Journal: Microbiology Spectrum

Article Title: Subcellular Distribution of BALF2 and the Role of Rab1 in the Formation of Epstein-Barr Virus Cytoplasmic Assembly Compartment and Virion Release

doi: 10.1128/spectrum.04369-22

Figure Lengend Snippet: The majority of BALF2 is detected in the nucleus, and a small portion of BALF2 localizes to the juxtanuclear concave region and colocalizes with assembly compartment components at the mid-stage of EBV reactivation in TW01-EBV cells. (A) The specificity of BALF2 monoclonal antibody (OT13B) was examined by immunoblotting of pSG5-Rta or pSG5 vector-transfected TW01-EBV cell lysates. A similar setting of transfected cell lysates of the parental TW01 cells served the negative control. BRLF1 (Rta) and BMRF1 (EA-D) expression was detected as lytic markers. GAPDH served as the loading control. The detection of slight signals of BMRF1 and BALF2 indicates that a small portion of TW01-EBV underwent spontaneous lytic reactivation. (B) The BALF2 expression levels were examined at 24, 48, and 72 h post-pSG5-Rta transfection into TW01-EBV cells. GAPDH served as the loading control. (C) The distribution of BALF2 in TW01-EBV cells at 24, 48, and 72 h post-Rta transfection was detected by OT13B Ab and observed with confocal microscopy. Cell numbers with nuclear only (N) or partial cytoplasmic (N+C) distribution of BALF2 are indicated at the bottom. (D) The subcellular distribution of BALF2 and components in the cytoplasmic assembly compartment in TW01-EBV cells was detected by immunofluorescence staining and observed by confocal microscopy. The slide-cultured TW01-EBV cells were harvested at 48 h post-pSG5-Rta or pSG5 transfection and stained with specific antibodies. BALF2 (green) was detected together with cis -Golgi marker GM130 (top panel), EBV tegument protein BBLF1 (middle panel), or BGLF4 (bottom panel). (E) EBV virions were purified from TPA/sodium-induced B95-8 cells and subjected to a sucrose gradient fractionation. (a) Virion components, including BLLF1 (gp350/220), BcLF1 (VCA), BALF2 (single-stranded DNA [ssDNA] binding protein), BVRF1 (capsid associated tegument protein, DNA packaging factor), and BGLF4 (protein kinase), were detected by immunoblotting with specific antibodies. (b) EBV genomic DNA was detected by qPCR amplification of BALF5 in each fraction (bottom panel). (c and d) Fractions F2 and F7 from panel a were fixed and stained with 1% uranyl acetate and then observed under TEM at 200 kV and ×12,000 magnification. In F2, the size of vesicle-like particles is small (~50 nm diameter). In F7, the virion size is around ~160 nm.

Article Snippet: Primary antibodies used in protein detection, including Western blotting (WB) and immunofluorescence assay (IF), included BALF2 (OT13B; 1:80,000 in bovine serum albumin [BSA] for WB and 1:800 in BSA for IF) , Rta (37-1H10; 1:1,000 in skim milk for WB) , GFP (GTX113617, GeneTex; 1:1,000 in skim milk for WB), gp350/220 (72A1, ATCC, Manassas, VA; 1:5 in BSA for WB and undiluted for IF), BMRF1 (88A9; 1:100 in skim milk for WB) , gp110 (L2) , BGLF4 (2224, 1:100 in BSA for IF; 2616, 1:1,000 for WB), and BVRF1 mouse antiserum (1:200 in BSA for WB).

