Evolution of broad host range in retroviruses leads to cell death mediated by highly cytopathic variants

Abstract

The ability of many retroviruses to cause disease can be correlated to their cytopathic effect (CPE) in tissue culture characterized by an acute period of cell death and viral DNA accumulation. Here, we show that mutants of a subgroup B avian retrovirus (Alpharetrovirus) cause a very dramatic CPE in certain susceptible avian cells that is coincident with elevated levels of apoptosis, as measured by nuclear morphology, and persistent viral DNA accumulation. These mutants also have a broadly extended host range that includes rodent, cat, dog, monkey, and human cells (31). Previously, we have shown that the mutants exhibit diminished resistance to superinfection. The results presented here have important implications for the process of evolution of retroviruses to use distinct cellular receptors.

FIG. 1.

Viral replication and cell growth. Cells were infected and passaged at equal density every 3 to 4 days. RT activity was measured and plotted to give viral growth curves for the DF1 cell line (A) and a CEF cell strain (B). The average of three viable cell counts was used to determine the relative increase in cell number for the DF1 cells (C) and CEF cells (D). Results are the average of three replicates (± standard error of the mean [SEM]) from a single experiment, representative of three independent experiments. The interval during which the strongest CPE was evident for DF1 cells infected with L154S and LT154/155SI is indicated (A and C).

FIG. 2.

Micrographs of infected cells. Cells were examined using phase-contrast microscopy and photographed with a charge-coupled device camera. Typical fields are shown for cells infected with each virus for DF1 (16 days) and CEF (14 days) cells. Black bars, 100 μm.

FIG. 3.

Apoptosis in infected cultures. Adherent and nonadherent cells were pooled and stained with Hoechst 33342 dye. Apoptotic nuclei were scored as a percentage of total nuclei by fluorescence microscopy. (A) DF1 cell line. The interval with the strongest CPE is indicated. (B) CEF cell strain. Results are the average of three counts (± SEM) from a single experiment, representative of two independent experiments. The area under the curve was determined to indicate cumulative apoptosis levels (arbitrary units) in infected cells and plotted versus time for DF1 (C) and CEF (D) cells.

FIG. 4.

Accumulation of viral DNA. Southern blots of total cellular DNA were hybridized to a labeled gag probe. Data from DF1 cells infected with PrB and L154S are shown (A), and these as well as the others were quantitated with a phosphorimager and plotted (B to E). Integrated high-molecular-weight (C and E) and unintegrated (B and D) DNA forms were measured for DF1 (B and C) and CEF (D and E) cells, and the DNA copy number was obtained by comparison to a standard curve present on every gel. Results are presented from a single experiment, representative of two independent experiments.

FIG. 5.

Induction of apoptosis by cycloheximide in chronically infected DF1 cells. Chronically infected cells (60 days postinfection) were treated with cycloheximide (10 μg/ml), harvested at indicated times, and scored for apoptosis as described in the legend to Fig. 3. Results are the average of three replicates (± SEM) from a single experiment, representative of three independent experiments.