Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: HSV-1 gD and gH/gL but not gB restricts the production of infectious HIV-1. 293 cells were co-transfected with either the empty pcDNA3.1(+) plasmid or plasmids expressing HSV-1 gD, gB, gH/gL, or UL47 proteins and pNL4-3. At 48 h, the culture supernatants were collected. The levels of infectious virus released into the culture supernatants was determined using TZM-bl cell assays and p24 in the culture supernatants determined using p24 antigen capture assays. a The level of infectious virus released into the culture medium from cells co-transfected with pcDNA3.1(+) or a plasmid expressing gD, gB, gH/gL, or UL47 and pNL4-3. b The levels of p24 protein produced from cells co-transfected with either the empty pcDNA3.1(+) plasmid or expressing gD, gB, gH/gL, or UL47 proteins and pNL4-3. c The cell lysates from the above restriction assays were analyzed for the presence of gD, gB, gH/gL, or UL47 using immunoblots and appropriate antibodies. The experiments were performed at least four times and statistical differences with the pcDNA3.1(+)/HIV-1 control evaluated using a two-tailed Student’s t -test, with p < 0.01 (filled triangle) considered significant

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Transfection, Plasmid Preparation, Expressing, Produced, Western Blot, Two Tailed Test

Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: HSV-1 gB, gD, and gH/gL do not significantly affect the synthesis and processing of HIV-1 viral proteins. 293 cells were transfected with either the empty pcDNA3.1(+) vector or one expressing HSV-1 gB, gD, or gH/gL and pNL4-3. At 30 h post-transfection, cells were starved for methionine/cysteine and radiolabeled with 35 S-methionine/cysteine for 16 h. The culture medium was collected and cell lysates prepared as described in the Materials and Methods section. HIV-1 proteins were immunoprecipitated using anti-HIV-1 antibodies while HSV-1 proteins were immunoprecipitated with gB, gD, gH, or myc (gL) antibodies. The immunoprecipitates were collected on protein-A-Sepharose, washed, and boiled in sample reducing buffer. The proteins were separated on 7.5% SDS-PAGE and visualized using standard radiographic techniques. a Immunoprecipitation of HIV-1 and gD proteins from cell lysates or culture medium from 293 cells co-transfected with either pcDNA3.1(+) and pNL4-3 or a vector expressing gD and pNL4-3. b Immunoprecipitation of HIV-1 and gB proteins from cell lysates or culture medium from 293 cells co-transfected with either pcDNA3.1(+) and pNL4-3 or a vector expressing gB and pNL4-3. c Immunoprecipitation of HIV-1 and gH/gL proteins from cell lysates or culture medium from 293 cells co-transfected with vectors pcDNA3.1(+) and pNL4-3 or vectors expressing gH/gL and pNL4-3. Shown below each cell lysate panel is the expression of β-actin as a loading control

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page

Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: HSV-1 gD and gB do not significantly affect the processing of HIV-1 Gag and gp160. 293 cells were co-transfected with empty pcDNA3.1(+) vector or one expressing gD and pNL4-3. At 24 h, cells were starved in medium lacking methionine/cysteine for 2 h followed by radiolabeling cultures with 35 S-methionine/cysteine. The radiolabel was removed and washed three times in medium containing 100X methionine/cysteine and chased in the same medium for 0, 1, 3, and 6 h. The culture medium was harvested and cell lysates prepared as described in the Materials and Methods. HIV-1 Env and Gag proteins were immunoprecipitated using the HIV-1 antibodies and HSV-1 gD was immunoprecipitated with an appropriate monoclonal antibody. The immunoprecipitates were collected on protein-A-Sepharose beads, washed, and boiled in sample reducing buffer. The proteins were separated on 7.5% SDS gels and visualized using standard radiographic techniques. a , b HIV-1 proteins immunoprecipitated from the cell lysates ( a ) and culture medium ( b ) of cells co-transfected with pcDNA3.1(+) and pNL4-3. c , d HIV-1 proteins immunoprecipitated from the cell lysates ( c ) and culture medium ( d ) of cells co-transfected with a vector expressing gD and pNL4-3. e , f HSV-1 gD protein immunoprecipitated from cell lysates ( e ) and culture medium ( f ) of cells co-transfected with a vector expressing gD and pNL4-3

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Transfection, Plasmid Preparation, Expressing, Radioactivity, Immunoprecipitation

Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: HSV-1 gD and gB are efficiently incorporated into HIV-1 particles. 293 cells were co-transfected with either empty pcDNA3.1(+) vector, one expressing the HSV-1 gD or HSV-1 gB and pNL4-3. At 30 h, the cells were starved for methionine/cysteine for 2 h and then radiolabeled for with 35 S-methionine/cysteine for 16 h. At 48 h post-transfection, the cell culture medium was harvested and subjected to low speed centrifugation to remove cellular debris and virus pelleted through a 20% sucrose cushion using ultracentrifugation. The cells were lysed in RIPA buffer on ice, centrifuged to remove the nuclei. The resulting supernatant was transferred to a new microfuge tube. The medium, cell lysates, and virus pellet were used in immunoprecipitation analysis using anti-HIV-1 antibodies (to immunoprecipitate Env and Gag proteins) and appropriate monoclonal antibodies to immunoprecipitate HSV-1 gD and gB. The immunoprecipitates were collected on protein-A-Sepharose beads, washed, and boiled in sample reducing buffer. The proteins were separated on 7.5% SDS-PAGE and visualized using standard radiographic techniques. a HIV-1 and gD proteins immunoprecipitated from cell lysates prepared from cells co-transfected with either empty vector and pNL4-3 or a vector expressing gD and pNL4-3. b Cells were co-transfected with either empty vector and pNL4-3 or a vector expressing gD and pNL4-3 followed by immunoprecipitation of HIV-1 and gD proteins from the culture medium and pellet after ultracentrifugation. c HIV-1 and gB proteins immunoprecipitated from cell lysates prepared from cells co-transfected with either empty vector and pNL4-3 or a vector expressing gB and pNL4-3. d Cells were co-transfected with either empty vector and pNL4-3 or a vector expressing gB and pNL4-3 followed by immunoprecipitation of HIV-1 and gB proteins from the culture medium and pellet after ultracentrifugation

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Transfection, Plasmid Preparation, Expressing, Cell Culture, Centrifugation, Immunoprecipitation, SDS Page

Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: Sucrose density gradient centrifugation purification of virus reveals the gp120 is not incorporated in viral particles in the presence of HSV-1 gD. 293 cells were co-transfected with either empty pcDNA3.1(+) vector and pNL4-3, a vector expressing gD and pNL4-3, or a vector expressing gB and pNL4-3. At 30 h, the cells were starved for methionine/cysteine, radiolabeled and the culture medium harvested at 48 h post-transfection. Following low speed centrifugation, the culture supernatants were layered onto a 20% sucrose cushion and virus pelleted by ultracentrifugation. The pelleted virus resuspended in DMEM without serum and layered on a discontinuous 20–60% sucrose gradient. The virus was subjected to ultracentrifugation for 20 h (76,000 x g, SW55Ti rotor), 12 fractions were collected, and subjected to immunoprecipitation analysis using anti-HIV-1 antibodies to immunoprecipitated HIV-1 Gag and Env) and appropriate monoclonal antibodies to immunoprecipitate HSV-1 gD or gB. The immunoprecipitates were collected on protein-A-Sepharose, washed, and boiled in sample reducing buffer. The proteins were separated on 7.5% SDS-PAGE and visualized using standard radiographic techniques. a Immunoprecipitation of HIV-1 proteins from gradient fractions of cells co-transfected with empty pcDNA3.1(+) vector and pNL4-3. b Analysis of virus infectivity from various fractions in ( a ). c Immunoprecipitation of HIV-1 proteins from gradient fractions of cells co-transfected with a vector expressing gD and pNL4-3. d Immunoprecipitation of HSV-1 gD from gradient fractions of cells transfected with a vector expressing gD and pNL4-3. e Immunoprecipitation of gD from gradient fractions of cells transfected with a vector expressing gD. f Analysis of virus infectivity from various fractions in ( c , d )

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Gradient Centrifugation, Purification, Transfection, Plasmid Preparation, Expressing, Centrifugation, Immunoprecipitation, SDS Page, Infection

Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: Over-expression of HSV-1 gD and HIV-1 Env still results in exclusion of HIV gp120/gp41 from particles. 293 cells were co-transfected with either the empty pcDNA3.1(+) vector and pNL4-3, a vector expressing Bal Env and pNL4-3, a vector expressing gD and pNL4-3, or vectors expressing gD, Bal Env and pNL4-3. At 30 h post-transfection, the cells were starved for 2 h with methionine/cysteine-free media and then radiolabeled for 16 h with 35 S-methionine/cysteine. The culture media was harvested and subjected to low speed centrifugation to remove cellular debris. The resulting supernatant was layered on a 20% sucrose cushion and subjected to ultracentrifugation to pellet viral particle as described in the Materials and methods. The pelleted virus was harvested, resuspended in 200 μl of DMEM without serum and layered on a discontinuous 20-60% sucrose gradient. The virus was subjected to ultracentrifugation for 20 h at 76,000× g in a SW55Ti at which time the fractions were collected and proteins immunoprecipitated using anti-HIV-1 antibodies to immunoprecipitate HIV-1 Gag and Env or a monoclonal antibody to immunoprecipitate HSV-1 gD. The immunoprecipitates of the HIV-1 proteins and HSV-1 gD from gradient fractions of cells co-transfected with pcDNA3.1(+) and pNL4-3 or gD and pNL4-3 were the same as in Fig. . a Immunoprecipitation of HIV-1 proteins and HSV-1 gD from cell lysates of cells co-transfected with pcDNA3.1(+) and pNL4-3, a vector expressing Bal Env and pNL4-3, a vector expressing gD and pNL4-3, or a vector expressing gD, Bal Env and pNL4-3. b Immunoprecipitation of HIV-1 proteins from supernatants prior to and after ultracentrifugation through a 20% sucrose cushion from cells co-transfected with pcDNA3.1 and pNL4-3, a vector expressing Bal Env and pNL4-3, a vector expressing gD and pNL4-3, or a vector expressing gD and Bal Env and pNL-3. c Immunoprecipitation of HSV-1 gD from viral pellet after ultracentrifugation through a 20% sucrose cushion from cells transfected with pcDNA3.1(+) and pNL4-3, a vector expressing gD and pNL4-3, a vector expressing Bal Env and pNL4-3, or a vector expressing gD, Bal Env, and pNL4-3. d Immunoprecipitation of HIV-1 proteins from gradient fractions of cells co-transfected with Bal Env and pNL4-3. e , f Immunoprecipitation of HIV-1 proteins ( e ) and HSV-1 gD ( f ) from gradient fractions of cells co-transfected with a vector expressing gD, Bal Env, and pNL4-3

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Centrifugation, Immunoprecipitation

Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: The gD protein from HSV-2 also restricts the release of infectious HIV-1. 293 cells were co-transfected with either the empty pcDNA3.1(+) vector or one expressing gD, HSV-2 gD (gD2), gB, or UL47 and pNL4-3. At 48 h, the culture supernatants were collected. The levels of infectious virus released into the culture supernatants was determined using TZM-bl cell assays and p24 in the culture supernatants determined using p24 antigen capture assays. a The level of infectious virus released into the culture medium from cells co-transfected with pcDNA3.1(+), gD, HSV-2 gD (gD2), gB, or UL47 and pNL4-3. b The levels of p24 protein produced from cells co-transfected with pcDNA3.1(+), gD, HSV-2 gD (gD2), gB, or UL47 and pNL4-3. c The cell lysates from the above restriction assays were analyzed for the presence of gD, HSV-2 gD (gD2), gB, or UL47 using Western blots and appropriate antibodies. d 293 cells were transfected with empty vector pcDNA3.1(+), pcDNA3.1(+) and pNL4-3, or the vector expressing gD2 and pNL4-3. At 30 h post-transfection, cultures were radiolabelled as per Fig. . At 48 h, the culture medium was harvested, centrifuged at low speed to remove cellular debris and layered onto a 20% sucrose cushion. The virus containing medium was subjected to ultracentrifugation to pellet the virus and HIV-1 proteins and gD2 immunoprecipitated using appropriate antibodies. The samples were analyzed by SDS-PAGE and standard radiographic techniques. e , f A companion experiment was performed exactly like d except the pelleted virus resuspended in DMEM without serum and layered on a discontinuous 20–60% sucrose gradient. The virus was subjected to ultracentrifugation for 20 h (76,000 x g, SW55Ti rotor), 12 fractions were collected, and subjected to immunoprecipitation analysis using anti-HIV-1 antibodies to immunoprecipitated HIV-1 proteins (Gag and Env) and an appropriate monoclonal antibody to immunoprecipitate HSV-1 gD2. The immunoprecipitates were collected on protein-A-Sepharose beads, washed, and boiled in sample reducing buffer. The proteins were separated on 7.5% SDS-PAGE and visualized using standard radiographic techniques. The experiments in a – c were performed at least four times and statistical differences with the pcDNA3.1(+)/HIV-1 control evaluated using a two-tailed Student’s t -test, with p < 0.01 (filled triangle) considered significant

