Avian Sarcoma and Leukosis Virus Envelope Glycoproteins Evolve to Broaden Receptor Usage Under Pressure from Entry Competitors †

Abstract

The subgroup A through E avian sarcoma and leukosis viruses (ASLV(A) through ASLV(E)) are a group of highly related alpharetroviruses that have evolved their envelope glycoproteins to use different receptors to enable efficient virus entry due to host resistance and/or to expand host range. Previously, we demonstrated that ASLV(A) in the presence of a competitor to the subgroup A Tva receptor, SUA-rIgG immunoadhesin, evolved to use other receptor options. The selected mutant virus, RCASBP(A)Δ155-160, modestly expanded its use of the Tvb and Tvc receptors and possibly other cell surface proteins while maintaining the binding affinity to Tva. In this study, we further evolved the Δ155-160 virus with the genetic selection pressure of a soluble form of the Tva receptor that should force the loss of Tva binding affinity in the presence of the Δ155-160 mutation. Viable ASLVs were selected that acquired additional mutations in the Δ155-160 Env hypervariable regions that significantly broadened receptor usage to include Tvb and Tvc as well as retaining the use of Tva as a receptor determined by receptor interference assays. A similar deletion in the hr1 hypervariable region of the subgroup C ASLV glycoproteins evolved to broaden receptor usage when selected on Tvc-negative cells.

Keywords: genetic selection inhibiting entry; receptor use expansion; the subgroup A through E avian sarcoma and leukosis viruses.

Figure 1

Comparison of the subgroup A and subgroup C avian sarcoma and leukosis viruses (ASLV) envelope glycoprotein hr1, hr2, and vr3 hypervariable regions. The protein sequence alignments were done using the ClustalW program in MacVector 14.5.3; gaps in the alignment are denoted by (−). The RCASBP(A) vector contains the envelope glycoprotein hypervariable regions from the Schmidt-Ruppin A subgroup A ASLV strain (SR-A Env WT) UniProt P03397; the RCASBP(C) vector contains the envelope glycoprotein hypervariable regions from the Prague-C subgroup C ASLV (Prague C Env WT) Genbank AAB59934.1. The RCASBP(A) Δ155–160 six residue deletion is highlighted in red.

Figure 2

Evolution of RCASBP(A) Δ155–160 virus variants resistant to cksTva-mIgG. (A) TF/cksTva-19 cells expressing high levels of chicken sTva-mIgG protein were infected with DF-1 infection supernatant (10 mL) containing RCASBP(A) Δ155–160 virus (~10⁵ ifu/mL) either neat (Δ155–160) or after preincubation with cksTva-mIgG containing supernatant (Δ155–160+sTva1), or supernatant (1.0 mL) containing the subgroup B ASLV, RCASBP(B), and the infected cell cultures passaged to allow virus replication and spread. Viral growth was monitored by ELISA for the ASLV CA protein. The day the Δ155–160 infected culture began a transient period of ASLV induced cytotoxicity is marked with an asterisk (*). (B) The selected virus pools (1.0 mL) from the day 32 supernatants above were re-passaged in TF/cksTva-19 cells either neat (Δ155–160+sTva1), the selected supernatant preincubated with cksTva-mIgG supernatant (Δ155–160+sTva2), and non-selected wild-type RCASBP(A) Δ155–160 as a control, and the infected cell cultures passaged to allow virus replication and spread. (C) The ASLV(A) env nucleotide sequences were determined from clones generated from PCR amplified ASLV(A) env sequences from DNA isolated from infected cells. Rd: Round.

Figure 3

Virus replication rates of Δ155–160 and the evolved Δ155–160 mutant viruses. Viral growth was monitored by ELISA for the ASLV CA protein. Plasmids encoding the ASLVs were transfected into parental DF-1 cells (A) and TF/cksTva-19 cells (B), and the transfected cell cultures passaged to allow virus replication. (C) TF/cksTva-19 cells were infected with Δ155–160 and Δ155–160 mutant viruses at a multiplicity of infection of 0.01. The infected cell cultures were passaged to allow virus replication.