Techniques: Western Blot, Plasmid Preparation, Transfection, Negative Control, Expressing, Control, Confocal Microscopy, Immunofluorescence, Staining, Cell Culture, Marker, Purification, Fractionation, Binding Assay, Amplification

Journal: Microbiology Spectrum

Article Title: Subcellular Distribution of BALF2 and the Role of Rab1 in the Formation of Epstein-Barr Virus Cytoplasmic Assembly Compartment and Virion Release

doi: 10.1128/spectrum.04369-22

Figure Lengend Snippet: Expression of dominant-negative GFP-Rab1A(N124I) diminishes the clustering of BALF2 to the viral assembly compartment. (A) TW01-EBV cells were cotransfected with pSG5-Rta or pSG5 plus GFP-C1, GFP-Rab1A, GFP-Rab1A N124I, or GFP-Rab1A Q70L. The cell lysates were harvested at 48 hpt. The expression levels of viral proteins BALF2, BMRF1 (EA-D), BLLF1 (gp350/220), and BALF4 (gp110) and cellular proteins GM130 and Rab1A were detected with BRLF1 antibody, BALF2 antibody, BMRF1 antibody, gp350/220 antibody, gp110 antibody, GM130 antibody, and Rab1A antibody in immunoblotting. Transfected GFP, GFP-Rab1A, and BRLF1 (Rta) were also detected in the blotting. GAPDH served as the loading control. Proteins in the top panel were displayed in regular 10% SDS-PAGE, whereas gp350/220 and gp110 were detected in immunoblotting after nonreducing 8% SDS-PAGE. Open arrowheads indicate mature gp350 or gp220, while the solid arrowhead indicates the precursor of gp350/220. (B) The distribution of BALF2 with different GFP-Rab1A constructs was observed by immunostaining and confocal microscopy at 48 h post cotransfection of Rta and GFP, GFP-Rab1A, GFP-Rab1A(N124I), or GFP-Rab1A(Q70L). The percentages listed next to each group were calculated to indicate cells expressing BALF2 localized to the viral assembly compartment or not over GFP + GM130 + cells.

Article Snippet: Primary antibodies used in protein detection, including Western blotting (WB) and immunofluorescence assay (IF), included BALF2 (OT13B; 1:80,000 in bovine serum albumin [BSA] for WB and 1:800 in BSA for IF) , Rta (37-1H10; 1:1,000 in skim milk for WB) , GFP (GTX113617, GeneTex; 1:1,000 in skim milk for WB), gp350/220 (72A1, ATCC, Manassas, VA; 1:5 in BSA for WB and undiluted for IF), BMRF1 (88A9; 1:100 in skim milk for WB) , gp110 (L2) , BGLF4 (2224, 1:100 in BSA for IF; 2616, 1:1,000 for WB), and BVRF1 mouse antiserum (1:200 in BSA for WB).

Techniques: Expressing, Dominant Negative Mutation, Western Blot, Transfection, Control, SDS Page, Construct, Immunostaining, Confocal Microscopy, Cotransfection

Journal: Microbiology Spectrum

Article Title: Subcellular Distribution of BALF2 and the Role of Rab1 in the Formation of Epstein-Barr Virus Cytoplasmic Assembly Compartment and Virion Release

doi: 10.1128/spectrum.04369-22

Figure Lengend Snippet: Expression of GFP-Rab1(N124I) interferes with glycoprotein gp350/220 maturation and targeting to the viral assembly compartment. (A) The distribution of EBV glycoprotein gp350/220 under the same setting as in Fig. 7B . The percentages listed under each group were calculated as cells expressing a shown pattern of gp350/220 over GFP + cells. (B) TW01-EBV cells were cotransfected with plasmids expressing Rta and GFP-C1, GFP-Rab1A, GFP-N124I, or GFP-Q70L. TW01-EBV cells transfected with the Rta-expressing plasmid or vector were treated with DMSO or brefeldin A (1 μg/mL) at 24 hpt, and cell lysates were harvested after another 24 h of incubation. Expression of BLLF1 (gp350/220) and BALF4 (gp110) were analyzed by nonreducing 8% SDS-PAGE followed by immunoblotting at 48 h posttransfection. Hypo-glycosylated gp350/220 (about 165 kDa) is indicated with a black arrowhead, while glycosylated gp350 and gp220 are indicated with open arrowheads. Lanes 3, 6, 9, and 12 were treated with brefeldin A (1 μg/mL) for Golgi disruption at 24 h post-Rta transfection.

