Journal: Virology

Article Title: Incorporation of the herpes simplex virus type 1 tegument protein VP22 into the virus particle is independent of interaction with VP16.

doi: 10.1016/j.virol.2007.07.020

Figure Lengend Snippet: Fig. 1. Characterization of the ability of VP22 truncation mutants to bind VP16 in a GST pulldown assay. (A) N-terminal andC-terminal truncations of VP22 fused to GST. A schematic representation of full length, N-terminal and C-terminal truncated forms of VP22 fused to the C-terminus of the GST protein. (B and C) GST pulldown from HSV-1-infected cell lysates using GST–VP22 and N-terminal and C-terminal truncation mutants respectively. The GST fusion proteins were expressed in Escherichia coli cells, purified on glutathione-Sepharose beads, and approximately equal amounts of each were added to NP-40 lysates of Vero cells that had been infected with HSV-1. Beads were washed extensively with lysis buffer and bound proteins were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using a rabbit monospecific polyclonal antibody raised against VP16. (D) VP16 interaction domain mutants of VP22 fused to GST. Schematic representation of VP22, deletion of amino acids 87–120 of VP22 or the predicted VP16 interaction domain of VP22, and the interaction domain alone fused to the C-terminus of the GST protein. (E) GST pulldown from HSV-1-infected cell lysates using VP16 interaction domain mutants of VP22. Purified fusion proteins were tested for their ability to bind VP16 from HSV-1 infected cells, as described in the legend to panels B and C. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

Article Snippet: To further control for the quantities of GST-fusion proteins used in the pulldown, nitrocellulose membranes were stripped (60 mM Tris–HCl [pH 8.0], 2% SDS, 0.75% β-mercaptoethanol [β-ME] for 45 min at 55 °C) and reprobed with a goat polyclonal antibody raised against the GST protein (Rockland).

Techniques: GST Pulldown Assay, Infection, Purification, Lysis, SDS Page, Western Blot

Journal: Virology

Article Title: Incorporation of the herpes simplex virus type 1 tegument protein VP22 into the virus particle is independent of interaction with VP16.

doi: 10.1016/j.virol.2007.07.020

Figure Lengend Snippet: Fig. 2. Coimmunoprecipitation of N-terminal and C-terminal VP22 truncation mutants with VP16. (A) N-terminal and C-terminal truncation mutants of VP22 fused to GFP. A schematic representation of full length, N-terminal and C-terminal truncated forms of VP22 fused to the N-terminus of the GFP protein. (B and D) Expression of VP22 N-terminal and C-terminal truncation mutants respectively, in transfected/infected cells. Vero cells expressing GFP, VP22–GFP constructs or mock transfected cells (Mock) were infected with HSV-1, and at 10 h post-infection were lysed with NP-40 lysis buffer. A fraction of each cell lysate was analyzed by Western blotting using a goat polyclonal antibody specific for GFP. (C and E) Coimmunoprecipitation of VP16 with N-terminal and C-terminal truncation mutants of VP22, respectively. The remainder of the transfected/infected cell lysate was incubated with a goat polyclonal antibody against GFP and the resulting antibody– antigen complexes were collected with protein G-agarose beads. After extensive washes with lysis buffer, material that immunoprecipitated with anti-GFP antibody was separated by SDS-PAGE and transferred to nitrocellulose. Coimmunoprecipitated VP16 was detected by immunoblot using a rabbit monospecific polyclonal antibody raised against a C-terminal peptide of VP16. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

Article Snippet: To further control for the quantities of GST-fusion proteins used in the pulldown, nitrocellulose membranes were stripped (60 mM Tris–HCl [pH 8.0], 2% SDS, 0.75% β-mercaptoethanol [β-ME] for 45 min at 55 °C) and reprobed with a goat polyclonal antibody raised against the GST protein (Rockland).

