RealTime PCR analysis of 293T cells transfected with LMP1 or ΔLMP1-MAVS.

<p>293T cells were transfected with pcDNA3.1, LMP1 plasmid, or ΔLMP1-MAVS plasmid and total RNA isolated following 36-hour culture. Normalized expression was determined relative to the pcDNA3.1 control.</p

ΔLMP1-MAVS enhances anti-Gag immune responses as an Ad5 viral vector vaccine adjuvant.

<p>BALB/c mice were left untreated or vaccinated with a combination of Ad5-Gag and either Ad5-GFP (control), Ad5-ΔLMP1-MAVS, or Ad5-LMP1. Two weeks following vaccination, mice were challenged with vaccinia-gag virus. Vaccinia titers were measured from ovaries after 5 days. NT: no treatment.</p

Gene array analysis of transfected 293T cells and primary CD4+ T cells cultured with 293T supernatant.

<p>Three independent wells of 293T cells were transfected with pcDNA3.1 empty vector or ΔLMP1-MAVS plasmid and total RNA isolated 36 hours later. Primary CD4+ T cells from 3 independent donors were cultured with 293T supernatant (collected 24 hours following pcDNA3.1 or ΔLMP1-MAVS transfection). Total CD4+ T cell RNA was isolated 36 hours later. (A) Venn Diagrams of the number of probe sets upregulated and down-regulated (>2-fold change) by ΔLMP1-MAVS. (B) Differential gene expression of 293T cells and CD4+ T cells. (C) List of genes upregulated by ΔLMP1-MAVS in both 293T cells and CD4+ T cells. Fold-change and P-values for each probe set are shown for CD4+ T cells following ΔLMP1-MAVS treatment. (D) List of genes upregulated by CD4+ T cells but not 293T cells. Fold-change between pcDNA3.1 and pΔLMP1-MAVS and P-values for each probe set are shown. (E) Gen Go networks analysis of pathways significantly upregulated by ΔLMP1-MAVS in transfected 293T cells, or CD4+ T cells cultured with ΔLMP1-MAVS transfected 293T supernatant.</p

Beta-interferon mediates inhibition of HIV-1 BaL replication.

<p>(A) The relative level of HIV-1 BaL strain viral replication in TZM-bl cells was measured following incubation with supernatant from 293T cells transfected with either pcDNA3.1 or pΔLMP1-MAVS plasmid, combined with 60 μg of isotype control antibody, anti-IFN-β antibody, or anti-IFN-α antibody. (B) Human CD4+ T cells were infected with HIV-1 BaL in the presence of increasing concentrations of interferon-α and compared to infection in the presence of 293T supernatant following transfection with either pcDNA3.1 or pΔLMP1-MAVS plasmid. (C) Supernatant from 293T cells transfected with pcDNA3.1 GFP, LMP1, or ΔLMP1-MAVS plasmid was assayed for IFN-α and IFN-β secretion by ELISA. NT: no treatment.</p

Inhibition of HIV and SIV viral infection of TZM-bl and primary human CD4+ T cells.

<p>The relative level of viral replication in TZM-bl cells was measured following incubation with supernatant from 293T cells transfected with pcDNA3.1 vector expressing GFP (control), LMP1, or ΔLMP1-MAVS plasmid. (A) Infection with HIV-1 BaL strain. (B) Infection with VSV-G pseudotyped single cycle SIV. (C) CD4+ T cells were isolated from a healthy donor by negative selection, activated, and cultured with 293T supernatant for 24 hours. Cells were then washed and infected with HIV-1 BaL at an MOI or 0.1 or 1. The concentration of p24 was measured 6 days later by ELISA assay. *p<0.05, **p<0.01, ***p<0.001.</p

Exosome-depleted 293T supernatant inhibits HIV and SIV replication.

<p>293T cells were transfected with pcDNA3.1 plasmids encoding GFP, LMP1, or ΔLMP1-MAVS and supernatant was isolated. Supernatant was depleted of exosomes by ultracentrifugation. (A) TZM-bl cells were cultured with isolated exosomes and infected with increasing concentrations of HIV-1 BaL. (B) TZM-bl cells were cultured with exosome-depleted supernatant, followed by infection with HIV-1 BaL. (C) TZM-bl cells were cultured with exosome-depleted supernatant, followed by infection with VSV-G pseudotyped single cycle SIV. *p<0.05, **p<0.01, ***p<0.001.</p

Mass spectrometry analysis of total cell lysate.

