Mechanisms of avian retroviral host range extension

Abstract

Alpharetroviruses provide a useful system for the study of the molecular mechanisms of host range and receptor interaction. These viruses can be divided into subgroups based on diverse receptor usage due to variability within the two host range determining regions, hr1 and hr2, in their envelope glycoprotein SU (gp85). In previous work, our laboratory described selection from a subgroup B avian sarcoma-leukosis virus of an extended-host-range variant (LT/SI) with two adjacent amino acid substitutions in hr1. This virus retains its ability to use the subgroup BD receptor but can also infect QT6/BD cells, which bear a related subgroup E receptor (R. A. Taplitz and J. M. Coffin, J. Virol 71:7814-7819, 1997). Here, we report further analysis of this unusual variant. First, one (L154S) of the two substitutions is sufficient for host range extension, while the other (T155I) does not alter host range. Second, these mutations extend host range to non-avian cell types, including human, dog, cat, mouse, rat, and hamster. Third, interference experiments imply that the mutants interact efficiently with the subgroup BD receptor and possibly the related subgroup E receptor, but they have another means of entry that is not dependent on these interactions. Fourth, binding studies indicate that the mutant SU proteins retain the ability to interact as monomers with subgroup BD and BDE receptors but only bind the subgroup E receptor in the context of an Env trimer. Further, the mutant SU proteins bind well to chicken cells but do not bind any better than wild-type subgroup B to QT6 or human cells, even though the corresponding viruses are capable of infecting these cells.

FIG. 1.

Schematic representation of the alpharetrovirus gp85 SU. Sequences shown to be important for host range determination are represented by boxes (black for subgroup B and hashed for subgroup E). vr1, vr2, and vr3 are variable regions, and hr1 and hr2 are highly variable host-range-determining regions. NTRE4 is a chimeric virus resulting in recombination between td-PrRSV-B and RAV-0. The location of the point mutations studied here is indicated at the bottom in the hr1 rectangle. WT subgroup E (RAV-0), subgroup B (td-PrRSV-B), and the mutant amino acid sequences are listed below. The host range phenotypes are listed on the right (43, 47).

FIG. 2.

A single mutation expands the host range of td-PrRSV-B virus. (A) Generation of pseudotyped egfp virus. Wild-type or mutant viruses were used to infect cells that contain a stably integrated replication-defective genome with the egfp gene and sequences necessary for packaging and reverse transcription. These cells then produced a mixture of viruses that bear the envelope and other viral proteins encoded by the infecting virus and contain the genome of the infecting virus, the integrated egfp element, or one copy of each. These stocks were used in subsequent experiments to score infection by egfp expression. (B) Quail cells resistant to infection by subgroup B and D alpharetroviruses (QT6/BD) and chicken cells resistant to infection by subgroup E alpharetroviruses (C/E) were infected with pseudotyped egfp viruses in triplicate, and infection was scored by flow cytometry 2 days later. Titers were determined as described in Materials and Methods. Error bars show the standard error of the mean for each determination. Titers below the limit of detection are graphed at the detection limit and marked with a “<.”

FIG. 3.

Interference patterns among wild-type, recombinant, and mutant viruses. Chicken cells resistant to infection by subgroup E alpharetroviruses (C/E) and quail cells resistant to infection by subgroup B and D alpharetroviruses (QT6/BD) were infected with the viruses indicated by the shading of the bars and passaged at least 5 times, and RT assays were conducted to determine that infection was complete. They were then superinfected with pseudotyped egfp viruses in triplicate, and infection was scored by flow cytometry 2 days later. Three independent titers were determined for each virus for each preinfected group (and for uninfected cells). Interference was calculated as the titer on uninfected cells/titer on preinfected cells for each replicate. These values were then averaged and graphed as in Fig. 2. (A) CEF cells expressing the endogenous TVB^s3; (B) QT6 cells expressing their endogenous TVB^Q.

FIG. 4.

Binding of immunoadhesins to CEF. Cells were removed from plates by using EDTA (no trypsin) and bound to 0, 0.01, 0.1, 0.3, and 1 μg of purified immunoadhesin in 100 μl of PBA buffer at 4°C. They were then incubated with fluorescein-labeled secondary antibody as described in Materials and Methods and analyzed by flow cytometry. (A) Subgroup E immunoadhesin. Median fluorescence intensities were 5.69 (no immunoadhesin), 5.72 (0.01 μg), 5.73 (0.1 μg), 5.85 (0.3 μg), and 5.83 (1 μg). (B) PrB immunoadhesin. Median fluorescence intensities were 5.69 (no immunoadhesin), 11.2 (0.01 μg), 18 (0.1 μg), 22.1 (0.3 μg), and 24.5 (1 μg).

FIG. 5.

Binding of SU immunoadhesins to avian cells. Purified immunoadhesins were bound to cells in triplicate in 100 μl of PBA buffer as described in Materials and Methods. The shift in median fluorescence intensity (FI) was determined by subtracting the background median FI (no immunoadhesin) from each replicate. The graphs show the average values ± standard errors of the means. (A) CEF cells expressing the endogenous TVB^s3; (B) QT6 cells expressing their endogenous TVB^Q.

FIG. 6.

Binding of SU immunoadhesins to 293 cells expressing ASLV receptors. The procedure was the same as that described for Fig. 5. (A) 293 cells, no ASLV receptors; (B) 293 cells expressing tv-b^s1; (C) 293 cells expressing tv-b^s3; (D) 293 cells expressing tv-b^T. Immunoadhesins: ▪, TVA; ▴, E; ▾, NTRE4; ♦, PrB; •, L154S; □, T1551; ▵, LT154/155SI.

FIG. 7.

Binding of receptor immunoadhesins to Q24 cells expressing alpharetrovirus Env glycoproteins. The procedure was the same as that described for Fig. 5. Q24 cells infected with the indicated viruses were bound by immunoadhesin proteins derived from TVA, TVB^s3, or TVB^T, and the level of binding was plotted as a function of immunoadhesin concentration. (A) Mock; (B) RCASBP(A); (C) RAV-60 (subgroup E); (D) NTRE4; (E) PrB (subgroup B); (F) L154S; (G) T155I; and (H) LT154/155SI.

FIG. 8.

Host range extension mutant viruses infect a broad range of species. Infectivity is expressed as percentage of viral titer on Q24 cells (all viral stocks were approximately 10⁵ IU/ml on these cells). (A) 293 human embryonic kidney cells; (B) D17 dog cells; (C) FEF; (D) CHO-K1 cells; (E) Rat-1 fibroblasts; and (F) NIH 3T3 mouse fibroblasts.