Article Snippet: Primary antibodies used in protein detection, including Western blotting (WB) and immunofluorescence assay (IF), included BALF2 (OT13B; 1:80,000 in bovine serum albumin [BSA] for WB and 1:800 in BSA for IF) , Rta (37-1H10; 1:1,000 in skim milk for WB) , GFP (GTX113617, GeneTex; 1:1,000 in skim milk for WB), gp350/220 (72A1, ATCC, Manassas, VA; 1:5 in BSA for WB and undiluted for IF), BMRF1 (88A9; 1:100 in skim milk for WB) , gp110 (L2) , BGLF4 (2224, 1:100 in BSA for IF; 2616, 1:1,000 for WB), and BVRF1 mouse antiserum (1:200 in BSA for WB).

Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, SDS Page, Western Blot, Disruption

Journal: Microbiology Spectrum

Article Title: Subcellular Distribution of BALF2 and the Role of Rab1 in the Formation of Epstein-Barr Virus Cytoplasmic Assembly Compartment and Virion Release

doi: 10.1128/spectrum.04369-22

Figure Lengend Snippet: Expression of GFP-Rab1(N124I) in TW01-EBV and 293/rM81 cells attenuates EBV release and infectivity. (A) TW01-EBV cells were transfected with pSG5-Rta or pSG5 plus GFP, GFP-Rab1A, GFP-Rab1A(N124I), or GFP-Rab1A(Q70L) using Lipofectamine 2000. The cell lysates were harvested for immunoblotting at 48 hpt. The expression levels of Rta, GM130, endogenous Rab1A, BALF2, and wild-type, dominant-active, or constitutive GFP-Rab1A in TW01-EBV cells were detected by GFP antibody, BRLF1 antibody, BMRF1 antibody, and Rab1A antibody. Gp110 and gp350/220 were analyzed by 8% SDS-PAGE (bottom panels). (B) The intracellular EBV genomic DNA copy numbers of TW01-EBV cells were analyzed by q-PCR at 48 hpt of Rta and GFP-C1 vector or GFP-Rab1A constructs. (C) The released EBV virion DNA in the medium in the same setting was analyzed by q-PCR at 48 hpt. The representative data from 4 independent experiments are displayed. (D) 293/rM81 cells were cotransfected with Zta and gp110 expression plasmids or pSG5 control plus GFP, GFP-Rab1A, GFP-Rab1A(N124I), or GFP-Rab1A(Q70L) using TransIT-LT1. Cell lysates were harvested for immunoblotting at 72 hpt. The expression levels of Zta, Rta, BMRF1, BALF2, and wild-type, dominant-active, or constitutive GFP-Rab1A in 293/rM81 cells were detected using specific antibodies. For detecting gp350/220, gp110, and BcLF1, lysates were displayed in nonreducing 8% SDS-PAGE. (E) The intracellular EBV genomic DNA copy number of 293/rM81 cells was analyzed by q-PCR at 72 hpt of Zta, gp110, and GFP-C1 vector or GFP-Rab1A constructs. (F) The released EBV virion DNA of the same setting was analyzed with q-PCR at 72 hpt. (G) Culture supernatants from the experiment in panel D were filtered through a 0.45-μm filter, and that same volume of filtered viral supernatant was added to Raji cells and incubated for 72 h in duplicated wells. The percentages of Raji cells expressing GFP were determined through flow cytometry. The results of one experiment are presented. The horizontal axis represents the forward scatter area (FSC-A), and the vertical axis indicates the intensity of GFP fluorescence (GFP-A). The GFP positivity threshold was determined according to the noninduction control (C1-pSG5). Experimental results are presented using the mean with standard deviation (SD) as the bar graph in panel H.

Article Snippet: Primary antibodies used in protein detection, including Western blotting (WB) and immunofluorescence assay (IF), included BALF2 (OT13B; 1:80,000 in bovine serum albumin [BSA] for WB and 1:800 in BSA for IF) , Rta (37-1H10; 1:1,000 in skim milk for WB) , GFP (GTX113617, GeneTex; 1:1,000 in skim milk for WB), gp350/220 (72A1, ATCC, Manassas, VA; 1:5 in BSA for WB and undiluted for IF), BMRF1 (88A9; 1:100 in skim milk for WB) , gp110 (L2) , BGLF4 (2224, 1:100 in BSA for IF; 2616, 1:1,000 for WB), and BVRF1 mouse antiserum (1:200 in BSA for WB).

