The Novel Avian Leukosis Virus Subgroup K Shares Its Cellular Receptor with Subgroup A

Abstract

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.

Keywords: Tva; avian leukosis virus K; host range; resistance/susceptibility to retrovirus; retrovirus receptor; superinfection interference.

FIG 1

Alignment of the amino acid sequences of the ALV subgroup A and subgroup K envelope glycoproteins. Amino acid sequences deduced from the available GenBank data are shown. Subgroup A is represented by the Schmidt-Ruppin A (SR-A) strain (GenBank accession no. NP040548), and subgroup K is represented by the Chinese and Taiwanese nonrecombinant strains JS11C1 (GenBank accession no. KF746200), JS13LY19 (GenBank accession no. AWO14321), GD14LZ (GenBank accession no. ANW72067), TW-3593 (GenBank accession no. ADP21276), Km_5845 (GenBank accession no. BAL70358), and GDFX0601 (GenBank accession no. AKP18446). Identical amino acids are highlighted in black. Gaps are indicated by dots. The main functional domains of envelope glycoproteins are delineated above the sequence. SU, surface subunit; TM, transmembrane subunit; vr1, vr2, and vr3, variable regions 1 to 3; hr1 and hr2, hypervariable regions 1 and 2; HR1 and HR2, heptad repeats 1 and 2.

FIG 2

Host range of RCASBP(JS11C1)GFP in galliform species and inbred lines of domestic chickens. (A) Time courses of RCASBP(JS11C1)GFP and RCASBP(A)GFP infection in DF-1 cells. The percentages of GFP-positive cells in the 3 consecutive days after infection are shown as means and standard deviations of triplicate assays. (B) Efficiency of RCASBP(JS11C1)GFP and RCASBP(A)GFP infection, measured as the percentages of GFP-positive cells 3 days after infection for cultured embryo fibroblasts of 14 galliform species (Reeve’s pheasant [SR], turkey [MG], silver pheasant [LN], white-crested kalij pheasant [LL], gray jungle fowl [GS], Mrs. Hume’s pheasant [SH], chukar [AC], northern bobwhite [CV], California quail [CC], Gambel’s quail [CG], gray partridge [PP], ringed-neck pheasant [PC], red jungle fowl [GG], and guinea fowl [NM]), QT6 cells representing the Japanese quail (JQ), and embryo fibroblasts of the domestic duck (AP). Results are shown as means ± standard deviations of duplicate assays. (C) Efficiency of RCASBP(JS11C1)GFP and RCASBP(A)GFP infection in cultured embryo fibroblasts of five inbred chicken lines. Results are shown as means ± standard deviations of duplicate assays.

FIG 3

Expression of the tva gene is sufficient and necessary for RCASBP(JS11C1)GFP infection. (A) Efficiency of RCASBP(JS11C1)GFP and RCASBP(A)GFP infection in the Syrian hamster cell line NIL-2 ectopically expressing the tva gene (NIL-Tva). DF-1 cells were used as a susceptible control. Viruses with B and C specificity, i.e., RCASBP(B)GFP and RCASBP(C)GFP, respectively, and NIL-2 cells ectopically expressing the tvc gene were used as independent controls. Infection efficiencies are shown as percentages of GFP-positive cells and are presented as means ± standard deviations of triplicate assays. (B) Efficiency of RCASBP(JS11C1)GFP infection in intact DF-1 cells (left) and DF-1 cells with a frameshift mutation in the tva gene (right), presented as FACS histograms of GFP-negative and GFP-positive cells. The relative GFP fluorescence is plotted against the cell count, and the percentage of GFP-positive cells is indicated. Typical results from triplicate assays are shown.

FIG 4

Superinfection interference between ALV-A and ALV-K subgroups. (A) Results of interference experiments shown as FACS dot plots. Preinfection of DF-1 cells with RCASBP(A)GFP blocks superinfection with RCASBP(JS11C1)dsRed (left), whereas preinfection with RCASBP(B)GFP (middle) or RCASBP(J)GFP (right) permits subsequent superinfection with RCASBP(JS11C1)dsRed. Horizontal axes, dsRed positivity; vertical axes, GFP positivity. (B) Efficiency of superinfection interference between RCASBP(A)GFP, RCASBP(B)GFP, and RCASBP(J)GFP as preinfection viruses and RCASBP(A)dsRed and RCASBP(JS11C1)dsRed as superinfection viruses. The results are shown as mean percentages of GFP-negative/dsRed-negative, GFP-positive/dsRed-negative, GFP-negative/dsRed-positive, and GFP-positive/dsRed-positive cells, measured in triplicate.

FIG 5

Soluble Tva interference with ALV-A and ALV-K infection in susceptible cells. RCASBP(A)GFP and RCASBP(JS11C1)GFP viruses were preincubated (filled columns) or mock-incubated (empty columns) with sTva-mIgG immunoadhesin and used for infection of DF-1 cells. Infection efficiencies are shown as means ± standard deviations of percentages of GFP-positive cells; the experiment was performed in triplicate.