Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 3. Subcellular localization of recycling endosomes (transferrin receptor), multivesicular bodies (CD63) and lysosomal (Lamp1) markers in HCMV-infected cells. BJ1 cells were either mock infected (upper panels) or infected with HCMV at an moi of 0.5 (lower panels). After 4 dpi, cells were fixed, permeabilized and stained with anti-transferrin receptor, TfR (green in left panels), anti-CD63 (green in middle panels), anti-Lamp1 (green in right panels), and anti-HCMV glycoproteins UL33 and gH (red) antibodies. In HCMV-infected cells TfR was recruited to the virus factory and showed colocalization with HCMV UL33 (yellow in merge lower panel), CD63 was recruited to the assembly site and Lamp1 was excluded from this site. Scale bars, 20 mm.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Infection, Staining, Virus

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 5. Examination of cellular markers in cell lysates and supernatants of HCMV-infected cells. A. Equal number of BJ1 cells mock infected or HCMV infected for 4 days at an moi of 3 were lysed and viral particles pelleted by centrifugation of the clarified supernatants were mixed with non-reducing SDS-PAGE sample buffer. Lysates were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were cut and incubated, or sequentially incubated, with antibodies against cellular markers and antibodies against clathrin heavy chain or actin to check protein loading in cell lysates, and antiviral tegument protein pp28 antibodies to check HCMV viral particles secreted into the supernatants. Autoradiography films were scanned; images were cropped and assembled with Adobe Photoshop. Molecular weights in kDa are indicated. We found CD63, TGN46, EEA1, TfR and CI-M6PR in the supernatants of HCMV-infected cells. B. Results of cell lysates in (A) were analysed by densitometry, normalized to the expression levels of loading controls and expressed as a percentage of the levels in uninfected cells. We observed a reproducible reduction in the levels of CD63, TGN46 and CD-M6PR together with an increase in the levels of TfR by comparison with uninfected cells.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Infection, Centrifugation, SDS Page, Incubation, Autoradiography, Expressing, Comparison

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 6. Examination of cellular markers in purified HCMV virions. A. Electron microscopy of purified HCMV virions negatively stained with 2% uranyl acetate. When viral particles were partially disrupted, uranyl acetate revealed the nucleocapsids. Scale bar, 200 nm. B. 35 ng of purified virions per lane were separated by SDS-PAGE under non-reducing conditions and transferred to PVDF membranes. Membranes were cut and incubated, or sequentially incubated, with antibodies against cellular markers and antiviral tegument protein pp28 antibodies. Autoradiography films were scanned; images were cropped and assembled with Adobe Photoshop. Molecular weights in kDa are indicated. We found CD63, TGN46, TfR and CI-M6PR in purified virions.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Electron Microscopy, Staining, SDS Page, Incubation, Autoradiography

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 7. Transcriptional regulation of cellular markers during HCMV infection. Equal number of BJ1 cells were mock infected or infected with HCMV at an moi of 3, and RNA was extracted at the indicated times. Gene expression of cellular markers and HCMV UL83 (viral protein pp65) was measured by qPCR, normalized to 18S rRNA expression and calibrated to the levels in uninfected cells. CD63, TGN46, HRS and annexin I transcripts were downregulated over the time-course of HCMV infection, while EEA1 and CI-M6PR transcripts levels changed slightly. TfR and CD-M6PR transcripts were transiently upregulated at 1 dpi and then downregulated with the mRNA levels of TfR increasing at 5 dpi and of CD-M6PR unaltered after 3 dpi. HCMV pp65 transcripts levels increased during the infection. Data corresponding to three biological replicates were averaged, bars represent the average normalized fold increase, and error bars represent standard errors of the means.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Infection, Gene Expression, Expressing

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 8. Immuno-gold localization of viral and cellular proteins on the viral envelope of isolated HCMV viral particles. Isolated viral particles were labelled with antibodies against HCMV viral glycoprotein gH (A, PAG10) and viral chemokine receptor-like protein UL33 (E, PAG15), TGN46 (B, PAG15), CD63 (C, PAG10), EEA1 (F, PAG10), annexin1 (G, PAG10), TfR lumenal domain (H, PAG10), CI-M6PR lumenal domain (I, PAG10) either without (A–D, H–I) or after permeabilization with saponin (E–G). (D) Double-labelling with antibodies against CD63 (PAG10) and TGN46 (PAG15). When viral particles were partially disrupted, uranyl acetate revealed the nucleocapsids (A, C, E–G). Scale bar, 50 nm.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Isolation

