Endothelial cells support persistent gammaherpesvirus 68 infection

Abstract

A variety of human diseases are associated with gammaherpesviruses, including neoplasms of lymphocytes (e.g. Burkitt's lymphoma) and endothelial cells (e.g. Kaposi's sarcoma). Gammaherpesvirus infections usually result in either a productive lytic infection, characterized by expression of all viral genes and rapid cell lysis, or latent infection, characterized by limited viral gene expression and no cell lysis. Here, we report characterization of endothelial cell infection with murine gammaherpesvirus 68 (gammaHV68), a virus phylogenetically related and biologically similar to the human gammaherpesviruses. Endothelial cells supported gammaHV68 replication in vitro, but were unique in that a significant proportion of the cells escaped lysis, proliferated, and remained viable in culture for an extended time. Upon infection, endothelial cells became non-adherent and altered in size, complexity, and cell-surface protein expression. These cells were uniformly infected and expressed the lytic transcription program based on detection of abundant viral gene transcripts, GFP fluorescence from the viral genome, and viral surface protein expression. Additionally, endothelial cells continued to produce new infectious virions as late as 30 days post-infection. The outcome of this long-term infection was promoted by the gammaHV68 v-cyclin, because in the absence of the v-cyclin, viability was significantly reduced following infection. Importantly, infected primary endothelial cells also demonstrated increased viability relative to infected primary fibroblasts, and this increased viability was dependent on the v-cyclin. Finally, we provide evidence for infection of endothelial cells in vivo in immune-deficient mice. The extended viability and virus production of infected endothelial cells indicated that endothelial cells provided a source of prolonged virus production and identify a cell-type specific adaptation of gammaherpesvirus replication. While infected endothelial cells would likely be cleared in a healthy individual, persistently infected endothelial cells could provide a source of continued virus replication in immune-compromised individuals, a context in which gammaherpesvirus-associated pathology frequently occurs.

Figure 1. Endothelial cells supported γHV68 replication.

In vitro growth assays of γHV68 in MB114, SVEC, and CD3 endothelial cells. Cell were infected at the indicated MOI with either wildtype γHV68 (A) or γHV68-GFP (B) and harvested at the indicated times. (A) Samples were multiply freeze-thawed prior to quantification by plaque assay on NIH 3T12 cells. Top panel, γHV68 titer 36 hours post-infection in 3T3 fibroblasts compared to MB114, SVEC, and CD3 endothelial cells. n = 2–3. (B) Cells infected with γHV68-GFP were analyzed by flow cytometry for GFP expression at 24 and 48 hours post-infection. Fluorescence was determined relative to cells infected with wildtype γHV68 (grey). Histograms are representative of two independent infections.

Figure 2. A population of endothelial cells remained intact following γHV68 infection.

Non-adherent cells were collected at six days post-infection (MOI of 5 PFU/cell), cultured, and viability determined at six and 12 days post-infection. Status of γHV68 replication was determined at six days post-infection. (A) Bright field images of 3T3 fibroblasts, MB114 endothelial cells, and SVEC endothelial cells prior to infection (top panel) and at six days after γHV68 infection (bottom panel). Magnification of 200X. Infected cells were harvested by centrifugation and resuspended in fresh media prior to imaging. (B) Percentage of cells infected that were non-adherent and viable at six days post-infection. Where indicated, cells were treated with PAA after one hour of infection. Non-adherent cells were collected at six days post-infection and viability determined as percentage of trypan blue excluding cells. A significantly lower percentage of all fibroblast lines analyzed were viable at six days post-infection as compared to all endothelial cell lines analyzed (*p<0.001). In the presence of PAA, a significantly lower percentage of endothelial cells were non-adherent and viable at six days post-infection (°p = 0.018). Data is from 2–3 independent experiments per cell line. (C) Following collection at six days post-infection, cells were centrifuged and stained for surface expression of gp150 (top panel) or with propidium iodine (PI) (bottom panel). Latent S11E cells do not express lytic viral proteins and are a negative control for gp150 expression, as well as a positive control for viable cells. Fluorescence was determined relative to unstained cells (grey) and numbers reflect percent positive staining. Histograms are representative of two independent experiments. (D) Cultures of non-adherent cells at 12 days post-infection were stained with PI and viability determined by flow cytometry as percent PI negative cells. n = 3 per cell line. Cultures of endothelial cells remained significantly more viable than fibroblasts at 12 days post-infection (*p<0.001). S11s were analyzed as a positive control for viable cells.

