Figure 1.
RSV-M protein expression inhibits cell proliferation.
Panel A, western blot analysis, with specific anti RSV-M monoclonal antibody (C781) in cells transfected with pSG-5 plasmid alone (Con) or pSG-5 plasmid carrying RSV-M cDNA (pSG-M). Panel B, sub-confluent monolayer (approximately 10% confluency) of A549 cells (left panel) and PHBE cells (right panel) were transfected with pSG-M and pSG-5 (vector control). Cells were treated with nutlin-3 (positive control) and captisol alone (vehicle control). Cell replication was then determined by counting the cell numbers using a hemocytometer. Error bars indicate standard error of the mean (SEM) (n=3).
Figure 2.
RSV-M protein induces cell cycle arrest in both A549 and PHBE cells.
Cells were transfected with pSG-M or pSG-5 (vector control). The distribution of cell cycle phases of the transfected A549 cells (panel A) and PHBE cells (panel B) were determined by flow cytometry at 2 days post-transfection. The cell cycle properties were analyzed using BrdU incorporation and 7-AAD staining. The left panels are FACS data, and the right panels are the statistical analysis of each cycle from 3 separate experiments. Error bars are the standard error of the mean (n=3). *p-values<0.05.
Figure 3.
RSV Infection and RSV-M protein expression induce p53 activation.
Panel A, A549 cells were infected with RSV at MOI of 5 PFU/cell. Cell extracts were prepared at indicated times and used in western blot using anti p53 mAb (DO-1). A549 cells (panel B, left) or PHBE cells (panel B, right) were transfected with pSG-M (M) or pSG-5 (V). At indicated time points, total cell extracts were prepared and were used in western blot analysis. The specific anti-total p53 (DO-1), anti-phospho-p53 at serine-15 residue, anti-p21, anti-phospho-Rb at residue 780 and anti-RSV-M protein antibody were used to identify the respective molecules. Anti-GAPDH antibody was used as an internal control. Panel C, qualification of p53, phospho-p53, p21, and phospho-Rb was performed by using ImageJ software. Error bars are the standard error of the mean (n=3). *p-values<0.05.
Figure 4.
p53 is necessary for RSV-M-induced cell cycle arrest.
Panel A, western blot analysis of H1299 cells transfected with a plasmid carrying the wild type p53 cDNA. After isolation of stable transformants, H1299 cells (panel B) and H1299 p53+/+ cells (panel C) were transfected with pSG-M (RSV-M) and pSG-5 (vector). The cycle phase distribution was then analysed by flow cytometry at 48 hr post transfection. The right panels are the statistical analysis of each cycle from 3 separate experiments. Error bars are the standard error of the mean (n=3). *p-values<0.05.
Figure 5.
p53 expression enhances RSV replication.
Panel A, H1299 and H1299 p53+/+ cells were infected with RSV at MOI of 1 PFU/cell. After 24 and 48 hours, total cellular RNA was isolated. The RSV gene transcription levels between the p53-expressing and non-expressing cells were normalized to GAPDH and compared at the respective time points. Panel B, cells were prepared as in panel A but to measure virus replication a standard plaque assay was performed (left panel). The number of plaques and the titer of the virus was then determined (right panel). Panel C, A549 and PHBE cells were treated with nutlin-3 or captisol alone (vehicle control). After 48 hours, cells were infected with RSV at MOI of 1 PFU/cell. After 24 hr, relative changes of NS1 and G genes were determined. The error bars are standard error of the mean from three independent assays.
Figure 6.
A working model of the effects of environmental factors on p53 expression and enhancement of virus replication.