Journal: PLoS ONE

Article Title: Rectal HSV-2 Infection May Increase Rectal SIV Acquisition Even in the Context of SIVΔnef Vaccination

doi: 10.1371/journal.pone.0149491

Figure Lengend Snippet: A) Schematic representation of the rectal challenges regimen. I. n = 9 animals were inoculated with 3000 TCID 50 of SIVΔNef, 12 weeks later challenged with 4x10 6 pfu of HSV-2 and 3 weeks later with 3000 TCID 50 of SIVmac239wt. II. n = 9 animals were challenged with 4x10 6 pfu of HSV-2 and 3 weeks later with 3000 TCID 50 of SIVmac239wt. III) n = 8 animals were inoculated with 3000 TCID 50 of SIVΔNef and 15 weeks later challenged with 3000 TCID 50 of SIVmac239wt. IV) 6 animals were challenged with 3000 TCID 50 of SIVmac239wt as a control. The results of each infection are shown in the . B) Plasma SIVgag RNA copies are shown for the SIVΔNef infected animals throughout the study. The dashed and full vertical lines indicate, respectively, the time of HSV-2 and SIVmac239wt challenges. C) Plasma SIVmac239wt (WT) RNA copies detected by discriminatory RT-qPCR are shown post-SIVmac239wt challenge for SIVmac239wt infected animals (WT; mean±SEM n = 12 animals at day 5 and 10, n = 5 at day 15), HSV-2/SIVmac239wt co-infected (HSV-2 WT; mean±SEM 7 animals) and for the 1 SIVΔNef/HSV-2/SIVmac239wt co-infected animal. All animals were necropsied 15 days after SIVmac239wt challenge. D-E) Blood CD4 + T cells counts 2 weeks post-WT challenge (D) and 2 weeks post-HSV-2 challenge (E) are shown for the animals that acquired HSV-2 or SIVΔNef infection compared to those that remained uninfected (Uninf). Bars represent mean ± SEM. Significant p values are shown (α<0.05).

Article Snippet: The generation of HSV-2 strain G stocks was performed on Vero cells (CCL-81; ATCC) [ ] and virus titers were determined by plaque-forming assay [ ].

Techniques: Infection, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Rectal HSV-2 Infection May Increase Rectal SIV Acquisition Even in the Context of SIVΔnef Vaccination

doi: 10.1371/journal.pone.0149491

Figure Lengend Snippet: A) The concentration of CXCL8 in rectal swabs at day 7 post-HSV-2 challenge is shown for the animals that acquired HSV-2 or SIVΔNef infection (or both) compared to those that remained uninfected (Uninf). B) The concentration of CXCL8 in rectal swabs at day 7 post-HSV-2 challenge is plotted against SIV plasma VL at week 2 post-WT challenge for the animals in Round 2 with the exclusion of the SIVΔNef+ ones (plasma VL at week 2 post-WT is missing for Round 1). Spearman correlation p value is shown (significance p<0.05). C) Blood cells were gated on singlets, live and CD3 + CD4 + cells. The frequency of CCR6 + CD4 + T cells on day 7 post HSV-2 (left) and FoxP3 + CD127 low CD4 + T cells on day 14 post-HSV-2 (right) is shown for the animals that acquired HSV-2 or SIVΔNef infection compared to those that remained uninfected (Uninf). D) DCs from inguinal LNs were gated on live, singlets, Lin − HLA-DR + cells. The expression of CD80 on total LNs DCs and the frequency of α 4 β 7 + DCs 10 days post-HSV-2 infection are shown for the animals that acquired HSV-2 or SIVΔNef infection compared to those that remained uninfected (Uninf). Bars represent mean±SEM. Significant p values from Mann-Whitney test are shown (α 4 <0.05).

Article Snippet: The generation of HSV-2 strain G stocks was performed on Vero cells (CCL-81; ATCC) [ ] and virus titers were determined by plaque-forming assay [ ].

Techniques: Concentration Assay, Infection, Expressing, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Rectal HSV-2 Infection May Increase Rectal SIV Acquisition Even in the Context of SIVΔnef Vaccination

doi: 10.1371/journal.pone.0149491

Figure Lengend Snippet: The concentration of cytokines and chemokines significantly modulated by HSV-2 infection in plasma 3 weeks post-HSV-2 infection (time of SIVmac239wt challenge) are shown for the animals that acquired HSV-2 or SIVΔNef infection compared to those that remained uninfected (Uninf). Bars represent mean±SEM. Significant p values from Mann-Whitney test are shown (α<0.05).

Article Snippet: The generation of HSV-2 strain G stocks was performed on Vero cells (CCL-81; ATCC) [ ] and virus titers were determined by plaque-forming assay [ ].

