The key amino acid sites 199-205, 269, 319, 321 and 324 of ALV-K env contribute to the weaker replication capacity of ALV-K than ALV-A

Abstract

Background: Avian leukosis virus (ALV) is an infectious retrovirus, that mainly causes various forms of tumours, immunosuppression, a decreased egg production rate and slow weight gain in poultry. ALV consists of 11 subgroups, A-K, among which ALV-K is an emerging subgroup that has become prevalent in the past 10 years. Most ALV-K isolates showed weak replication ability and pathogenicity. In this study, the weak replication ability of ALV-K was explored from the perspective of the interaction between ALV-K gp85 and the Tva receptor.

Methods: Fourteen soluble recombinant ALV-A/K gp85 chimeric proteins were constructed by substituting the sequence difference regions (hr1, hr2 and vr3) of the ALV-A gp85 protein with the skeleton ALV-K gp85 protein for co-IP and competitive blocking tests.

Results: The binding capacity of ALV-K gp85 to Tva was significantly weaker than that of ALV-A gp85 (P < 0.05) and the key amino acid sites 199-205, 269, 319, 321 and 324 of ALV-K env contributed to the weaker replication capacity of ALV-K than ALV-A.

Conclusions: This is the first study to reveal the molecular factors of the weak replication ability of ALV-K from the perspective of the interaction of ALV-K gp85 to Tva, providing a basis for further elucidation of the infection mechanism of ALV-K.

Keywords: Avian leukosis virus K subgroup; Binding capacity; Env; Recombinant chimaeras; Tva receptor.

Fig. 1

Comparison of replication capacity between RCASBP(A)-EGFP and RCASBP(K)-EGFP. a The replication capacity of RCASBP(A)-EGFP and RCASBP(K)-EGFP was observed by fluorescence microscopy. b The percentages of GFP-positive DF-1 cells transfected with RCASBP(A)-EGFP and RCASBP(K)-EGFP were determined by flow cytometry. Three independent experiments were performed, and the data are shown as the mean ± SD of triplicate samples from a representative experiment. Statistical analysis (two-way analysis of variance) was performed using GraphPad Prism 7; ****P < 0.0001

Fig. 2

Comparison of replication competitive advantages between ALV-A and ALV-K based on RCASBP. a, c The replication capacities of RCASBP(A) and RCASBP(K) were observed by fluorescence microscopy. b, d The percentages of GFP/mCherry-positive DF-1 cells transfected with RCASBP(A)-EGFP and RCASBP(K)-EGFP were determined by flow cytometry. Three independent experiments were performed, and the data are shown as the means of triplicate samples

Fig. 3

Analysis of the binding of the ALV-A and ALV-K gp85 proteins to the Tva receptor. a, b The co-IP test results and grey value analysis results for the ALV-A and ALV-K gp85 protein interactions with the Tva receptor. c Evaluation of the competitive blocking effect of the ALV-A and ALV-K gp85 proteins on RCASBP(A)-EGFP infection. Three independent experiments were performed, and the data are shown as the mean ± SD of triplicate samples from a representative experiment. Statistical analysis (two-way analysis of variance) was performed using GraphPad Prism 7; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

Analysis of the binding of a recombinant chimeric ALV-A/K gp85 protein for the Tva receptor. a Homology comparison between ALV-A RSA and ALV-K GDFX0602 gp85 protein sequences; b technology roadmap of co-IP and competitive blocking tests; c, d binding of recombinant chimeric fragments s1–s5 of the Agp85 protein to the Tva receptor and relative grey values; e, f binding of recombinant chimeric fragments s1–s5 of the Agp85 protein to the Tva receptor and relative grey values; g Competitive blocking of a recombinant chimeric ALV-A/K gp85 protein. Three independent experiments were performed, and the data are shown as the mean ± SD of triplicate samples from a representative experiment. Statistical analysis (two-way analysis of variance) was performed using GraphPad Prism 7; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Analysis of the binding of recombinant gp85 s3 protein point mutants for the Tva receptor. a, b Co-IP test results and grey value analysis results for the interactions between soluble recombinant chimeric gp85 proteins and the Tva receptor; c evaluation of the efficiencies of soluble recombinant chimeric gp85 proteins in competitively blocking RCASBP(A)-EGFP infection. Three independent experiments were performed, and the data are shown as the mean ± SD of triplicate samples from a representative experiment. Statistical analysis (two-way analysis of variance) was performed using GraphPad Prism 7; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

Analysis of the binding of recombinant gp85 s8 protein point mutants for the Tva receptor. a, b Co-IP test results and grey value analysis results for the interactions between soluble recombinant chimeric gp85 proteins and the Tva receptor; c evaluation of the efficiency of soluble recombinant chimeric gp85 proteins in competitively blocking RCASBP(A)-EGFP infection. Three independent experiments were performed, and the data are shown as the mean ± SD of triplicate samples from a representative experiment. Statistical analysis (two-way analysis of variance) was performed using GraphPad Prism 7; *P < 0.05, ****P < 0.0001, ns P > 0.05

Fig. 7

Analysis of the binding of recombinant gp85 vr3 protein point mutants for the Tva receptor. a, b Results of the co-IP test and grey value analysis for the interactions between soluble recombinant chimeric gp85 proteins and the Tva receptor; c evaluation of the efficiencies of soluble recombinant chimeric gp85 proteins in competitively blocking RCASBP(A)-EGFP infection. Three independent experiments were performed, and the data are shown as the mean ± SD of triplicate samples from a representative experiment. Statistical analysis (two-way analysis of variance) was performed using GraphPad Prism 7. Statistical analysis (two-way analysis of variance) was performed using GraphPad Prism 7; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 8

The percentages of GFP positive DF-1 cells fot the point mutants RCASBP(A)-EGFP G199S, V200I, P201D, W202T, Y203L, L204S, G205D, R274V, G324P, I326T and 328–329insP were analysed by flow cytometry

Fig. 9

Recovery test of ALV-K gp85 protein point mutants. a, b Results of the co-IP test and grey value analysis of the interactions between soluble recombinant chimeric gp85 vr3 proteins and the Tva receptor. c, d Results of the co-IP test and grey value analysis of the interactions between restored soluble recombinant chimeric gp85 protein point mutants and the Tva receptor; e the percentages of GFP-positive cells for point mutation recovery mutants were analysed by flow cytometry. Three independent experiments were performed, and the data are shown as the mean ± SD of triplicate samples from a representative experiment. Statistical analysis (two-way analysis of variance) was performed using GraphPad Prism 7; *P < 0.05, **P < 0.01