Infection and persistence of rhesus monkey rhadinovirus in immortalized B-cell lines

Abstract

Similar to its close relative human herpesvirus 8, rhesus monkey rhadinovirus (RRV) persists predominantly in B cells of its natural host. Rhesus monkey B-cell lines immortalized by the Epstein-Barr-related virus from rhesus monkeys (rhEBV) were used as targets for infection by RRV. These cultured B cells were susceptible to infection by RRV and continued to produce low titers of RRV for months of continuous culture. Infection by RRV did not detectably alter the growth rates of these B-cell lines when it was measured at standard or reduced serum concentrations. Depending on the cell line, 5 to 40% of the B cells stained positive for the RRV genome by fluorescence in situ hybridization (FISH). Most RRV-positive cells showed a fine punctate nuclear staining pattern consistent with latent infection, while a small minority of cells (0.2 to 1%) contained large, intensely staining nuclear foci consistent with productive, replicative infection. Greater than 90% of the cells were rhEBV genome positive in a pattern consistent with latent infection, and again only a small minority of cells showed a productive, replicative staining pattern. Dual, two-color FISH staining revealed coinfection of numerous cells with both RRV and rhEBV, but productive replication of RRV and rhEBV was always observed in separate cells, never in the same cell. Thus, productive replication of RRV is unlinked to that of rhEBV; factors that influence activation to productive replication act separately on RRV and rhEBV, even within the same cell. The percentage of B cells expressing green fluorescent protein (GFP) early after infection with a recombinant RRV containing a GFP reporter gene was dose dependent and at a low multiplicity of infection increased progressively over time until 14 to 17 days after infection. These results establish a naturalistic cell culture system for the study of infection and persistence by RRV in rhesus monkey B cells.

FIG. 1.

Influence of RRV infection on growth properties of B-cell lines 211-98 (A), 260-98 (B), and 309-98 (C). (Top) Immortalized rhesus B-lymphocyte cell lines were infected with RRV and cultured in the presence of 10% or 2% FBS starting with a cell density of 0.1 × 10⁶ cells per ml of medium. Live-cell counts were determined for each culture up to day 15 postinfection. On day 7 (arrows), cultures were arbitrarily diluted 1:6 for cultures supplemented with 10% FBS and 1:3 for cultures supplemented with 2% FBS. (Bottom) Following 4 weeks of culture postinfection, the same cell lines were diluted again to a density of 0.1 × 10⁶ cells per ml of medium supplemented with 10% or 2% FBS and growth properties were analyzed as outlined above.

FIG. 2.

Expression of RRV proteins in persistently infected B-cell lines. (A) RRV-infected rhesus B-cell lines and their parental cell lines (211-98, 260-98, and 309-98) were lysed in RIPA buffer, and 20 μg of total cellular protein was loaded per lane. As controls, uninfected rhesus fibroblast cells (Rf 388-93) and fibroblast cells that had been infected for 81 h were lysed in parallel and 10 μg of total cellular protein was loaded per lane. Following electrophoresis, proteins were transferred to membrane filters and RRV proteins were detected by Western blotting with an RRV-specific rabbit serum. (B) Detection of RRV proteins in virus particle preparations. Column-purified RRV particles were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RRV proteins were detected by Western blotting with an RRV-specific rabbit serum (α-RRV) or normal rabbit serum (NRS) as a control.

FIG. 3.

Detection of RRV genomes in persistently infected B-cell lines by FISH. RRV-infected B-cell lines and corresponding parental cell lines were fixed and hybridized in situ with a SpectrumRed-labeled RRV pooled-cosmid probe that covered 90% of the unique coding region. Cell nuclei were counterstained with DAPI (blue). Results are shown for the RRV-infected 211-98 cell line (A and B) and the parental RRV-negative cell line (C). Staining patterns observed were consistent with latent persistence of RRV (A) and, at a lower frequency, productive replication (B). No RRV-specific staining was observed in the RRV-negative 211-98 parental cell line (C).

FIG. 4.

Productive replication of RRV is independent of that of rhEBV. RRV-infected B-cell lines and corresponding parental cell lines were fixed and hybridized in situ simultaneously with a SpectrumRed-labeled RRV probe and a SpectrumGreen-labeled rhEBV probe. Cell nuclei were counterstained with DAPI (blue). Results are shown for the RRV-infected 211-98 cell line. Panels: A, latent coinfection of RRV and rhEBV; B, productive replication of RRV in the presence of latent rhEBV; C, productive replication of rhEBV in the presence of latent RRV infection. Note that in panel B, three rhEBV-positive cells are present that are apparently not infected by RRV.

FIG. 5.

Dose and time dependence. (A) The percentage of GFP-positive LCL211-98 cells after infection with RRV-GFP is MOI dependent. LCL211-98 cells were infected with increasing MOIs of RRV-GFP. The number of PFU of RRV-GFP was determined on rhesus monkey fibroblasts. At day 4 postinfection, cultures were examined for GFP expression by FACS analysis. The results show the percentage of viable GFP-positive cells along with the standard deviation at the indicated number of PFU per cell. Where no error bar is shown, the error falls within the size of the symbol. (B) The percentage of GFP-positive LCL211-98 cells increases over time postinfection (p.i.). Triplicate cultures of LCL211-98 cells were mock infected or infected with RRV-GFP at 0.037 PFU/cell. The number of PFU of RRV-GFP was determined on rhesus monkey fibroblasts. Each day postinfection, 1 ml of the suspension culture was removed and replaced with 1 ml of medium. The aliquoted cells were pelleted by centrifugation and examined by FACS analysis to determine the percentage of GFP-positive cells.

FIG. 6.

Neutralization of RRV-GFP infection. RRV-GFP (MOI = 0.3 PFU/cell) was incubated with either medium alone (No Antibody) or sera from RRV-negative rhesus monkeys (Mm 288-94 and 232-03) or rhesus monkeys naturally infected with RRV (Mm 526-91, 488-03, 541-03, 140-83, and 288-03) at a dilution of 1:20 or 1:100. The number of PFU of RRV-GFP was determined on rhesus monkey fibroblasts. After incubating of the virus-serum mixture for 3 h at 37°C with gentle rocking, LCL211-98 cells were inoculated with either medium alone (No Virus) or the virus-serum mixture. At day 4 postinfection, cultures were examined for GFP expression by FACS analysis. The results shown are the percentage of GFP-positive cells along with the standard deviation for each serum dilution tested.