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 9. Immunoprecipitation of virus particles with anti-TGN46 and anti-CD63 antibodies. Viruses secreted into the cell medium were incubated at 4°C overnight with antibodies against HSV-1 gD (negative control), HCMV gH (positive control), TGN46 and CD63. The virus-antibodies mixture was incubated with Pansorbin cells and viruses were precipitated. Supernatans were added to fresh BJ1 cells to determine the number of infectious unprecipitated viruses as described in Experimental procedures. To calculate the percentage of infectivity, the infectivity of samples treated with anti-HSV-1 gD antibodies was set at 100%. Data corresponding to three experiments were averaged, bars represent the average relative units and error bars represent standard errors of the means.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Immunoprecipitation, Virus, Incubation, Negative Control, Positive Control, Infection

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 10. Model of HCMV envelopment and secretion. Nucleocapsids assembled in the nucleus are released into the cytoplasm through the nuclear membranes. In the perinuclear region, where virus factories are established, nucleocapsids acquire tegument before completing their final envelopment in a hybrid compartment or in the transport vesicles between the TGN and endosomes. During envelopment viral glycoproteins and TGN46, CD63, annexin 1, EEA1, TfR and CI-M6PR are incorporated into the viral membrane of numerous particles. Mature virions are transported inside a vacuole towards the plasma membrane to be secreted into the extracellular environment. N: nucleus, INM: inner nuclear membrane, ONM: outer nuclear membrane, ER, endoplasmic reticulum; TGN, trans-Golgi network; MVB, multivesicular body; EE, early endosome; V, vacuole; PM, plasma membrane.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Virus, Membrane, Clinical Proteomics

Journal: Viruses

Article Title: Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

doi: 10.3390/v12040429

Figure Lengend Snippet: Construction of stable cell lines expressing bovine herpesvirus 1 (BoHV-1), pseudorabies virus (PRV), or herpes simplex 1 (HSV-1) glycoprotein B (gB), and characterization of size exclusion chromatography (SEC)-isolated extracellular vesicles (EVs). ( A ) The list of constructed cell lines expressing individual viral gB homologs. Human melanoma Mel JuSo (MJS) and Madin–Darby bovine kidney (MDBK) produce endogenous major histocompatibility (MHC) class II. The Human Jurkat T cell line and swine kidney SK6 cells are MHC II-negative. ( B – E ) Immunoblotting detection of EVs markers Alix and flotillin-2 (flot-2) in cell lysates (cl) of constructed cell lines or SEC-isolated EVs. Calnexin (CNX) and mitochondrial Tom 40 were used as non-EVs control proteins. MJSpuro, MDBKpuro, SK6puro, or untransduced Jurkat cells (Ø) were used as negative controls. ( F ) Representative transmission electron microscopy images of EVs preparations from gB-expressing species-specific cells; scale bar 100 nm.

Article Snippet: Other antibodies used for immunoblotting were mouse anti-HLA-DRα mAb (clone G-7, Santa Cruz Biotechnology), mouse anti-HLA-DRβ mAb (clone TAL14.1, Santa Cruz Biotechnology), mouse anti- HSV-1/2 glycoprotein C mAb (clone 3G9, Santa Cruz Biotechnology), mouse anti-HSV-1/2 glycoprotein D mAb (clone DL6, Santa Cruz Biotechnology), mouse anti-HSV-1/2 ICP5 mAb (clone 3B6, Santa Cruz Biotechnology), rabbit polyclonal anti-HSV-1 gamma 34.5 protein (ICP34.5) antibody (Bioss Antibodies, Woburn, MA, USA), mouse anti-BoHV-1 glycoprotein C mAb (clone F2, VMRD) and mouse anti-BoHV-1 glycoprotein D mAb (VMRD).

Techniques: Stable Transfection, Expressing, Size-exclusion Chromatography, Isolation, Construct, Western Blot, Transmission Assay, Electron Microscopy

Journal: Viruses

Article Title: Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

doi: 10.3390/v12040429

Figure Lengend Snippet: Immunoblotting detection of BoHV-1 ( A ), PRV ( B ), and HSV-1 gB ( C ) in cell lysates (cl) of constructed cell lines or SEC-isolated EVs. (+): gB-expressing cells; (−): MJSpuro, MDBKpuro, SK6puro, or untransduced Jurkat cells (Ø) were used as negative controls. Virus-infected cell lysates (cl) were analyzed as positive controls; in the case of BoHV-1, gB was immunoprecipitated (IP) with H2 antibody before immunoblotting with anti-BoHV-1 serum. Size markers are in kilodaltons. gBa: uncleaved BoHV-1 and PRV gB precursor; gBb: cleaved N-terminal gB subunit; gBc: cleaved C-terminal gB subunit.