Figure 3. Endothelial cells which were viable after γHV68 infection were productively infected.

(A) MB114 cells contained viral gene transcripts as far as 12 days post-infection. RT-PCR analysis of viral gene transcripts. 100 ng of total RNA from mock infected and infected MB114 and 3T3 cells and from S11 cells was added to each of the RT reactions along with primers specific for the viral transcripts polymerase III-1 and M2, and the cellular transcripts β-actin and 18S. No RT and no template controls are indicated. (B) MB114 cells contained viral proteins as far as 12 days post-infection. Immunoblot of lytic viral protein expression. 10 µg of total protein from mock infected and infected MB114 and 3T3 cells and from S11 cells were loaded per lane and blots probed with antibodies to the lytic γHV68 proteins M3 (top) and gB (middle), and mouse β-actin (bottom) as a loading control. Mock infected cells were collected at 24 hours. Latent S11 cells do not express lytic viral proteins and served as a negative control. (C) At the indicated times post-infection, MB114 cells were analyzed for gp150 surface expression by flow cytometry. Fluorescence was determined relative to unstained cells (grey). Histograms are representative of two independent infections. (D) Six days post-infection with GFP-γHV68, MB114 cells were collected and analyzed by flow cytometry for GFP expression. Cells were cultured and brightfield (top) and fluorescent (bottom) images taken at 20 days post-infection, 100X magnification. Fluorescence was determined relative to cells infected with wildtype γHV68 (grey), and histogram is representative of two independent infections.

Figure 4. Viable γHV68-infected endothelial cells produced and released new infectious virus particles.

(A) MB114 cells contained virions at various stages post-infection throughout the nucleus and cytoplasm. Transmission electron micrographs (TEM) of 3T12 cells and MB114 cells infected with MOI of 5. 36 hours post-infection, adherent 3T12 cells were collected and combined with non-adherent material in media (left panel). At six days post-infection, only non-adherent MB114 cells remained (middle and right panel). These cells were collected and cultured until 12 days post-infection. Pelleted cells were fixed in glutaraldehyde for TEM. Black arrows indicate virions. (B) MB114 cells released significantly more virus into the media as far as 12 days post-infection than 3T3 fibroblasts (p = 0.001). Non-adherent 3T3 fibroblasts and MB114 cells were collected at six days post-infection, washed twice, and resuspended in complete RPMI. At 12 days post-infection, cell-free supernatant titers were determined by plaque assay. Supernatant titer from latently infected S11 B cells, cultured in the same manner as infected 3T3 cells and MB114 cells was below the limit of detection for the plaque assay (0.5 log₁₀ PFU/mL, indicated by dashed line).

Figure 5. Endothelial cells exhibited prolonged viability and proliferated after γHV68 infection.

Viability and proliferation of an endothelial cell line collected at six days post-infection (MOI of 5 PFU/cell) were determined every three days of culture. (A) Non-adherent MB114 endothelial cells collected at six days post-infection remained viable as far as 30 days post-infection. Viability (left axis) and live cell number (right axis) by trypan blue exclusion counts were measured every three days of culture. n = 3. (B) MB114 cells proliferated after infection. MB114 cells were stained with CFSE and analyzed by flow cytometry for green fluorescence prior to infection (day 0), and at six and 12 days post-infection. Unstained cells analyzed in parallel were consistent in autofluorescence throughout the analysis. S11 B cells, harboring latent γHV68 genome, were stained and analyzed in parallel as a positive control. n = 2 per cell line. (C) Analysis of viability in MB114 cells (top panel) and 3T12 cells (bottom panel) following γHV68 infection. Viability was determined by double staining of PI and annexin V, and gated on unstained cells. The results are representative of 2–4 independent experiments.

Figure 6. Infected and viable endothelial cells were altered in size, shape, and surface protein expression.

