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1.
FIG. 3.

FIG. 3. From: Phosphorylation of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable for Virus Growth .

Examination of phosphorylation status of P proteins in infected cells. Parallel cultures of BHK-21 cells were infected with wt or mutant viruses. Cells were labeled with [32P]orthophosphate or were left unlabeled. Equal proportions of cytoplasmic extracts were analyzed by electrophoresis in parallel gels. 32P incorporation into the P protein (top) was detected by autoradiography. The total P proteins were examined by Western blotting (bottom). Arrows in the panels identify P proteins. Numbers at the bottom represent average ratios of 32P/P from three independent experiments.

Subash C. Das, et al. J Virol. 2004 June;78(12):6420-6430.
2.
FIG. 1.

FIG. 1. From: Phosphorylation of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable for Virus Growth .

(A) Domain structure of P protein. The P protein, with three functionally defined domains (I, II, and III) and a hinge region, is shown. The phosphate acceptor sites Ser-60, Ser-64, Ser-226, and Ser-227 are indicated by solid circles and Thr-62 is indicated by a solid square. (B) Various P protein domain I mutants used for the present study. Substitutions of alanine (A) and glutamic acid (E) residues for serine (S) and threonine (T) residues in domain I are shown. (C) Domain II P protein mutants. (D) Genome organization of VSV. The protein coding regions of VSV genes N, P, M, G, and L are shown in rectangular boxes. E, EcoRV sites flanking the P gene.

Subash C. Das, et al. J Virol. 2004 June;78(12):6420-6430.
3.
FIG. 6.

FIG. 6. From: Phosphorylation of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable for Virus Growth .

Analysis of DI particles generated by high-multiplicity passages of wt or mutant viruses. BHK-21 cells in 60-mm-diameter dishes were infected with 50 μl of undiluted cell culture supernatants from the indicated high-multiplicity passages of wt or mutant viruses. At 2 h postinfection, the cells were labeled for 4 h with [3H]uridine and [3H]adenosine in the presence of actinomycin D. Cytoplasmic extracts were prepared as described in Materials and Methods, and one-third of the extracts was used for the isolation of total RNAs. Two-thirds of the extract was incubated with an anti-VSV antibody to immunoprecipitate the nucleocapsids. Total RNAs (A) or RNAs recovered from immunoprecipitated nucleocapsids (B) were analyzed by electrophoresis as described above. DI RNAs seen as closely migrating doublets are identified in rectangular boxes. The positions of full-length genomic and antigenomic (+/−) RNAs as well as the five VSV mRNAs are indicated on the left.

Subash C. Das, et al. J Virol. 2004 June;78(12):6420-6430.
4.
FIG. 5.

FIG. 5. From: Phosphorylation of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable for Virus Growth .

(A) Phase-contrast microscopic images of BHK-21 cells infected with VSVPwt or VSVP227. BHK-21 cells were infected with each virus at an MOI of 0.5. At 12 h postinfection, live cells were observed under a Nikon microscope at a magnification of ×20, and images were processed through a camera (Optronics) attached to the microscope. (B) Expression of VSV proteins in VSVPwt- or VSVP227-infected cells. BHK-21 cells were infected with each virus at an MOI of 0.5 in 12-well plates. At 12 h postinfection, the cells were fixed and incubated with a mouse anti-VSV polyclonal antibody followed by a secondary antibody (anti-mouse Alexa-488). The fluorescence was observed at a magnification of ×40. The top panel shows cells under phase-contrast microscopy and the bottom panel shows the same field under fluorescence microscopy. (C) Quantitative analysis of apoptosis. BHK-21 cells infected with each virus at an MOI of 0.5 were trypsinized at 12 h postinfection and stained for apoptotic and necrotic cells with annexin V-fluorescein isothiocyanate and propidium iodide. The fluorescing cells were analyzed by flow cytometry, and the means of three independent experiments are presented, with error bars representing standard deviations.

Subash C. Das, et al. J Virol. 2004 June;78(12):6420-6430.
5.
FIG. 4.

FIG. 4. From: Phosphorylation of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable for Virus Growth .

(A) Single-cycle growth kinetics of viruses with substitutions in domain II phosphate acceptor sites. The experiment was performed as described in the legend for Fig. 2B. Error bars represent standard deviations. (B) Phosphorylation status of P proteins of domain II mutant viruses. Data were obtained as described in the legend for Fig. 3. Arrowheads in the panels identify P proteins. (C) Kinetics of RNA synthesis in cells infected with VSVPwt and VSVP227. BHK-21 cells infected with VSVPwt (lanes 1 and 3) or VSVP227 (lanes 2 and 4) at an MOI of 100 (lanes 1 and 2) or 10 (lanes 3 and 4) were radiolabeled with [3H]uridine and [3H]adenosine in the presence of actinomycin D for 1 h at 1 or 7 h postinfection as described in Materials and Methods. Total RNAs were isolated and analyzed by electrophoresis. The positions of full-length as well as the five VSV mRNAs are indicated on the left. Numbers at the bottom show the average levels of total viral mRNAs from three experiments. (D) Analysis of viral proteins in cells infected with VSVPwt and VSVP227. BHK-21 cells were infected with VSVPwt (lanes 1 and 3) or VSVP227 (lanes 2 and 4) at an MOI of 100 (lanes 1 and 2) or 10 (lanes 3 and 4). The proteins were radiolabeled with 35S, and equal proportions of cell lysates were analyzed by electrophoresis as described in Materials and Methods. The positions of the viral proteins are shown on the left. The levels of viral protein represent average values from three independent experiments.

Subash C. Das, et al. J Virol. 2004 June;78(12):6420-6430.
6.
FIG. 2.

FIG. 2. From: Phosphorylation of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable for Virus Growth .

(A) Yield of various mutant viruses. BHK-21 cells were infected with mutant viruses at an MOI of 1, and the yield of virus in clarified supernatants from infected cells at 16 h postinfection was determined by plaque assay. Error bars showing standard deviations for five independent experiments are shown. (B) Single-cycle growth kinetics of viruses with substitutions in domain I residues. BHK-21 cells were infected with wt or mutant viruses at an MOI of 20, and culture supernatants were collected at the indicated time points. The viruses in the supernatants were quantitated by plaque assay. The averages of three independent experiments are presented. (C) Analysis of VSV mRNAs in cells infected with domain I mutants. BHK-21 cells were infected with VSVPwt, VSVP60, VSVP62, and VSVP64 at an MOI of 10. RNAs were radiolabeled at 7 to 8 h postinfection in the presence of actinomycin D, analyzed by electrophoresis, and detected by fluorography as described in Materials and Methods. The positions of the VSV mRNAs are indicated on the right. Numbers at the bottom show the average levels of mRNA from three independent experiments. (D) Confirmation of identity of mutant viruses. Total RNAs isolated from wt or mutant virus-infected cells were subjected to RT-PCR amplification of the P coding region. The products were directly sequenced by cycle sequencing. A chromatogram showing the presence of the desired mutations is presented. Specific nucleotide changes corresponding to the mutant proteins are marked with arrowheads.

Subash C. Das, et al. J Virol. 2004 June;78(12):6420-6430.

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