<p>HEK293T cells, GALE KO (clone 4), GALK1 KO (clone 10), GALE+GALK1 KO (clone 12), GALE+GALK2 KO (clone 7), and GALE+GALK1 KO cells +galactose were grown in standard DMEM-10% FBS media. Cells were washed 3 times in cold PBS before analysis. <b>A)</b> MALDI-TOF mass spectra of permethylated O-glycans isolated from total cell lysates. All molecular ions are [M+Na]+. The sugar symbols are those as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179949#pone.0179949.ref028" target="_blank">28</a>]. Structural assignments are based on monosaccharide composition (obtained by MALDI-TOF MS), fragmentation analyses (MALDI-TOF/TOF MS/MS), and knowledge of glycan biosynthetic pathways. All peakes not labeled with am/z value are not glycans and are either matrix or general chemical background peaks. <b>B)</b> MALDI-TOF MS profiles of the permethylated N-linked glycans from total cell lysate of HEK293T cells, GALE KO, GALK1 KO, GALE+GALK1 KO, GALE+GALK2 KO, and GALE+GALK1 KO cells +galactose. All molecular ions are present in sodiated form [M +Na]⁺.</p

Cell growth curve.

<p>HEK293T cells, GALE KO (clone 4), GALK1 KO (clone 10), GALE+GALK1 KO (clone 12), GALK2 KO (clone 7), and GALE+GALK2 KO (clone 7) were analyzed to determine if disrupting key enzymes of the Leloir pathway impacted cell growth. 4x10^4 HEK293T cells were plated in triplicate in 6-well plates starting on day 0. Cells were harvested and counted using a Beckman Coulter Z1 Coulter Particle Counter every 24 hours.</p

Validation of GALE knockout cell lines.

<p>Following single cell sort, potential GALE KO clones were allowed to grow until they reached confluence in 6 well plates. <b>A)</b> Cell lysate was harvested by incubating cells in RIPA buffer. Clarified lysate was loaded onto a 4–12% SDS-PAGE gel and probed with an anti-GALE antibody. Wild-type HEK293T cell lysate was loaded in the first lane as a control. <b>B)</b> GALE enzymatic activity was analyzed on the protein level. GALE KO cells (clone 4) were transfected with expression vectors for SIVmac239 gp120 made as a truncated secreted product with a C-terminal polyhistidine tag. Secreted gp120 was purified from supernatant 48 hours post transfection using nickel-NTA columns. 3μg of purified gp120 was run on three 4–12% SDS-PAGE gels in triplicate. The first was analyzed by Coomassie Blue staining. The second gel was probed for gp120 using the rhesus anti-gp120 monoclonal antibody 3.11H. The third gel was probed for O-glycosylation using an HRP-labeled Jacalin lectin. For protein production, cells were grown in 10% FBS, 3% LDFBS, or 3% LDFBS with galactose and GalNAc (+sugars) until 12 hours post-transfection, at which point cells were washed and changed to serum free media. For further detail on cell culture conditions, refer to the materials and methods. <b>C)</b> Western blots were repeated as in <b>B</b>, with one exception. Galactose and GalNAc were added to the cell culture media separately to identify whether galactose or GalNAc was the limiting precursor for O-glycosylation in our cell culture conditions.</p

Leloir pathway of galactose metabolism.

<p>Illustrated are the 2 different pathways in which galactose and GalNAc can be salvaged or synthesized for use in glycosylation. Galactose and GalNAc can be taken up and converted to UDP-galactose and UDP-GalNAc, respectively, via the salvage pathway. UDP-galactose and UDP-GalNAc can also be interconverted from UDP-glucose and UDP-GlcNAc respectively by the enzyme UDP-galactose-4-epimerase (GALE). UDP-galactose and UDP-GalNAc can then be used for glycosylation.</p