Techniques: Concentration Assay, Infection, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Rectal HSV-2 Infection May Increase Rectal SIV Acquisition Even in the Context of SIVΔnef Vaccination

doi: 10.1371/journal.pone.0149491

Figure Lengend Snippet: A) The levels of cytokines and chemokines significantly modulated by HSV-2 co-infection in rectal swabs on day 7 post-SIVmac239wt challenge are shown for all animals in round 1 and 2 that acquired HSV-2, SIVΔNef or SIVmac239wt infection (all 3 or any combination of them) compared to those that remained uninfected (Uninf). B-C) Cells from iliac LNs were gated on live, singlets and CD3 + CD4 + (B) or Lin - HLA-DR + (C) cells. The expression of CCR6 on CD4 + T cells and the frequencies of α 4 β 7 + DCs and CD103 + DCs are shown for all animals in round 2 that acquired HSV-2, SIVΔNef and/or SIVmac239wt infection compared to the one that remained uninfected (Uninf) D-E) Cells from MLNs were gated on live, singlets and CD3 + CD4 + (D) or Lin - HLA-DR + (E) and the frequency of CCR5 + cells within CD95 + CD4 + T cells (D) and CCR7 + DCs and the expression of CD80 on DCs (E) are shown for all animals in round 2 that acquired HSV-2, SIVΔNef and/or SIVmac239wt infection compared to the one that remained uninfected (Uninf). Bars represent mean±SEM. Significant p values from Mann-Whitney test are shown (α<0.05). p<0.125 are also shown to indicate a tendency toward a significant difference.

Article Snippet: The generation of HSV-2 strain G stocks was performed on Vero cells (CCL-81; ATCC) [ ] and virus titers were determined by plaque-forming assay [ ].

Techniques: Infection, Expressing, MANN-WHITNEY

Journal: PLoS ONE

Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

doi: 10.1371/journal.pone.0088803

Figure Lengend Snippet: A (upper panel). Polarized tonsil epithelial cells were treated with active or inactive recombinant HIV tat and gp120 in combination for 5 days, and TER was then measured. A (lower panel). The same cells were used to evaluate paracellular permeability after 5 days of treatment, as determined by leakage of IgG (Fab’) 2 from the apical chamber to the basolateral chamber. OD, optical density. B. The same cells were immunostained for ZO-1 (green). Cell nuclei are stained in blue. C. HSV-1 at an MOI of 10 PFU per cell was added to the upper chamber of polarized cells, and culture medium was collected from the basolateral chamber after 1, 2, or 4 h. HSV-1 paracellular spread was confirmed by detection of ICP4 protein in Vero cells (green) 4 h after infection. Cell nuclei were stained with propidium iodide (red). Yellow indicates colocalization of ICP4 with the nuclear marker. D. HSV-1 paracellular spread was quantified by counting of HSV-1-infected Vero cells in 10 random microscopic fields and determining the percentage of cells positive for ICP4. A, D: Error bars indicate SEM (n = 3).

Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

Techniques: Recombinant, Permeability, Staining, Infection, Marker

Journal: PLoS ONE

Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

doi: 10.1371/journal.pone.0088803

Figure Lengend Snippet: A. Polarized tonsil epithelial cells were incubated with dual X4- and R5-tropic HIV-1 SF33 for 5 days. One set of cells was exposed to UV-inactivated virions. Culture medium was changed daily to add fresh virus, and TER was measured. B. HSV-1 was added to the apical surface of polarized cells upon complete disruption of TJs at 5 days. HSV paracellular spread at 1, 2, and 4 h after incubation was examined in Vero cells grown in the basolateral chamber of filter inserts by immunostaining of ICP4 protein. HSV-1 paracellular spread was quantified by counting HSV-1-infected Vero cells, and the percentage of cells positive for ICP4 was determined. A, B: Error bars indicate SEM (n = 3).

Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

Techniques: Incubation, Immunostaining, Infection

Journal: PLoS ONE

Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

doi: 10.1371/journal.pone.0088803

Figure Lengend Snippet: A. Polarized tonsil epithelial cells were coimmunostained for nectin-1 and E-cadherin. Yellow in the merged panel indicates colocalization of nectin-1 and E-cadherin. B. Polarized tonsil cells were treated with active or inactive tat and gp120 in combination for 5 days. In parallel experiments, cells were exposed to HIV-1 SF33 for 5 days. Cells were then immunostained for E-cadherin and nectin-1. C. Polarized cells were treated with active or inactive tat and gp120 in combination or with cell-free HIV-1 SF33 for 5 days. Cells were then extracted, and E-cadherin and nectin-1 were detected by Western blot assay. D. Apical or basolateral membranes of polarized epithelial cells were treated with inactive or active HIV tat and labeled with sulfo-NHS-LC-biotin. Nectin-1 was detected in the avidin-precipitated total membrane proteins by Western blot assay. E. HSV-1 gD(306t) at 20 µg/ml was added to apical or basolateral membranes of polarized epithelial cells treated with inactive or active HIV tat/gp120. After 30 min the cell surface was labeled with sulfo-NHS-LC-biotin. Proteins biotinylated at the cell surface were precipitated with streptavidin–agarose beads, and gD was detected by Western blot assay. AP, apical; BL, basolateral.

Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

Techniques: Western Blot, Labeling, Avidin-Biotin Assay

Journal: PLoS ONE

Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

doi: 10.1371/journal.pone.0088803

Figure Lengend Snippet: A. Polarized tonsil cells were treated with HIV tat/gp120 or HIV virions for 5 days and infected with HSV-1. After 24 h, cells were fixed and immunostained using anti-HSV-1 gB antibodies (red). Cell nuclei are stained in blue. B. HSV-1 infection was quantitatively evaluated, and the percentage of cells positive for gB was determined. Error bars indicate SEM. C. Cells were incubated with antibodies against nectin-1 for 1 h and then infected with HSV-1. Cells were fixed after 24 h, HSV-1 infection was confirmed by detection of goat anti-HSV-1 immune serum, and the number of infected cells was counted. ab, cells incubated with antibodies. c, control cells without antibodies. Error bars indicate SEM. *P<0.01, **P<0.001, all compared with the control group.

Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

Techniques: Infection, Staining, Incubation

Journal: PLoS ONE

Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

doi: 10.1371/journal.pone.0088803

Figure Lengend Snippet: A. Polarized tonsil cells were treated with active or inactive tat/gp120 and HIV virions for 5 days. Disrupted cells were infected with HSV-1 at 0.01 PFU per cell from basolateral membranes of polarized cells. After 3 days, cells were fixed and immunostained using goat anti-HSV immune serum (green). Cell nuclei are stained in red. Yellow represents colocalization of HSV proteins and nuclei. B. (upper panel) Plaque numbers were counted from 3 independent filter inserts and data are presented as the average number of HSV-infected plaques per insert. (lower panel) Cell-to-cell spread of HSV-1 was quantitatively evaluated by counting HSV-infected cells in the plaques. Results are presented as the average number of HSV-infected cells per plaque. Error bars indicate SEM. C. Polarized cells were infected with HSV-1. After 4 h, antibodies to nectin-1 and gD were added separately and in combination. Cell medium was changed daily to add fresh antibodies. Cells were fixed and immunostained for HSV-1, and the plaque numbers (upper panel) and the number of HSV-1-positive cells in plaques were counted (lower panel). Error bars indicate SEM. *P<0.05, *P<0.01, **P<0.001, all compared with the control group.

Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

Techniques: Infection, Staining

Journal: PLoS ONE

Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

doi: 10.1371/journal.pone.0088803

Figure Lengend Snippet: The oral mucosal epithelium consists of stratified squamous epithelial cells. Each layer of epithelial cells forms lateral intercellular junctional complexes, including AJs and TJs. HSV-1 gD receptor nectin-1 is sequestered within the intact AJs area of lateral membranes of epithelial cells (left panel). HIV-induced disruption of AJs exposes nectin-1 from its sequestered areas (right panel), which binds to HSV gD and thereby promotes HSV infection and cell-to-cell spread within the oral epithelium. HIV-induced disruption of TJs leads to paracellular spread of HSV virions, which may facilitate penetration of virus from the apical to the basolateral direction into the deeper part of the epithelium, and from the basolateral to the apical direction leading to release of virus into saliva. Thus, HIV-induced disruption of epithelial junctions may facilitate the spread of HSV-1 infection within the mucosal epithelium, leading to the rapid progression of HSV-mediated mucosal lesions and ulcers.

Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

Techniques: Infection

Journal: Life Science Alliance

Article Title: Attenuation of cGAS/STING activity during mitosis

doi: 10.26508/lsa.201900636

Figure Lengend Snippet: (A) Cells were treated with vehicle, nocodazole (NOC), golgicide A (GCA), or brefeldin A (BFA) for 1 h prior and during a 90-min transfection with HSV-60 oligonucleotide, calf-thymus DNA (CTD), or plasmid pGL3. (B, C, D) Transfection of vehicle- or GCA-treated cells with pGL3 or pIC, and (C, D) densitometric quantification of pIRF3 blots. ** P < 0.001, n = 5 biological replicates. (E) Vehicle- or GCA-treated cells were treated with H 2 O or 12.5 μg cGAMP for 90 min before SDS–PAGE and Western blot for cGAS/STING pathway components. (F) cGAMP production in vehicle- and GCA-treated cells upon pGL3 transfection. n = 3 biological replicates, with n = 2 technical replicates each. Source data are available for this figure.

Article Snippet: Cells were transfected with 500 ng dsDNA oligonucleotide HSV-60 (tlrl-hsv-60n, 60 bp; InvivoGen), calf-thymus DNA (D4764, >20 kb; Sigma-Aldrich), or endotoxin-free pGL3 (E1751, 5.3 kb; Promega), or 500 ng high molecular weight poly(I:C) (tlrl-pic, 1.5–8 kb; InvivoGen) using Lipofectamine 2000 (11668; Thermo Fisher Scientific) in OptiMEM (Life Technologies).

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot