 568-conjugated goat anti-rat IgG. Immunofluorescence observations were made with a Zeiss LSM510 confocal microscope equipped with a 100×, 1.4 NA oil immersion objective. Orthogonal sections were made in a z stack of 30 images (1-μm interval) according to the LSM510 operating manual. Distribution of nectin-1 and E-cadherin before and after disruption of adherens junctions.MDCK cells and MDCK cells stably transfected to express Flag-tagged human nectin-1α (referred to here as MDCK-nectin-1 cells) were grown to confluence in medium containing standard levels of Ca2+ (2 mM; NC medium) and then either maintained in this medium or switched to medium with greatly reduced levels of Ca2+ (∼2 μM; LC medium). (40). 1A to C).

The success of antiviral drug combinations depends on two essential factors: the drug combination used and the dose of each drug used. Specifically, 0.5 mL of a DMPEI suspension (or the supplemented PBS buffer) and 0.5 mL containing 210±10 PFU of HSV-1 were mixed in a T25 flask and gently agitated for 1 h at 37°C. RPA reactions were also incubated using the same method in a cold room with an ambient temperature of 10 degrees Celsius. E-cadherin was no longer detectable after 2 h (Fig. Cells were transiently transfected in 6-well tissue culture dishes, using Lipofectamine 2000 (Invitrogen) with plasmids expressing HSV-1 entry receptors (necitn-1, HVEM and 3-OST-3 expression plasmids) at 1.5 μg per well in 1 ml. The RNA expression levels of RCL1, the hsa-mir-101-2 precursor and mature miR-101 were found to be highly induced or reduced under ICP4 overexpression or knockdown conditions respectively (). 1H) as previously described (1).

Orthogonal sectional views demonstrated that nectin-1 was localized only at cell-cell junctions in NC medium and redistributed over large areas of the cell surface after Ca2+depletion (Fig. SAS cells were plated on 96-well plates and grown. Localization of Flag-tagged nectin-1 and E-cadherin in MDCK-nectin-1 cells before and after a switch to medium depleted of Ca2+. Cells were maintained in NC medium (A to C) or switched to LC medium for 1 h (D to F) or 2 h (G to I) and then double stained with mouse anti-Flag (B, E, H, J, and K) and rat anti-E-cadherin (C, F, and I) antibodies. (A, D, and G) Phase-contrast images. (J and K) Orthogonal sections of anti-Flag-stained MDCK-nectin-1 cells maintained in NC medium (J) or in LC medium for 2 h (K). Assay to induce cell resistance.Vero cells were incubated with various concentrations of the β-peptides for 1 h at 37°C.

The antibody used to detect nectin-1 in Fig. For a non-optimal … Since HSVs are transmitted by direct contact with viral lesions, an antiviral formulation should ideally be available in a form that can intimately interact with infected tissues. Figure 2 shows the temperature traces of mock RPA reactions incubated by 5 volunteers at 4 body locations. 1. All cells were incubated at 4 °C for 1 hr, washed five times to remove unbound virus, and finally replaced with warm medium for further incubation. A band shift was observed in the infected group but not in the uninfected cells control group ().
Disruption of Adherens Junctions Liberates Nectin-1 To Serve as Receptor for Herpes Simplex Virus and Pseudorabies Virus Entry

Mammalian nectin-1 is much more highly conserved in sequence than are nectin-2 (9, 23, 29) and HVEM (14, 22). Enhanced binding of gD to epithelial cells depleted of Ca2+.To assess the presence and distribution of gD receptors, MDCK cells and MDCK-nectin-1 cells were maintained in NC medium or switched to LC medium for 2 h and then were incubated with soluble forms of HSV-1 gD and PRV gD (gD:Fc hybrid molecules), followed by fixation and addition of labeled anti-Fc antibodies. SAS cells were exposed to ultrasound at first. Also, PRV, but not HSV-1, gD:Fc bound to the untransfected MDCK cells. PRV gD is known to bind to human nectin-1 with higher affinity than HSV-1 gD (8). One explanation for the results in Fig. 2A, C, E, and G is that PRV gD also binds with higher affinity to the endogenous dog receptor than does HSV-1 gD and that the latter interaction is too weak to be detected by the use of soluble gD:Fc.

After the cultures were blocked for 1 h, polyclonal rabbit anti-HSV type 1 antibody (1:100 dilution in 0.05% BSA in S−; Dako North America, Inc., Carpinteria, CA) was added. Binding of HSV-1 gD:Fc and PRV-gD:Fc to MDCK cells (A, C, E, and G) and MDCK-nectin-1 cells (B, D, F, and H). The search algorithm was the module of FSC that directed the tested drug combinations towards an optimal treatment for the biocomplex system. Moreover, most of the observed antiviral activity was due to the DMPEI particles larger than 0.45 μm because incubation with DMPEI suspension filtrate did not have a marked anti-HSV effect (). 3C) reached an average temperature of 33.2±1.6, 32.9±1.2, and 33.5±0.7 degrees Celsius, respectively. The binding of both gD:Fcs was localized to the junctions of cells maintained in NC medium (Fig. A group of multinucleate cells (10–15 joint cells) were scored positive for polykaryocytes formation as previously described (Tiwari et al., 2007).

HeLa cells were transfected with pri-miR-101 or ASO-miR-101, and total RNA and protein were extracted after 48 h to assess endogenous GRSF1 expression. This localization and subsequent redistribution are indistinguishable from those observed for human nectin-1 (compare with Fig. 1). In this situation, the virus entry estimated by plaque number was increased and 40-50% of inoculated virus entered the cells [11]. There was no stable binding of HSV-1 gD:Fc to MDCK cells, regardless of the conditions (Fig. 3), consistent with the immunofluorescence observations in Fig. 2.

Greater amounts of HSV-1 gD:Fc bound to MDCK-nectin-1 cells incubated for 2 h in LC medium than to cells maintained in NC medium. These β-peptides were designed to adopt a 14-helix conformation, i.e., a conformation defined by 14-membered-ring hydrogen bonds between backbone amide groups [N-H(i)—O = C(i + 2)] (11). 3). (B) After twelve iterations, the average dosage of ribavirin in 16 combinations increased, while the … Note that a control aqueous solution containing the same concentration of HEC alone had no appreciable influence on viral titer. To demonstrate that body heat may be harnessed to incubate RPA reactions, RPA reactions were secured with a strip of cotton fabric and incubated in the axilla of ten volunteers in an office or laboratory at room temperature and in a cold room at 10 degrees Celsius, while control RPA reactions were incubated in a heat block. Values are optical densities at 370 nm; means of triplicate determinations with standard deviations for one representative experiment of five replicates are shown.

HeLa cells express all known gD receptors while CF exclusively express HVEM and 3-OST-3 (Tiwari et al., 2005; Tiwari et al., 2006; Tiwari et al., 2008). Real-time PCR was used with primers that targeted a sequence from the HSV-1 DNA gD gene for viral quantification. This antibody completely inhibited the enhanced binding of HSV-1 gD:Fc to MDCK-nectin-1 cells depleted for Ca2+ whereas the anti-nectin-2 antibody was without effect (Fig. 4A). In the present study, the enhancing effect was lost after 20 min. 4B). Thus, the enhanced binding of HSV-1 and PRV gD:Fcs after Ca2+ depletion was due to increased availability of human nectin-1 or an endogenous gD receptor that is antigenically related to human nectin-1, probably dog nectin-1.

Anti-nectin-1 antibody blocks the enhanced binding of HSV-1 gD:Fc and PRV gD:Fc to MDCK cells and MDCK-nectin-1 cells. Cells in 96-well plates were maintained in NC medium or switched to LC medium for 2 h and incubated with serial dilutions of monoclonal antibodies against nectin-1 (R1.302.12) or nectin-2 (R2.477.1) for 1 h prior to addition of a single dose of HSV-1 gD:Fc (24 μg/ml) (A) or PRV gD:Fc (4 μg/ml) (B). 2). Values are optical densities at 370 nm; means of triplicate determinations with standard deviations for one representative experiment of five replicates are shown. The values of α and β reflect the relative importance of Vi and Rc. As to a possible prophylactic use, both optimized noncovalent painting of, and covalent attachment of hydrophobic polycations to, latex (polyisoprene) condoms should be explored and the resultant coated condoms tested in terms of their long-term stability and safety. As RPA is tolerant to sample impurities, simple lysis methods such as boiling may adequately prepare samples for amplification [14].

Viral entry assays were performed using cells maintained in NC medium or switched for 2 h to LC medium prior to incubation with serial dilutions of HSV-1(KOS)tk12 or PRV-gH−. Because NBE blocked HSV-1 entry, we next tested its ability to affect viral binding to the cells. A RIP assay was then performed to confirm the interaction between the GRSF1 protein and p40 mRNA. Expression of β-galactosidase signals the entry of virus into cells and expression of viral and reporter genes. Depletion of Ca2+ from the culture medium significantly enhanced the entry of both HSV-1 and PRV into MDCK cells and MDCK-nectin-1 cells. HSV-1-injected tumors should be exposed to ultrasound after an incubation time of virus with tumor cells or exposed repeatedly to ensure the entry of virus by receptor-mediated entry as well as pore-mediated entry of HSV-1. 5A show that viral entry was more efficient for the cells depleted of Ca2+ than for cells maintained in NC medium at each of the input doses of virus tested.

The levels of β-galactosidase activity were related to the numbers of cells infected at each virus dose, as shown by the X-Gal-stained cells in Fig. 5B to I. Thus, the disruption of cell junctions and release of junctional nectin-1 to larger areas of the cell surface correlated with enhanced susceptibility of the cells to viral entry. As shown in Fig. Enhanced entry of HSV-1 and PRV after a switch to LC medium. Both DE1- and DE2-treated samples sustained low levels of viral infection through day 4 (). After 6 h, cells were processed for incubation with the β-galactosidase substrate ONPG (A) or X-Gal (B to I).

Signal-to-background ratios of lateral flow strips. Values are optical densities at 410 nm; means of triplicate determinations with standard deviations for one representative experiment of five replicates are shown. Our results indicate that the presence of NBE significantly reduced the viral attachment and penetration. The binding specificity was confirmed by the displacement of the bound fraction with an excess of unlabeled RNA probe. Even though HSV-1 infection of the untransfected MDCK cells was enhanced by Ca2+ depletion, we were not able to detect a gD receptor using HSV-1 gD:Fc (Fig. 2 and 3). It seems likely that HSV-1 and PRV used the same endogenous MDCK receptor for entry.

Nectin-1 is the most highly conserved of all the alphaherpesvirus entry receptors and the only one that, in human, mouse, and pig forms, has been shown to serve as an entry receptor for both HSV-1 and PRV (6, 12, 20, 21, 29). The MDCK cells clearly express a PRV receptor related to human nectin-1, based on the ability of the antibody specific for human nectin-1 (R1.302) to inhibit the binding of PRV gD:Fc to the cells. Concentrations of this antibody that effectively blocked the binding of gD:Fc to the MDCK cells and MDCK-nectin-1 cells (Fig. 4), up to 2.5 μg/ml, preincubated with the cells at 37°C for 30 min, failed to inhibit infection of the cells by either HSV-1 or PRV before or after Ca2+ depletion (data not shown). The EC50 values for β-peptides 1, 2, and 3 were 1, 5, and 1 μM, respectively, in HK320 cells. In this study, preincubation at 4°C was not done, so as to maintain normal cell architecture. Both the availability of an endogenous dog receptor and the particular conditions used here for treating cells with the R1.302 antibody may explain the failure to protect MDCK cells from infection.

Insights into viral entry of epithelial cells.Recently, two cell surface glycoproteins, the coxsackievirus and adenovirus receptor (CAR) and the junction adhesion molecule, were identified as transmembrane components of the tight junction in epithelial cells as well as entry receptors for coxsackie virus and adenovirus and for reovirus, respectively (3, 7). Patel VB, Theron G, Lenders L, Matinyena B, Connolly C, et al. Sequestration of CAR in tight junctions apparently limits viral infection, similar to our findings that the localization of nectin-1 to adherens junctions actually impairs its ability to mediate HSV-1 and PRV entry. Our results showed that HSV-1 entry was significantly blocked in CHO-K1 cells expressing either protein receptors (nectin-1 and or HVEM) or a sugar receptor (3-OST-3 modified 3OS HS). Because the expression of miR-101 is induced at an early stage of HSV-1 infection, ICP4 of HSV-1 which is a transactivator of the promoter required for the expression of early and late genes of HSV-16,7,33,34 was considered to be involved in this process. Alternatively, other gD receptors, including forms of nectin-1 that do not bind to l-afadin (nectin-1β), could mediate entry of virus into the first cells of an intact epithelium to be infected. We thank Y.

Takai for the MDCK cells expressing Flag-tagged human nectin-1, R. Eisenberg and G. Cohen for anti-nectin-1 antibodies (CK6 and CK8), R. Goldman for use of his confocal microscope, and N. Susmarski and A. Infectivity was determined after 6 h at 37°C by measuring β-galactosidase (β-gal) activity. ↵*Corresponding author.

Mailing address: Northwestern University, The Feinberg School of Medicine, Department of Microbiology-Immunology, Mail Code S213, Room Ward 6-241, 320 E. Superior St., Chicago, IL 60611. Available: http://intranet.adm.wiener-lab.com.ar/areas/Investigacion y Asuntos Regulatorios/biblioteca-cibio/PLoS ONE 2011 6 e19738.pdf/view. Fax: (312) 503-1339. E-mail: p-spear{at}northwestern.edu.

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