Article Snippet: Other antibodies used for immunoblotting were mouse anti-HLA-DRα mAb (clone G-7, Santa Cruz Biotechnology), mouse anti-HLA-DRβ mAb (clone TAL14.1, Santa Cruz Biotechnology), mouse anti- HSV-1/2 glycoprotein C mAb (clone 3G9, Santa Cruz Biotechnology), mouse anti-HSV-1/2 glycoprotein D mAb (clone DL6, Santa Cruz Biotechnology), mouse anti-HSV-1/2 ICP5 mAb (clone 3B6, Santa Cruz Biotechnology), rabbit polyclonal anti-HSV-1 gamma 34.5 protein (ICP34.5) antibody (Bioss Antibodies, Woburn, MA, USA), mouse anti-BoHV-1 glycoprotein C mAb (clone F2, VMRD) and mouse anti-BoHV-1 glycoprotein D mAb (VMRD).

Techniques: Western Blot, Construct, Isolation, Expressing, Infection, Immunoprecipitation

Journal: Viruses

Article Title: Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

doi: 10.3390/v12040429

Figure Lengend Snippet: Analysis of interaction between MHC II and HSV-1, BoHV-1 or PRV gB, and the effect of alphaherpesvirus gB on MHC II incorporation in EVs. ( A ) Comparison of amino acid sequences representing potential HSV-1, BoHV-1, and PRV gB and MHC II biding site. HSV-1 gB contains an allotypic HLA-DR-binding region homologous to the human invariant chain (Ii, CD74), consisting of the promiscuous and the MHC II groove-binding sites. Whereas bovine and porcine Ii sequences are highly conserved, their corresponding regions in BoHV-1 and PRV gB (two candidate sequences) are variable. Amino acids (aa) that are identical to those in human Ii are in red frames. Boxes highlight predicted MHC II peptides. The groove-binding positions are in bold. ( B ) Complex formation between HSV-1 gB and HLA-DR or bovine MHC II from cell lysates was evaluated by co-immunoprecipitation (IP) using anti-MHC II mAb. Immunoprecipitated proteins were separated by SDS-PAGE and detected by immunoblotting (IB) with anti-gB or anti-DRα antibodies (for human cells). ( C ) and ( D ) Co-IP with anti HLA-DR or bovine MHC II (in C) mAb from MJS BoHV-1/PRV gB or MDBK BoHV-1 gB lysates. gB or DRβ were detected in SDS-PAGE-separated complexes by IB. Size markers are in kilodaltons. *LC: light chain of antibodies; HC: heavy chain of antibodies. Arrows indicate gB species. ( E ) Detection of DRα, DRβ and flotillin-2 (flot-2) as an EVs marker in SEC-purified EVs by IB. β-actin and DRβ were detected in cell lysates for comparison. Size markers are in kilodaltons.

Article Snippet: Other antibodies used for immunoblotting were mouse anti-HLA-DRα mAb (clone G-7, Santa Cruz Biotechnology), mouse anti-HLA-DRβ mAb (clone TAL14.1, Santa Cruz Biotechnology), mouse anti- HSV-1/2 glycoprotein C mAb (clone 3G9, Santa Cruz Biotechnology), mouse anti-HSV-1/2 glycoprotein D mAb (clone DL6, Santa Cruz Biotechnology), mouse anti-HSV-1/2 ICP5 mAb (clone 3B6, Santa Cruz Biotechnology), rabbit polyclonal anti-HSV-1 gamma 34.5 protein (ICP34.5) antibody (Bioss Antibodies, Woburn, MA, USA), mouse anti-BoHV-1 glycoprotein C mAb (clone F2, VMRD) and mouse anti-BoHV-1 glycoprotein D mAb (VMRD).

Techniques: Binding Assay, Immunoprecipitation, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Marker, Purification

Journal: Viruses

Article Title: Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

doi: 10.3390/v12040429

Figure Lengend Snippet: HSV-1 and BoHV-1 gB do not affect internalization but contribute to a retarded appearance of MHC II at the cell surface. ( A ) An internalization assay was performed on MJS HSV-1 gB (dark brown line), MJS BoHV-1 gB (pink line), or control MJSpuro (black line) cells stained on ice with anti-MHC class II L243 antibody and PE-conjugated IgG. The cells were then shifted to 37 °C for 20 or 40 min. The mean fluorescence intensities of remaining MHC II were assessed by flow cytometry for triplicate samples and compared with MHC II at time point 0 (set as 100%). The differences between MHC II levels were statistically insignificant. ( B ) For the MHC II export assay, the cells were stained on ice with a saturating amount of anti-MHC class II L243 antibody and incubated at 37 °C for 20 or 40 min in the presence of allophycocyanin (APC)-conjugated L243 MAb detecting newly arrived MHC II. The mean fluorescence intensities of MHC II staining were normalized to the values for time point 0 samples and depicted as a percentage of the increase in MHC II appearance on MJSpuro (black line) or MJS-gB cells. Results are representative of three independent experiments. The statistical significance of differences between MHC II on MJSpuro and gB-expressing cells at 20 or 40 min was estimated by a t -test; p ≤ 0.01.

