Article Figures & Data
Figures
- Fig. 1.
NV1066 induces apoptosis in infected cells as demonstrated by nuclear staining (A–C) and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL; D–F). OCUM cells were infected with NV1066 (MOI = 0.1) and examined under separate filters for GFP expression and apoptosis. Representative fields are depicted here and quantitatively described in Table 1<$REFLINK> . A, infected cells stained with Hoechst DNA fluorochrome express enhanced green fluorescent protein (eGFP) as seen under a GFP filter at ×630 magnification. The eGFP protein can be found within nuclei and cytoplasm. B, under a 4′,6-diamidino-2-phenylindole filter, Hoechst staining illustrates high resolution of nuclear morphology to assess nuclear morphology. C, an overlay image is created by superimposing the GFP and 4′,6-diamidino-2-phenylindole filter images to concurrently evaluate nuclear morphology and eGFP expression. Characteristic morphology of apoptotic cells, including pyknosis, nuclear fragmentation, and apoptotic bodies, is seen in the cells at the white arrows. These cells do not express eGFP; that is, they are apoptotic but are not infected. The inset taken from another field illustrates that an apoptotic cell can express eGFP. Characteristic apoptotic bodies are shown within the eGFP-filled cytoplasm. D, infected cells are stained by TUNEL and examined under a GFP filter at ×945 magnification. Expression of eGFP represents viral infection. E, using phase-contrast, bright-field microscopy, TUNEL staining is evaluated. Several apoptotic cells are depicted by brown-stained nuclei. F, an overlay image is created to compare TUNEL positivity and eGFP expression. TUNEL-positive cells do not express eGFP, confirming that apoptosis occurs in noninfected cells.
- Fig. 2.
Inhibition of apoptosis increases expression of the viral eGFP gene. Cells were infected with NV1066 at a multiplicity of infection (MOI) of 0.1 and a MOI of 0.01 and exposed to 10 mm N-acetylcysteine (NAC) or 1 mm NAC or left untreated. Cells were harvested and examined by flow cytometry for enhanced green fluorescent protein expression each day for 4 days after infection. Inhibition of apoptosis with NAC increases enhanced green fluorescent protein expression at both MOIs when compared with infection alone in this representative experiment (P < 0.001, z-test).
- Fig. 3.
Inhibition of apoptosis increases viral cytotoxicity and proliferation. OCUM cells are infected in vitro at multiplicities of infection of 0.1 and 0.01 and treated or not treated with 10 mm N-acetylcysteine (NAC). All samples were performed in triplicate and are presented as the mean ± SE. A, cells were harvested every day for a week, and cell survival was determined in a lactate dehydrogenase release assay. Cytotoxicity of viral treatment is enhanced in the presence of NAC at all time points until maximal lysis is achieved. B, supernatants from cell cultures were assayed in a standard viral plaque assay for viral count. By day 4, there is an increase of approximately 3× and 19× in viral production in the presence of NAC in the multiplicity of infection = 0.1 and 0.01 cultures, respectively.
Tables
- Table 1
NV1066 induces apoptosis in uninfected and infected cells
Random microscopic fields of NV1066-treated cells were assessed for nuclear morphology and eGFP expression, counted, and averaged from three experiments. Cells are either apoptotic and do not express eGFP, not apoptotic and express eGFP, apoptotic and express eGFP, or not apoptotic and do not express eGFP. The last group is not represented in the table. There was a significantly higher rate of apoptosis in the infected cells compared with uninfected cells (P < 0.005, Fisher’s exact test). Untreated samples represent the spontaneous rate of apoptosis in cultured cells. Actinomycin D was used as a positive control for the induction of apoptosis.
Cell phenotype (%) A+/G−a A−/G+ A+/G+ Untreated 0.8 ± 0.1b 0.1 ± 0.1 0.0 ± 0.0 MOI of 0.1 9.6 ± 2.8b 12.8 ± 2.4 4.4 ± 1.9 Actinomycin D (1.2 μm) 18.4 ± 4.7 0.0 ± 0.0 0.0 ± 0.0 -
a A, apoptotic; G, enhanced green fluorescent protein; MOI, multiplicity of infection.
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b P = 0.009.
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Volume 10, Issue 9