Techniques: Expressing, Transfection, Infection, Construct, Lysis, Western Blot, Incubation, Immunoprecipitation, SDS Page

Journal: Virology

Article Title: Incorporation of the herpes simplex virus type 1 tegument protein VP22 into the virus particle is independent of interaction with VP16.

doi: 10.1016/j.virol.2007.07.020

Figure Lengend Snippet: Fig. 4. Analysis of the VP22 domain that facilitates interaction with VP16 in a GST pulldown assay. (A) Expression of the VP22 truncation mutants represented in Fig. 3A, in transfected cells. Vero cells were transfected with the indicated constructs. At 20 h post-transfection, the transfected monolayers were lysed with NP-40 lysis buffer. A fraction of each cell lysate was analyzed for expression of the VP22-GFP fusion proteins by Western blotting using a rabbit polyclonal antibody specific for GFP. (B) GST pulldown from transfected cell lysates using GST–VP16. The GST fusion proteins were expressed in Escherichia coli cells, purified on glutathione- Sepharose beads, and approximately equal amounts of each were added to the remainder of the transfected cell lysates. Beads were washed extensively with lysis buffer and bound proteins were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using a rabbit polyclonal antibody raised against the GFP protein. The positions of molecular mass markers (in kilodaltons) are indicated on the left. (C) Binding efficiency of VP22 truncation mutants to VP16. Using densitometry, binding efficiency was quantitated by dividing the amount of VP22–GFP protein detected in the pulldown assay (normalized for the amount of GST–VP16 present) by the amount in the cell lysate (normalized for the amount of actin present). In each experiment, the wild-type VP22–GFP construct was set at 100% binding efficiency. Error bars represent standard deviations for four replicate experiments.

Article Snippet: To further control for the quantities of GST-fusion proteins used in the pulldown, nitrocellulose membranes were stripped (60 mM Tris–HCl [pH 8.0], 2% SDS, 0.75% β-mercaptoethanol [β-ME] for 45 min at 55 °C) and reprobed with a goat polyclonal antibody raised against the GST protein (Rockland).

Techniques: GST Pulldown Assay, Expressing, Transfection, Construct, Lysis, Western Blot, Purification, SDS Page, Binding Assay

Journal: Virology

Article Title: Incorporation of the herpes simplex virus type 1 tegument protein VP22 into the virus particle is independent of interaction with VP16.

doi: 10.1016/j.virol.2007.07.020

Figure Lengend Snippet: Fig. 5. Membrane flotation of the VP22 domains that facilitate interaction with VP16 and gE. Vero cells were transfected with the VP22 mutants represented in Fig. 3A. At 24 h post-transfection, the transfected monolayers were scraped into PBS, washed and resuspended in hypotonic buffer. Swollen cells were disrupted by passage through a 25 gauge needle and nuclei were removed by low-speed centrifugation. The resulting supernatants were added to 85% (wt/vol) sucrose in NTE buffer to yield a final sucrose concentration of 72%. A discontinuous gradient was formed by overlaying this mixture with 65% sucrose followed by 10% sucrose. After centrifugation at 100,000×g for 18 h at 4 °C in a Beckman SW41 rotor, 12 fractions were collected from the bottom of the tube. After TCA precipitation of each fraction, proteins were separated by SDS-PAGE and transferred to nitrocellulose. GFP-tagged proteins were detected by Western blotting using a goat polyclonal antibody raised against the GFP protein. Fractions 1–3 contain non-membrane associated proteins while fractions 10 and 11 correspond to the 65–10% sucrose interface and contain cellular membranes.

Article Snippet: To further control for the quantities of GST-fusion proteins used in the pulldown, nitrocellulose membranes were stripped (60 mM Tris–HCl [pH 8.0], 2% SDS, 0.75% β-mercaptoethanol [β-ME] for 45 min at 55 °C) and reprobed with a goat polyclonal antibody raised against the GST protein (Rockland).

