Gp37 Regulates the Pathogenesis of Avian Leukosis Virus Subgroup J via Its C Terminus

Abstract

Different from other subgroups of avian leukosis viruses (ALVs), ALV-J is highly pathogenic. It is the main culprit causing myeloid leukemia and hemangioma in chickens. The distinctiveness of the env gene of ALV-J, with low homology to those of other ALVs, is linked to its unique pathogenesis, but the underlying mechanism remains unclear. Previous studies show that env of ALV-J can be grouped into three species based on the tyrosine motifs in the cytoplasmic domain (CTD) of Gp37, i.e., the inhibitory, bifunctional, and active groups. To explore whether the C terminus or the tyrosine motifs in the CTD of Gp37 affect the pathogenicity of ALV-J, a set of ALV-J infectious clones containing different C termini of Gp37 or the mutants at the tyrosine sites were tested in vitro and in vivo Viral growth kinetics indicated not only that ALV-J with active env is the fastest in replication and ALV-J with inhibitory env is the lowest but also that the tyrosine sites essentially affected the replication of ALV-J. Moreover, in vivo studies demonstrated that chickens infected by ALV-J with active or bifunctional env showed higher viremia, cloacal viral shedding, and viral tissue load than those infected by ALV-J with inhibitory env Notably, the chickens infected by ALV-J with active or bifunctional env showed significant loss of body weight compared with the control chickens. Taken together, these findings reveal that the C terminus of Gp37 plays a vital role in ALV-J pathogenesis, and change from inhibitory env to bifunctional or active env increases the pathogenesis of ALV-J.IMPORTANCE ALV-J can cause severe immunosuppression and myeloid leukemia in infected chickens. However, no vaccine or antiviral drug is available against ALV-J, and the mechanism for ALV-J pathogenesis needs to be elucidated. It is generally believed that gp85 and LTR of ALV contribute to its pathogenesis. Here, we found that the C terminus and the tyrosine motifs (YxxM, ITIM, and ITAM-like) in the CTD of Gp37 of ALV-J could affect the pathogenicity of ALV-J in vitro and in vivo The pathogenicity of ALV-J with Gp37 containing ITIM only was significantly less than ALV-J with Gp37 containing both YxxM and ITIM and ALV-J with Gp37 containing both YxxM and ITAM-like. This study highlights the vital role of the C terminus of Gp37 in the pathogenesis of ALV-J and thus provides a new perspective to elucidate the interaction between ALV-J and its host and a molecular basis to develop efficient strategies against ALV-J.

Keywords: C terminus; Gp37; avian leukosis virus; gp37; pathogenesis; tyrosine motif; tyrosine motifs.

FIG 1

Strategy for construction of the recombinant ALV-J with different gp37 genes. (A) The original backbone infectious clone of ALV-J J1 is shown. The other infectious clones, EAV-HP, 4817, NY-EAV-HP, YF-EAV-HP, YF-J1, YN-J1, YN-4817, 2YF-4817, and NFF-4817, were cloned by recombination through replacing the C terminus of Gp37 (aa 74 to 197, J1) or mutating the tyrosine sites. Sequences which are identical are shown in the same color. (B) Sequence alignment for the Gp37 sequences of the ALV-J infectious clones used in this study. The sequences in green, purple, and light blue frames are the C terminus of Gp37 in this study and the YxxM motif and ITAM-like motif in the CTD of Gp37, respectively. The sequence with red line below it is ITIM.

FIG 2

Rescue of the recombinant ALV-J expressing different gp37 genes. DF-1 cells were transfected with the indicated ALV-J infectious clones for 7 days, and the virus-containing supernatants were blindly passaged in DF-1 cells. DF-1 cells were infected with the rescued EAV-HP (A), NY-EAV-HP (B), YF-EAV-HP (C), 4817 (D), YN-4817 (E), 2YF-4817 (F), NFF-4817 (G), J1 (H), YF-J1 (I), and YN-J1 (J) viruses, and the uninfected DF-1 cells were set as control (K). These infected cells were identified by IFA using JE9 MAb against ALV-J Gp85.

FIG 3

Growth curves of the recombinant ALV-J in DF-1 cells. The rescued EAV-HP, 4817, J1, NY-EAV-HP, YF-EAV-HP, YF-J1, YN-J1, YN-4817, 2YF-4817, and NFF-4817 viruses were inoculated into DF-1 cells at an MOI of 0.01, and the virus-containing supernatants were collected at the indicated time points for virus titration. (A) Growth curves for EAV-HP, NY-EAV-HP, and YF-EAV-HP viruses in DF-1 cells. (B) Growth curves for 4817, YN-4817, 2YF-4817, and NFF-4817 viruses in DF-1 cells. (C) Growth curves for J1, YF-J1, and YN-J1 viruses in DF-1 cells.

FIG 4

Viral cloacal shedding and viral tissue load in infected chickens. The blue, red, green, and yellow spots or columns represent the EAV-HP, 4817, J1, and control groups, respectively. (A and B) Comparison of viral P27 antigen levels (A) or viral positive rate (B) of the cloacal swabs assayed by ELISA from chickens infected with EAV-HP, 4817, and J1 viruses. (C to E) Comparison of viral loads in organs from chickens infected with EAV-HP, 4817, and J1 at 7, 21, and 35 dpi, respectively. The data were analyzed with a Student t test. A P value of <0.05 was considered significant. LOD, limit of detection.

FIG 5

Body weight loss, viremia, and antibody against ALV in infected chickens. The blue, red, green, and yellow columns or curves represent the EAV-HP, 4817, J1, and control groups, respectively. (A) Comparison of body weights measured at different time points. (B) Comparison of the viral positive rates in plasma from chickens infected with EAV-HP, 4817, and J1 viruses. (C) Comparison of positive rates for antibody against ALV from chickens infected with EAV-HP, 4817, and J1 viruses, determined by IFA. (D) Comparison of levels of antibody against ALV P27 from chickens infected with EAV-HP, 4817, and J1 viruses, determined by ELISA. Statistical analysis was performed with one-way ANOVA using GraphPad 5 software. A P value of <0.05 was considered significant. *, **, and *** indicate P values of less than 0.05, 0.01, and 0.001, respectively.