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Transfection, Plasmid Preparation, Expressing, Produced, Western Blot, Immunoprecipitation, SDS Page, Two Tailed Test

Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: The HSV-1 gD co-localizes with Gag-EGFP. 293 cells were transfected with either the vector expressing gD, Gag-EGFP, or co-transfected with gD and Gag-EGFP. At 24 h, cells on cover slips were washed, fixed, permeabilized and blocked as described in the Materials and Methods section. The cover slips were reacted with a mouse monoclonal antibody against gD and an appropriate secondary antibody and counterstained with DAPI (1 μg/ml) for 5 min. The cover slips were mounted and examined using a Leica TCS SPE confocal microscope using a 63X objective with a 2X digital zoom using the Leica Application Suite X (LAS X, LASX) software package. A 405 nm filter used to visualize the DAPI, 488 nm filter was used to visualize the Gag-EGFP, and a 594 nm filter to visualize gD staining. a – d Cells transfected with vector expressing gD. e – h Cells transfected with vector expressing Gag-EGFP. i – l Cells co-transfected with vectors expressing gD and Gag-GFP. a , e , i Visualization of DAPI staining. b , f , j Visualization of gD staining. c , g , k Visualization of Gag-EGFP. d , h , l Merge of the three panels to the left

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Transfection, Plasmid Preparation, Expressing, Microscopy, Software, Staining

Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: The HSV-1 gB co-localizes with Gag-GFP at the cell surface. COS-7 cells were transfected with either the vector expressing gB or co-transfected with gB and Gag-EGFP. At 24 h, cells on cover slips were washed, fixed, permeabilized and blocked as described above. The cover slips were reacted with a mouse monoclonal antibody against HSV-1 gB and an appropriate secondary antibody and counterstained with DAPI (1 μg/ml) for 5 min as described in the Materials and Methods section. The cover slips were mounted and examined using a Leica TCS SPE confocal microscope using a 63 × objective with a 2 × digital zoom using the Leica Application Suite X (LAS X, LASX) software package. A 405 nm filter used to visualize the DAPI, 488 nm filter was used to visualize the Gag-GFP, and a 594 nm filter to visualize gB staining. a – d Cells transfected with vector expressing gB. e – h Cells co-transfected with vectors expressing gB and Gag-GFP. a , e Visualization of Gag-EGFP. b , f Visualization of gB staining. c , g Visualization of DAPI staining. d , h Merge of the three panels to the left. The inset below Fig. h is a region of the cell plasma membrane showing co-localization of Gag-EGFP and gB. The results shown are representative of examining 50 transfected cells

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Transfection, Plasmid Preparation, Expressing, Microscopy, Software, Staining

Journal: Retrovirology

Article Title: Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles

doi: 10.1186/s12977-019-0470-5

Figure Lengend Snippet: Cell lines expressing gD also restrict HIV-1. 293 and TZM-bl cell lines were transduced with lentivirus vectors expressing HSV-1 gD as described in the Materials and Methods section. Transduced cells were selected using puromycin and gD expressing cell lines were stained for gD and sorted. The number of cells expressing gD was greater than 98%. Cells lines in 6 well plates were inoculated with HIV-1 (NL4-3, complete genome) pseudotyped with the VSV G-protein at an M.O.I of 0.5. At 48 h post-inoculation, the medium was collected and the levels of p24 determined with commercially available p24 ELISA kits and infectious virus released determined using TZM-bl assays. a – d Parental 293 and TZM-bl cell lines and those expressing HSV-1 gD (293-gD, TZM-bl-gD) were fixed and immunostained for gD and examined by confocal microscopy. e – h Parental 293 and TZM-bl cell lines and those expressing EGFP (293-EGFP, TZM-bl-EGFP) were examined for the expression of EGFP by confocal microscopy using a 488 nm filter. i The levels of the p24 released from cells inoculated with VSV-G pseudotyped HIV-1. j The levels of infectious virus released from cells inoculated with VSV-G pseudotyped HIV-1. The experiments were performed at least three times and statistical differences with the 293 or TZM-bl/HIV-1 controls were evaluated using a two-tailed Student’s t -test, with p < 0.01 (filled triangle) considered significant. The numbers above the bars represent the percentage of p24 or infectious virus compared to the control

Article Snippet: A plasmid expressing the HSV-1 gL (myc-DDK-tagged at C-terminus) was obtained from Origene (gL: catalog number, VC100788; CD8: catalog number RC206608).

Techniques: Expressing, Transduction, Staining, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Two Tailed Test