Flow cytometric analysis of morphology and surface protein expression of non-adherent endothelial cells collected at six days post-infection. (A) MB114 cells were altered in size and granularity by γHV68 infection. Flow cytometric determination of forward and side scatter properties of MB114 cells collected at six days post-infection. MB114 cells treated with phophonoacetic acid (PAA) one hour after infection were collected and analyzed at six days post-infection. n = 2. (B) MB114 cells were altered in surface protein expression after γHV68 infection. Uninfected and infected MB114 cells harvested at six days post-infection were analyzed for cell surface expression of Thy1, ICAM-1, and VCAM-1 by flow cytometry. Fluorescence was determined relative to unstained cells (grey). Results are representative from three independent experiments.

Figure 7. Optimal viability of endothelial cells after γHV68 infection requires the viral cyclin.

(A) MB114 cells contained the γHV68 v-cyclin transcript. RT-PCR analysis of the γHV68 v-cyclin transcript. Total RNA was isolated from infected MB114 cells and 3T3 cells at the indicated times post-infection, as well as from latently infected S11 B cells. 100 ng of RNA from each sample was subjected to RT-PCR analysis with primers specific for the v-cyclin transcript. As a loading control, we also amplified the cellular transcript β-actin. No RNA could be isolated from cultured 3T3 cells at 12 days post-infection. No RT and no template controls are indicated. (B & C) Viability and persistent viral replication in the presence or absence of the v-cyclin. MB114 cells were infected with wildtype (black) or v-cyclin.STOP γHV68 (grey) at an MOI of 5 PFU/cell. Cells were harvested at six days post-infection by centrifugation from supernatant, and cultured in complete RPMI. Every six days cells were centrifuged to remove supernatant for titer (C) and stained with propidium iodine (PI) every three days (B). Viability of post-infection cultures was determined as percent PI negative cells by flow cytometry. Significant differences in viability were observed between wildtype γHV68 and v-cyclin.STOP infections at day six (p<0.001), day nine (p<0.001), day 12 (p<0.001), and day 15 (p = 0.002). n = 3 for wildtype infection and n = 4 for v-cylin.STOP infection. (C) Cell free virus titer of supernatants from wildtype γHV68 (black) and v-cyclin.STOP γHV68 (grey) infected cells was determined by plaque assay at the indicated times post-infection. Significant differences observed at day 12 (p = 0.008) and day 24 (p = 0.042). n = 2.

Figure 8. Primary endothelial cells were infected in vivo, and demonstrated prolonged viability while supporting γHV68 growth ex vivo.

Multi-step growth assay of γHV68 in primary C57/BL6 lung endothelial cells (A) and corresponding cell viability (B). Primary cells were infected at an MOI of 0.05 PFU/cell with either wildtype γHV68 or v-cyclin.STOP γHV68, harvested at the indicated times, and trypan blue exclusion counts performed (B). (A) Samples were multiply freeze-thawed prior to quantification by plaque assay on NIH 3T12 cells. n = 3. (B) At 0, 48, 96, and 120 hours post-infection, trypan blue exclusion counts were performed on primary lung endothelial cells and primary MEFs infected with either wildtype or v-cyclin.STOP γHV68 at an MOI of 0.05 PFU/cell. n = 3. Limiting dilution-PCR of viral DNA (C) and RT-PCR (D) analysis of endothelial enriched (E) and depleted (D) lung cells from CD8-alpha knockout mice at six days post-intranasal infection. (C) Frequency of viral genome-positive cells was determined by LD-PCR. Percentage of positive PCR reactions are indicated on the y axis and the number of cells analyzed is indicated on the x axis. For each cell dilution, 12 PCR reactions were analyzed. The frequency of viral genome positive cells was determined by Poisson distribution indicated by the dashed line at 63.2%. Data represent the averages of three separate infected animals. Error bars represent standard errors of the mean. (D) Total RNA was isolated from D and E populations and 100 ng of RNA subjected to RT-PCR analysis with primers specific for the γHV68 pol III-1 transcript and the cellular transcripts CD31 and β-actin. Results are shown from three infected and one mock infected mouse.