Figure 4

Analysis of receptor usage of the parental Δ155–160 and Δ155–160 mutant viruses. Infectious titers were determined using 10-fold serial dilutions of wild-type RCASBP(A), RCASBP(B), and RCASBP(C) viruses, the parental Δ155–160 virus, and the Δ155–160 mutant virus supernatants produced using DF-1 cells. The infectious titer was determined by the AP assay. No infectious units detected are denoted with (∗). The results shown are an average of three different experiments; error bars show standard deviations. (A) ASLV receptor interference patterns of the ASLVs infecting parental DF-1 cells, and DF-1 cells chronically infected with ASLV(A), ASLV(B), ASLV(C), or subgroup J ASLV, HPRS103. (B) The infectious titers of the ASLV viruses were determined using Line C CEFs that do not express a functional Tva receptor. (C) The infectious titers of the ASLV viruses were determined using Line 15I5 CEFs that do not express a functional Tvc receptor.

Figure 5

Analysis of the binding affinities and antiviral effects of receptor immunoadhesins on the evolved RCASBP(A) Δ155–160 virus variants. DF-1 cells infected with either control and variant ASLVs were fixed and incubated with different amounts of either the CKsTva-mIgG (A), QsTva-mIgG (C), or sTvb-mIgG (E) soluble ASLV receptor immunoadhesins and the envelope glycoprotein:soluble receptor complexes bound to sheep anti-mouse Ig antibody linked to fluorescein. The levels of fluorescein bound to the infected cells were quantitated by fluorescence-activated cell sorting (FACS) and the mean level of fluorescence was plotted as a function of soluble ASLV receptor concentration. Representative experiments are shown. Infectious titers were determined using 10-fold serial dilutions of control and variant ASLVs supernatants directly on DF-1 cells, or after each dilution was preabsorbed with a set amount of CKsTva-mIgG (B), QsTva-mIgG (D), or sTvb-mIgG (F) to allow the soluble receptor time to bind and inhibit infection of DF-1 cells. The infectious titer was determined by the AP assay.

Figure 6

Genetic selection of RCASBP(C) mutants capable of efficient replication using Line 15I5 CEFs that lack a functional Tvc receptor. (A) Chicken embryo fibroblasts (CEFs) derived from Line 15I5 were infected with RCASBP(C) supernatant at 0.003, 0.03, and 0.3 M.O.I., or the positive control RCASBP(A) supernatant (0.1 mL), produced using DF-1 cells. The infected cells were passaged to allow virus replication. Viral growth was monitored by ELISA for the ASLV CA protein. (B) Supernatant (1.0 mL) harvested from the RCASBP(C) 0.3 M.O.I. infected culture on day 37, and supernatant (0.1 mL) harvested from day 37 RCASBP(A) culture, were used to infect fresh Line 15I5 CEFs in a second round of genetic selection. (C) In a third round of genetic selection, second-round day-25 supernatants from RCASBP(C) 0.3 M.O.I. and RCASBP(A) cultures were again used to infect fresh Line 15I5 CEFs; unselected RCASBP(C) virus was used as a “negative” control. (D) Cells were harvested from Rd 3 RCASBP(C) 0.3 M.O.I. infected culture, and clones were generated from PCR amplified ASLV(A) env sequences from isolated DNA from infected cells, and the ASLV(A) env nucleotide sequences determined. (E) ASLV receptor interference patterns of the ASLVs infecting parental DF-1 cells, and DF-1 cells chronically infected with ASLV(A), ASLV(B), ASLV(C), or subgroup J ASLV, HPRS103, were determined as described in Figure 4 legend. Rd: Round.

Figure 7

Comparison of the subgroup A and subgroup C ASLV envelope glycoprotein hr1, hr2, and vr3 hypervariable regions. The protein sequence alignments were done using the ClustalW program in MacVector 14.5.3; gaps in the alignment are denoted by (-). The RCASBP(A) vector contains the envelope glycoprotein hypervariable regions from the Schmidt-Ruppin A subgroup A ASLV strain (SR-A Env WT) UniProt P03397; the RCASBP(C) vector contains the envelope glycoprotein hypervariable regions from the Prague-C subgroup C ASLV (Prague C Env WT) Genbank AAB59934.1. The mutations selected and characterized in this study are highlighted in orange; the original RCASBP(A) Δ155–160 mutation is highlighted in red. The results from this study are compared to the ASLV(B) hr1, hr2, and vr3 regions of Rous associated virus-2 (RAV-2) Genbank AAA87241. Selection of a mutant RAV-2 that was able to infect avian cells that lacked a Tvb receptor had a deletion in hr1 residues 136–142 (Δ136–142) highlighted in green [32].