Soluble Forms of the Subgroup A Avian Leukosis Virus [ALV(A)] Receptor Tva Significantly Inhibit ALV(A) Infection In Vitro and In Vivo

Abstract

The interactions between the subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and soluble forms of the ALV(A) receptor Tva were analyzed both in vitro and in vivo by quantitating the ability of the soluble Tva proteins to inhibit ALV(A) entry into susceptible cells. Two soluble Tva proteins were tested: the 83-amino-acid Tva extracellular region fused to two epitope tags (sTva) or fused to the constant region of the mouse immunoglobulin G heavy chain (sTva-mIgG). Replication-competent ALV-based retroviral vectors with subgroup B or C env were used to deliver and express the two soluble tv-a (stva) genes in avian cells. In vitro, chicken embryo fibroblasts or DF-1 cells expressing sTva or sTva-mIgG proteins were much more resistant to infection by ALV(A) (∼200-fold) than were control cells infected by only the vector. The antiviral effect was specific for ALV(A), which is consistent with a receptor interference mechanism. The antiviral effect of sTva-mIgG was positively correlated with the amount of sTva-mIgG protein. In vivo, the stva genes were delivered and expressed in line 0 chicken embryos by the ALV(B)-based vector RCASBP(B). Viremic chickens expressed relatively high levels ofstva and stva-mIgG RNA in a broad range of tissues. High levels of sTva-mIgG protein were detected in the sera of chickens infected with RCASBP(B)stva-mIgG. Viremic chickens infected with RCASBP(B) alone, RCASBP(B)stva, or RCASBP(B)stva-mIgG were challenged separately with ALV(A) and ALV(C). Both sTva and sTva-mIgG significantly inhibited infection by ALV(A) (95 and 100% respectively) but had no measurable effect on ALV(C) infection. The results of this study indicate that a soluble receptor can effectively block infection of at least some retroviruses and demonstrates the utility of the ALV experimental system in characterizing the mechanism(s) of viral entry.

Identification of Key Residues in Subgroup A Avian Leukosis Virus Envelope Determining Receptor Binding Affinity and Infectivity of Cells Expressing Chicken or Quail Tva Receptor

“…Vector constructions. The construction of the quail stva-mIgG gene in the CLA12NCO adapter plasmid and the RCASBP(C)stva-mIgG retrovirus has been described previously (38). The stva-mIgG gene encodes the signal peptide and 83-amino-acid extracellular domain of the quail pg950 Tva cDNA, fused to the constant region of the mouse IgG heavy chain.…”

“…A soluble form of the chicken Tva receptor, encoding the leader and first six amino acids of quail Tva fused to the extracellular region of chicken Tva (residues 7 to 83), was described previously (16). The chicken stva-mIgG (ckstva-mIgG) gene was constructed by replacing the region encoding the quail sTva domain with the region encoding the chicken sTva domain, isolated as a NcoI-EagI fragment from pLC126, into the NcoI and EagI sites of CLA12NCO/stva-mIgG (38). The ckstva-mIgG gene cassette was isolated as a ClaI fragment and subcloned into the ClaI site of RCASBP(C).…”

“…The ALV CA protein was detected in culture supernatants by ELISA as previously described (59). The levels of sTva-mIgG and cksTva-mIgG were determined in culture supernatants by ELISA for the mouse IgG tag as previously described (38). The linear range for a standard experiment was between 0.5 and 50 ng of ImmunoPure mouse IgG Fc fragment per ml.…”

“…In a preadsorption AP challenge assay, the 10-fold viral serial dilutions were first mixed for 3 h at 4°C with 2 ml of supernatant containing quail sTva-mIgG and then assayed as above. The assay for AP activity was described previously (38).…”

“…Similarly, a soluble form of the 83-amino-acid extracellular domain of the quail Tva receptor, produced and purified with a baculovirus expression system, efficiently bound ALV(A) envelope glycoproteins and specifically blocked ALV(A) infection (5). We recently showed that soluble forms of the chicken and quail Tva receptors, expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells (ϳ200-fold antiviral effect) and chickens (Ͼ98% of the birds were not infected) (38). The antiviral effect was specific for ALV(A), consistent with a receptor interference mechanism.…”