Techniques: Membrane, Transfection, Centrifugation, Concentration Assay, TCA Precipitation, SDS Page, Western Blot

Journal: Virology

Article Title: Incorporation of the herpes simplex virus type 1 tegument protein VP22 into the virus particle is independent of interaction with VP16.

doi: 10.1016/j.virol.2007.07.020

Figure Lengend Snippet: Fig. 6. Virion incorporation of the VP22 domain that facilitates interaction with VP16. Vero cells were transfected with the indicated VP22–GFP constructs, and 20 h later, they were infected with a VP22-null virus (UL49−). After an additional 20-h incubation, cell lysates were prepared (A) and virions were collected from the media by centrifugation through a 30% sucrose cushion (B). Cell lysates and extracellular virus were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using a rabbit polyclonal antibody specific for GFP. As a loading control, the blot was stripped and reprobed with a rabbit polyclonal antibody raised against the HSV-1 major capsid protein VP5. The positions of molecular mass markers (in kilodaltons) are indicated on the left. (C) Packaging efficiency. Using densitometry, packaging efficiency was quantitated by dividing the amount of VP22–GFP protein detected in extracellular virus particles (normalized for VP5) by the amount in the cell lysate (normalized for VP5). In each experiment, the wild-type VP22–GFP construct was set at 100% packaging efficiency. Error bars represent standard deviations for four replicate experiments.

Article Snippet: To further control for the quantities of GST-fusion proteins used in the pulldown, nitrocellulose membranes were stripped (60 mM Tris–HCl [pH 8.0], 2% SDS, 0.75% β-mercaptoethanol [β-ME] for 45 min at 55 °C) and reprobed with a goat polyclonal antibody raised against the GST protein (Rockland).

Techniques: Transfection, Construct, Infection, Virus, Incubation, Centrifugation, SDS Page, Western Blot, Control

Journal: Virology

Article Title: Incorporation of the herpes simplex virus type 1 tegument protein VP22 into the virus particle is independent of interaction with VP16.

doi: 10.1016/j.virol.2007.07.020

Figure Lengend Snippet: Fig. 9. Characterization of the ability of VP22 dileucine motif mutants to bind to the cytoplasmic tail of gE in a GST pulldown assay. (A) Expression of VP22 dileucine motif mutants in transfected cells. Vero cells were transfected with the indicated constructs and at 20 h post-transfection, the transfected monolayers were lysed with NP-40 lysis buffer. A fraction of each cell lysate was analyzed for expression of the VP22–GFP fusion proteins by Western blotting using a rabbit polyclonal antibody specific for GFP. (B) GST pulldown from transfected cell lysates using GST–gECT. The GST fusion proteins were expressed in Escherichia coli cells, purified on glutathione-Sepharose beads, and approximately equal amounts of each were added to the remainder of the transfected cell lysates. Beads were washed extensively with lysis buffer and bound proteins were separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using a rabbit polyclonal antibody raised against the GFP protein. The positions of molecular mass markers (in kilodaltons) are indicated on the left. (C) Binding efficiency of VP22 dileucine motif mutants to the cytoplasmic tail of gE. Using densitometry, binding efficiency was quantitated by dividing the amount of VP22–GFP protein detected in the pulldown assay (normalized for the amount of GST–gECT present) by the amount in the cell lysate (normalized for the amount of actin present). In each experiment, the wild-type VP22–GFP construct was set at 100% binding efficiency. Error bars represent standard deviations for four replicate experiments.

Article Snippet: To further control for the quantities of GST-fusion proteins used in the pulldown, nitrocellulose membranes were stripped (60 mM Tris–HCl [pH 8.0], 2% SDS, 0.75% β-mercaptoethanol [β-ME] for 45 min at 55 °C) and reprobed with a goat polyclonal antibody raised against the GST protein (Rockland).

Techniques: GST Pulldown Assay, Expressing, Transfection, Construct, Lysis, Western Blot, Purification, SDS Page, Binding Assay