Journal: Journal of Virology

Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

doi: 10.1128/JVI.01441-10

Figure Lengend Snippet: At 2 h postinfection with SIN/nsP3GFP and ns polyprotein cleavage mutants, virus-specific dsRNAs are located at the plasma membrane. BHK-21 cells were infected with the indicated viruses at an MOI of 20 PFU/cell. At 2 h postinfection, they were fixed, permeabilized, and stained with dsRNA-specific MAb. The 3D image stacks were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz sections are presented as multiple-intensity projections of 2-μm section. Bars correspond to 5 μm.

Article Snippet: They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software.

Techniques: Virus, Membrane, Infection, Staining, Microscopy, Software

Journal: Journal of Virology

Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

doi: 10.1128/JVI.01441-10

Figure Lengend Snippet: At early times postinfection of mosquito cells, nsP3-GFP and dsRNAs are distributed at both the plasma membrane and endosomes. C710 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. After 4 h of incubation at 30°C, cells were fixed, permeabilized, and stained with dsRNA-specific MAb and Alexa Fluor 555-labeled secondary Ab as described in Materials and Methods. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective, and image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz sections are presented as multiple-intensity projections of a 2-μm section. White arrowheads indicate positions of stained dsRNAs having a cytoplasmic, endosome-like distribution. White arrows indicate plasma membrane-associated dsRNAs. Bars correspond to 5 μm.

Article Snippet: They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software.

Techniques: Membrane, Infection, Incubation, Staining, Labeling, Microscopy, Software

Journal: Journal of Virology

Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

doi: 10.1128/JVI.01441-10

Figure Lengend Snippet: At late times postinfection with SIN/nsP3GFP and ns polyprotein cleavage mutants, SINV-specific dsRNAs retain mostly plasma membrane-specific localization. BHK-21 cells were infected with the indicated viruses at an MOI of 20 PFU/cell. At 8 h (and 16 h) postinfection, cells were fixed, permeabilized, and stained with a dsRNA-specific MAb. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz sections are presented as a multiple-intensity projection of 2-μm section. Bars correspond to 5 μm. White arrowheads indicate positions of stained dsRNAs demonstrating cytoplasmic localization.

Article Snippet: They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software.

Techniques: Membrane, Infection, Staining, Microscopy, Software

Journal: Journal of Virology

Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

doi: 10.1128/JVI.01441-10

Figure Lengend Snippet: Re-localization of spherules from the plasma membrane into the cytoplasm. (A) Plasma membrane invaginations and vacuole formation in BHK-21 cells, infected with SINV/nsP3GFP, at 3 h postinfection. The bar corresponds to 200 nm. (B) CPV1 in BHK-21 cells, infected with SINV/nsP3GFP, at 6 h postinfection. The bar corresponds to 100 nm. (C and D) Distribution of nsP1, nsP3-GFP, and dsRNA in the cells at 2 and 8 h postinfection with SINV/nsP3GFP, respectively. BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At the indicated times, cells were fixed, permeabilized, and stained with dsRNA- and nsP1-specific Abs. Panels a, xy panels represent multiple-intensity projections of a 1-μm xy section of the cell fragment, and xz and yz panels represent multiple-intensity projections of a 2-μm xy section as denoted on the xy panel. Panels b, c, and d, enlarged single xz section of the cell presented in panels a. Panels b show the distributions of nsP1 (red) and dsRNA (blue). A magenta color indicates colocalization of dsRNA and nsP1. Panels c show the distributions of nsP3 (green) and dsRNA (blue). A cyan color indicates colocalization of dsRNA and nsP3. Panels d demonstrate the distributions of nsP1 (red), nsP3 (green), and dsRNA (blue). A white color indicates colocalization of dsRNA and nonstructural proteins nsP1 and nsP3. The white arrowhead indicates the position of one of the endosomes stained with nsP1- and dsRNA-specific Ab and positive for the presence of nsP3-GFP. The red arrowhead indicates one of the nsP1-positive cytoplasmic complexes that lack nsP3-GFP and dsRNA. White arrows point to the cytoplasmic dsRNA-positive foci lacking nsP3-GFP and nsP1 association. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. Bars correspond to 5 μm.

Article Snippet: They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software.

Techniques: Membrane, Infection, Staining, Microscopy, Software

Journal: Journal of Virology

Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

doi: 10.1128/JVI.01441-10

Figure Lengend Snippet: At early times postinfection, dsRNAs are located in membrane spherules presented on the external surface of the plasma membrane. (A) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At 2 h postinfection, cells were fixed with 4% PFA, permeabilized with either 0.02% or 0.5% saponin, treated with dsRNA-specific MAb and rabbit anti-SINV nsP1 Ab, and further stained with appropriate secondary Abs. Images were acquired on a Zeiss LSM700 META confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. Bars correspond to 10 μm. (B) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At 3 h postinfection, cells were fixed with 4% PFA, permeabilized with 0.02% saponin, treated with dsRNA-specific MAb and gold-labeled secondary Abs, and further processed for EM. (C) The same cell sample as in panel B was processed for EM without permeabilization and immunostaining.

Article Snippet: They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software.

Techniques: Membrane, Infection, Staining, Microscopy, Software, Labeling, Immunostaining

Journal: Journal of Virology

Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

doi: 10.1128/JVI.01441-10

Figure Lengend Snippet: In situ PLA demonstrates colocalization of dsRNA with SINV nsP1, nsP2, and nsP3 proteins. (A and B) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At 2 h postinfection, cells were fixed with 4% PFA, permeabilized with either 0.02% (A) or 0.5% (B) saponin, treated with dsRNA-specific MAb and rabbit anti-SINV nsP1 Ab, and further processed for in situ PLA. Red signals indicate colocalization of nsP1 and dsRNA. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stacks, and xz sections are presented as multiple-intensity projections of a 2-μm section. Bars correspond to 5 μm. (C) Numbers of PLA-positive foci detected in the cells by using dsRNA MAbs and nsP-specific, affinity-purified Abs. The nsP1-, nsP2-, or nsP3-specific Abs used and the concentrations of saponin are indicated. The dsRNA-positive foci in 3D images were quantitated using a Spot function in Imaris 3D rendering software. The P values were calculated using the Mann-Whitney test. Average values are indicated in red. (D) Results of PLA performed using dsRNA-specific MAb and nsP1-specific rabbit Ab with mock-infected cells.

Article Snippet: They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software.

Techniques: In Situ, Infection, Microscopy, Software, Affinity Purification, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

doi: 10.1128/JVI.01441-10

Figure Lengend Snippet: The plasma membrane is the site of viral RNA synthesis. BHK-21 cells were infected with SINV/nsP3GFP at an MOI of ca. 20 PFU/cell. At 4 h postinfection, they were treated with digitonin (1 μg/ml) in the presence of ActD, and an in vitro transcription reaction was performed as described in Materials and Methods. Cells were then stained with BrU-specific and dsRNA-specific antibodies. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz and yz sections are presented as multiple-intensity projections of a 0.06-μm section. Bars correspond to 5 μm. White arrowheads indicate sites of colocalization of dsRNA and incorporated BrU. The white arrow points to one of the BrU-positive foci that are not associated with dsRNA staining. Mock-infected cells demonstrated no detectable staining under these experimental conditions; therefore, their images are not presented.

Article Snippet: They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software.

Techniques: Membrane, Infection, In Vitro, Staining, Microscopy, Software