Figure 1.
Productive infection of HUVEC with low passage wtRV.
(A–B) Kinetics of RV replication in HUVEC, Vero and A549 cells. Cells were infected with RV-Dz at an MOI of 0.05 or 5. Cell culture supernatants (A) or cell lysates (B) were titered in duplicate on Vero cells. Data are presented as a mean value +/- standard deviation of two independent experiments each performed in duplicate. The data were analyzed by two-way ANOVA with the Bonferroni posttests for correcting for multiple comparisons (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Quantitation of intracellular rubella genomic RNA. HUVEC, Vero and A549 cultures were infected with RV-Dz at an MOI of 5. Genomic RNA was quantitated by RT-qPCR. GAPDH mRNA was used for normalization in the comparative threshold cycle method. Data are presented relative to the genomic RNA amount at 4 hpi. The results represent the mean of at least two independent experiments each done in duplicate. (D) Phase contrast pictures of cells at 5 dpi either mock infected or RV-Dz infected at MOI=5. Note cytopathic effect of wtRV in A549. (E) Representative images of rubella virions observed by TEM in HUVEC infected with RV-Dz at MOI=50 at 24 hpi. (F) Representative images of rubella virions and (G) replicative complexes observed by TEMin Vero cells infected with RV-Dz at MOI=50 at 24 hpi. Inserts represent enlarged images from the replicative complex and virions that are marked with the red arrows. Bars, 100 nm.
Figure 2.
Expression of viral structural proteins in infected HUVEC.
(A) Kinetics of viral protein synthesis. HUVEC were mock infected (M) or infected with RV-Dz at an MOI=5. Proteins were separated by 4-12% NuPage gel, either nonreducing (E1, C, β-actin) or reducing (E2), and then the blots were probed with rubella E1, E2 and C-specific MAb and β-actin MAb.
(B) Spatial distribution of E1, E2 and C proteins in infected cells. HUVECs were infected with RV-Dz at an MOI=5 on chamber slides and processed for indirect immunofluorescence at 2 dpi using E1, E2 and capsid-specific MAb. Nuclei were counterstained with DAPI.
Figure 3.
Lack of global cellular protein synthesis shut-off in infected HUVEC.
HUVEC and A549 cells were mock infected or infected with RV-Dz at MOI=5. The cultures were metabolically pulse-labeled with non-radioactive protein-labeling reagent AHA at the indicated times postinfection. Equal amounts (5 μg/lane for detection of AHA-labeled proteins and β-actin and 20 μg/lane for capsid) of each sample were separated by 4-12% NuPage and blotted onto nitrocellulose membrane. (A–B) Blots were probed as described in Material and Methods to reveal newly synthesized proteins. (C) The blots were also probed with β-actin MAb to demonstrate equal protein loading and with capsid MAb to confirm RV infection. Representative results of two independent experiments are shown.
Figure 4.
Effects of RV infection on cell proliferation and mitosis.
(A) Growth curves of mock-infected and RV-infected HUVECs. HUVEC were mock infected or infected with RV-Dz at MOI=10 and then counted daily. The data are results of 2 independent experiments each performed in duplicate. (B) Mitosis in infected HUVEC. At 2 dpi the mock infected and infected HUVEC (RV-Dz, MOI=10) were immunostained by IFA with capsid MAb and DAPI to quantitate infected cells and mitotic figures. Mitotic indexes (MI) were calculated as % cells with mitotic figures in two duplicate wells. Note RV-antigen positive mitotic cell in red circle. (C) Cell cycle analysis of infected HUVEC. Serum-starved HUVECs were mock-infected or infected with RV-Dz at MOI=10. Histograms of cell cycle analysis at 1 dpi show DNA content of propidium iodide-stained cells by flow cytometry and % of cells in each phase of the cell cycle. The representative results of two independent experiments are shown.
Figure 5.
(A) Growth curves of RV-Dz at different MOI. Media were collected every 2-3 days and the extracellular viruses were titered on Vero cells in duplicate. The adherent cells on the flasks at 35 dpi were collected by trypsinization into 4 ml of media and counted (cells/ml) using a Scepter cell counter. The representative results of two independent experiments are shown. (B) Western blot analysis of RV capsid protein expression in persistently infected HUVEC. The blot was re-probed with β-actin MAb to confirm equal protein loading. (C) Phase contrast images of cells at different times postinfection showing the lack of CPE in HUVEC persistently infected with RV-Dz strain.
Figure 6.
RV persistence after sequential passages of infected HUVEC.
Cells infected at low and high MOI were passaged 1:4 at 3, 5 and 9 dpi (indicated by the arrows). (A) Media were changed every 2-3 days and titer of extracellular virus was determined by titration on Vero cells. The data are representative results of 2 independent experiments. (B) After each passage, the number of the infected cells was determined by counting E1-positive cells after IFA staining of infected cells for E1, endothelial cell marker (vWF) and counterstaining for nuclei (DAPI). (C) The representative results of IFA staining of the infected cells immediately after the third passage.