Article Figures & Data
Figures
- Fig. 1.
DNA and RT-PCR analyses of HHV8-infected DMVEC. (a) DNA PCR amplification of the HHV8-specific KS330233 sequence from serial dilutions of HHV8-exposed DMVEC (+HHV8). DNA was amplified from 2 × 103, 4 × 102, and 2 × 102 and cell equivalents at 7 and 14 days PI (d7 PI and d14 PI, respectively). For positive and negative controls, PCR was performed on genomic DNA prepared from 2 × 103 BCBL-1 cells and mock-infected DMVEC. (b) RT-PCR detection of ORF 29 mRNA using cDNA from 2 × 103 HHV8-exposed DMVEC (+HHV8) at 7 and 14 days PI (d7 PI and d14 PI, respectively) to verify the authenticity of HHV8 infection. cDNA prepared from BCBL-1 cells was used as a positive control. No signal was obtained from mock-infected cells or samples prepared in the absence of RT (−RT). Cellular HPRT was simultaneously amplified from all +RT samples as a control for cDNA synthesis (data not shown). Amplification products from reverse-transcribed BCBL-1 cell cDNA and genomic DNA (BCBL-1 gDNA) demonstrate that the RT-PCR product from spliced mRNA is smaller than the product from genomic DNA. Products amplified from HHV8-infected DMVEC show only the smaller band, indicating lack of contamination from the viral inoculum or replicated virus.
- Fig. 2.
Expression of latency-associated genes in HHV8-infected DMVEC. (a) RT-PCR detection of HHV8 kaposin (ORF K12) mRNA by RT-PCR from HHV8-infected (+HHV8) but not mock-infected DMVEC. cDNA from uninduced BCBL-1 cells was included as a positive control. Products amplified from cDNA prepared from 2 × 103, 4 × 102, and 2 × 102 and cell equivalents at day 14 PI are shown. No signal was detected in samples prepared in the absence of RT (−RT). Cellular HPRT was simultaneously amplified from all +RT samples as a control for cDNA synthesis (data not shown). (b) Nuclear expression of latent antigen ORF 73 in HHV8-infected DMVEC at day 7 PI visualized with a polyclonal antibody against ORF 73 and a goat anti-rabbit FITC conjugate.
- Fig. 3.
Expression of lytic-cycle-associated proteins in HHV8-infected DMVEC. (a) Nuclear expression of ORF 59 visualized with MAb 11D1 and a goat anti-mouse FITC conjugate at 1, 4, and 8 weeks PI in HHV8-infected DMVEC cultures that were not induced (No TPA). Cells were subcultured at weekly intervals. The increase in ORF 59-positive cells indicates maintenance of the HHV8 genome and a complete viral replication cycle allowing infection spread. (b) ORF 59 expression in duplicate DMVEC cultures at 1, 4, and 8 weeks PI that had been pretreated with TPA for 48 h (+TPA). (c) ORF 59 expression in HHV8-infected DMVEC at 4 weeks PI without (No Dex) or with (+Dex) pretreatment with dexamethasone for 5 days. Treatment with exogenous induction stimuli (TPA or dexamethasone) increased the percentage of ORF 59-positive cells approximately 10-fold. (d) Expression of glycoprotein ORF K8.1A/B, a late gene product, on the surface of HHV8-infected DMVEC visualized with a MAb against K8.1A/B and a goat anti-mouse FITC conjugate.
- Fig. 4.
Detection of HHV8 particles in infected DMVEC by electron microscopy. (a) View of a DMVEC nucleus showing herpesvirus-like capsid structures. The insert shows an enlarged view of a single virion (bar = 100 nm). (b) View of a cytoplasmic vesicle containing mature virions sectioned within the nuclear plane (bar = 120 nm).
- Fig. 5.
Induction of spindle morphology in DMVEC cultures following HHV8 infection. (a) Phase-contrast microscopy of mock-infected DMVEC, demonstrating the typical cobblestone appearance. (b) Phase-contrast microscopy of HHV8-infected DMVEC at 1 week PI, demonstrating the appearance of cells with a spindle shape within the monolayer. (c) Phase-contrast microscopy of HHV8-infected DMVEC at 4 weeks PI, demonstrating the increase in morphologic change with time PI. (d) Fluorescence (left) and phase-contrast (right) microscopy fields of HHV8-infected DMVEC, demonstrating a correlation between ORF 59 expression and severity of phenotypic change. (e) Fluorescence (left) and phase-contrast (right) microscopy fields, demonstrating strong expression of ORF K8.1A/B in DMVEC exhibiting cell rounding.
- Fig. 6.
Expression of endothelial cell phenotypic markers by HHV8-infected DMVEC. (a) IFA demonstrating maintenance of VE-cadherin at cell junctions in spindle-shaped, HHV8-infected DMVEC (+HHV8) and mock-infected, cobblestone DMVEC. CD31 expression was similarly retained following HHV8 infection (data not shown). (b) IFA demonstrating loss of vWF expression in DMVEC cultures following HHV8 infection (+HHV8). In contrast, mock-infected cultures express high levels of vWF in characteristic rod-shaped Weibel-Palade bodies. Cultures were uninduced and were stained at 4 weeks PI when approximately 80% of cells expressed ORF 73 (data not shown).
- Fig. 7.
Loss of contact inhibition and growth in soft agar by HHV8-infected DMVEC. (a) Phase-contrast microscopy of an HHV8-infected DMVEC monolayer demonstrating loss of contact inhibition and piling up of cells into foci following culture postconfluency for 5 and 10 days. (b) Phase-contrast microscopy of cultures grown on soft agar 2 weeks after seeding of cells from mock-infected and HHV8-infected (+HHV8) DMVEC. Colonies were formed exclusively by HHV8-infected cells.