Techniques: Expressing, Infection, Transfection, Western Blot, SDS Page, Plasmid Preparation, Construct, Control, Incubation, Flow Cytometry, Fluorescence, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Subcellular Distribution of BALF2 and the Role of Rab1 in the Formation of Epstein-Barr Virus Cytoplasmic Assembly Compartment and Virion Release

doi: 10.1128/spectrum.04369-22

Figure Lengend Snippet: A hypothetical diagram of the BALF2 subcellular distribution during virus replication and the role of Rab1A in the assembly of EBV viral particles. The top part, above the dotted line, indicates early steps of EBV reactivation. (a) BALF2 is translated from the ER and transported into the nucleus through a canonical importin-β-dependent mechanism. (b) At the mid- to late stage of EBV replication, viral DNAs are encapsidated into procapsids, and the nucleocapsids are egressed from the nucleus through either a primary envelopment and deenvelopement process (b) or a nuclear egress complex and ESCRT-dependent mechanisms (b′). (c) After synthesis on the ER membrane, gp350/220 was transported through Rab1-mediated vesicle transport to the cis -Golgi apparatus for proper glycosylation. (d) Fragmented Golgi and Golgi-derived vesicles with membrane-associated gp350/220 may target the viral assembly compartment. (e) Rab1 interacts with GM130 and anchors on the cis -Golgi membranes and Golgi-derived vesicles. The membrane-associated Rab1 helps the engulfment of nucleocapsids or nucleocapsids containing vesicles through interaction with a BALF2-associated protein-protein interaction. (f) The mature virion with tegument (yellow regions) may be released through exocytosis.

Article Snippet: Primary antibodies used in protein detection, including Western blotting (WB) and immunofluorescence assay (IF), included BALF2 (OT13B; 1:80,000 in bovine serum albumin [BSA] for WB and 1:800 in BSA for IF) , Rta (37-1H10; 1:1,000 in skim milk for WB) , GFP (GTX113617, GeneTex; 1:1,000 in skim milk for WB), gp350/220 (72A1, ATCC, Manassas, VA; 1:5 in BSA for WB and undiluted for IF), BMRF1 (88A9; 1:100 in skim milk for WB) , gp110 (L2) , BGLF4 (2224, 1:100 in BSA for IF; 2616, 1:1,000 for WB), and BVRF1 mouse antiserum (1:200 in BSA for WB).

Techniques: Virus, Membrane, Glycoproteomics, Derivative Assay

Journal: Cell Reports

Article Title: Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms

doi: 10.1016/j.celrep.2023.113608

Figure Lengend Snippet: SHH levels remain normal in patients with MPNs, while GLI1 RNA and protein level increases are linked to the JAK2 V617F mutation (A) Plasma from patients with MPNs (n = 12, IDs MPN1–MPN12) and healthy donors (HDs) was isolated, and SHH protein levels were determined by ELISA. (B) GLI1 expression in megakaryocytic cell lines was quantified with qPCR relative to GAPDH , and the JAK2 V617F -containing SET2 cell line was normalized to the JAK2 WT MEG-01 cell line (n = 3). (C) GLI1 expression in undifferentiated patient-derived iPSC lines was quantified with qPCR relative to GAPDH , normalized to respective JAK2 WT clones (n = 3). (D) PBMCs isolated from patients with MPNs compared with HD samples (HDs, n = 4, n = 5; MF0, n = 3; MF1, n = 4; MF2 and MF3, n = 3, IDs MPN13–MPN22). Shown are MFI quantification of GLI1 levels in CD66b HI granulocytes per group normalized to fluorescence minus one (FMO) controls and a representative GLI1 histogram, with count normalized to mode. (E and F) MFI quantification of GLI1 levels per group normalized to FMO controls in CD88 + CD89 + HLADR + monocytes (E), CD3 + T cells, and CD19 + B cells (F). (G) MFI quantification of GLI1 levels in CD66b + granulocytes after PBMCs from HDs (n = 3) were stimulated with 10 ng/mL rTGFb or 50 ng/mL rIL-6 for 72 h, normalized to FMO controls. Bar chart data are shown as mean ± SEM with unpaired Student’s t test. Boxplot data are shown as minimum (min) to maximum (max) with line at mean, one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ = p < 0.01, ∗∗∗∗ = p < 0.0001. See also Figure S1 and Table S1 .