Article Snippet: Other antibodies used for immunoblotting were mouse anti-HLA-DRα mAb (clone G-7, Santa Cruz Biotechnology), mouse anti-HLA-DRβ mAb (clone TAL14.1, Santa Cruz Biotechnology), mouse anti- HSV-1/2 glycoprotein C mAb (clone 3G9, Santa Cruz Biotechnology), mouse anti-HSV-1/2 glycoprotein D mAb (clone DL6, Santa Cruz Biotechnology), mouse anti-HSV-1/2 ICP5 mAb (clone 3B6, Santa Cruz Biotechnology), rabbit polyclonal anti-HSV-1 gamma 34.5 protein (ICP34.5) antibody (Bioss Antibodies, Woburn, MA, USA), mouse anti-BoHV-1 glycoprotein C mAb (clone F2, VMRD) and mouse anti-BoHV-1 glycoprotein D mAb (VMRD).

Techniques: Staining, Fluorescence, Flow Cytometry, Incubation, Expressing

Journal: Viruses

Article Title: Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

doi: 10.3390/v12040429

Figure Lengend Snippet: Alphaherpesvirus gB localizes to EVs-enriched fractions isolated from virus-infected MJS cells and BoHV-1-infected MDBK. OptiPrep gradient-purification of EVs-enriched and virion-enriched fractions from HSV-1-infected MJS ( A ), BoHV-1-infected MJS ( B ), PRV-infected MJS ( C ), and BoHV-1-infected MDBK ( D ) supernatants. MJS cells were infected with HSV-1 at a multiplicity of infection (moi) of 0.1, with BoHV-1/PRV at an moi of 0.5; MDBK cells were infected at an moi of 0.1. After 50 h post-infection, 0.5 mL fractions were collected from 6%–18% iodixanol (Optiprep) gradient by ultracentrifugation. The 40 µL samples were analyzed in denaturing (for viral glycoprotein C-gC, glycoprotein B-gB, glycoprotein D-gD, for HSV-1, glycoprotein E-gE, for BoHV-1 and PRV or EVs marker Alix) or non-denaturing (for EVs markers CD63 and CD9) SDS-PAGE, and immunoblotted with specific mAbs or polyclonal anti-PRV gB serum. Uninfected (Ø) or virus-infected cell lysates (cl) were analyzed as controls. Bottom panels represent virus titres in each fraction measured by plaque assay. ( E ) Proteins from the indicated fractions (40 μL) were separated by SDS-PAGE, and immunoblotted with antibodies specific to the ICP5/VCP5 homolog of HSV-1 and BoHV-1, virulence factor γ34.5 from HSV-1, immunomodulatory UL49.5 protein of BoHV-1 and envelope glycoproteins gD, and gM, as indicated. HLA-DRβ was detected in the fractions as an EVs marker. Cell lysates (cl) and virus-infected cell lysates (30 μg) were analyzed as controls. ( F ) Comparison of HLA-DRβ levels between EVs from virus-infected and uninfected MJS. DRβ and flotillin-2 (flot-2, EVs marker) from F11 fractions were immunoblotted with specific mAb. Numbers indicate the optical density of DRβ bands from virus-infected MJS EVs, normalized to flot-2 from the same samples, relative to DRβ in EVs from uninfected MJS (set as 1).

Article Snippet: Other antibodies used for immunoblotting were mouse anti-HLA-DRα mAb (clone G-7, Santa Cruz Biotechnology), mouse anti-HLA-DRβ mAb (clone TAL14.1, Santa Cruz Biotechnology), mouse anti- HSV-1/2 glycoprotein C mAb (clone 3G9, Santa Cruz Biotechnology), mouse anti-HSV-1/2 glycoprotein D mAb (clone DL6, Santa Cruz Biotechnology), mouse anti-HSV-1/2 ICP5 mAb (clone 3B6, Santa Cruz Biotechnology), rabbit polyclonal anti-HSV-1 gamma 34.5 protein (ICP34.5) antibody (Bioss Antibodies, Woburn, MA, USA), mouse anti-BoHV-1 glycoprotein C mAb (clone F2, VMRD) and mouse anti-BoHV-1 glycoprotein D mAb (VMRD).

Techniques: Isolation, Infection, Purification, Marker, SDS Page, Plaque Assay