Article Snippet: For SHH concentration, Plasma samples were used undiluted and quantified using the Human Sonic Hedgehog/Shh N Terminus DuoSet ELISA (R&D).

Techniques: Mutagenesis, Clinical Proteomics, Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Derivative Assay, Clone Assay, Fluorescence

Journal: Cell Reports

Article Title: Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms

doi: 10.1016/j.celrep.2023.113608

Figure Lengend Snippet: MIF protein levels are relevant in patients with MPNs, and genetic or pharmacological perturbation of the MIF-CD74 axis reduces disease phenotype (A) MIF protein level assessed by ELISA in patients with MPNs (n = 54) was normalized to HD (n = 10) samples (IDs MPN23–MPN76). Unpaired Student’s t test with Welch’s correction. Shown is correlation of MIF protein levels and PLT levels in patients with MPNs; simple linear regression testing. (B) MIF protein levels divided according to genotype (Jak2 V617F , n = 33; CALR mut , n = 21). One-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C) JAK2 V617F mutant HEL cells were treated with 500 nM ruxolitinib for 24 h or 250 nM BEZ235 for 4 h, and MIF expression levels relative to GAPDH were quantified by qPCR and normalized to the DMSO-treated control (n = 3). Unpaired Student’s t test with Welch’s correction. (D) C57BL/6 recipient mice received lethal irradiation followed by transplantation with either WT or MIF −/− cKit + HSPCs transduced with either ThPO or empty vector (EV) control lentiviral vectors (n = 5/group). Mice were sacrificed 32 days after transplantation. Monocytes (CD11b + GR1 − ) and macrophages (F4/80 + GR1 − ) in the PB were analyzed via FACS over the course of the experiment. Mean ± SEM, two-way ANOVA followed by Tukey’s post hoc test; significance for values at sacrifice is indicated. (E) Monocytes (CD11b + GR1 − ) and macrophages (F4/80 + GR1 − ) were analyzed in the BM at sacrifice via FACS. Mean ± SEM, one way ANOVA followed by Tukey’s post hoc test. (F) Representative images of H&E staining of the femur. Scale bar, 60 μm. (G) Representative images of reticulin staining. Scale bar, 200 μm. (H) Quantification of myelofibrosis grade based on reticulin staining of the femur. (I) BM cellularity (total cell count) and viability (percent trypan blue negative) at sacrifice. (J) Human BM-MSC cell line cultured with conditioned medium collected from spin embryoid bodies (EBs) generated from either JAK2 WT or JAK2 V617F patient-derived IPSCs (n = 2 each) and treated with 40 μ M ISO-1 or DMSO for 48 h. Col1a1 expression was quantified with qPCR relative to HPRT1, normalized to respective JAK2 WT conditioned medium, untreated conditions (n = 3). Mean ± SEM, one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also Figure S5 .

Article Snippet: For SHH concentration, Plasma samples were used undiluted and quantified using the Human Sonic Hedgehog/Shh N Terminus DuoSet ELISA (R&D).

Techniques: Enzyme-linked Immunosorbent Assay, Mutagenesis, Expressing, Control, Irradiation, Transplantation Assay, Transduction, Plasmid Preparation, Staining, Cell Counting, Cell Culture, Generated, Derivative Assay

Journal: Cell Reports

Article Title: Non-canonical Hedgehog signaling mediates profibrotic hematopoiesis-stroma crosstalk in myeloproliferative neoplasms

doi: 10.1016/j.celrep.2023.113608

Figure Lengend Snippet:

Article Snippet: For SHH concentration, Plasma samples were used undiluted and quantified using the Human Sonic Hedgehog/Shh N Terminus DuoSet ELISA (R&D).

Techniques: Virus, Recombinant, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Retroviral, Software