Residues E124, K129, and R134 in eEF1A domain I promote ALV-J replication via association with reverse transcriptase

Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus posing significant threats to the global poultry industry. While host factors are known to regulate retroviral reverse transcription, the specific host proteins that support this critical step in ALV infection have remained completely unknown. In this study, we confirmed that overexpression or knockdown of eEF1A significantly enhanced or suppressed ALV-J proliferation, respectively. Co-immunoprecipitation and molecular docking demonstrated an association between eEF1A and the ALV-J reverse transcriptase (RT). Furthermore, three conserved residues in eEF1A domain I, including glutamate 124 (E124), lysine 129 (K129), and arginine 134 (R134), were identified as critical for the eEF1A-RT association. Alanine substitution at these EKR residues disrupted the eEF1A-RT association, markedly impairing viral reverse transcription and infectious virion production. From the viral perspective, a conserved tryptophan (W250) in the RT thumb domain acts as the key counterpart to the eEF1A EKR motif, and its mutation similarly abolishes the association and viral replication. Notably, this eEF1A-RT association and its functional significance are conserved across ALV subgroups A, B, K, and J. Together, our work elucidates that three residues E124, K129, and R134 within domain I of eEF1A mediate the association with ALV-J RT, promoting viral replication by enhancing reverse transcription efficiency. This finding provides the first mechanistic insight into host-dependent regulation of reverse transcription in ALV infection, broadens the understanding of how retroviruses co-opt host factors to facilitate this critical process, and provides a rational basis for genome editing of eEF1A to generate broad-spectrum ALV-resistant poultry lines. IMPORTANCE Avian leukosis viruses (ALVs) cause severe economic losses to the global poultry industry, and current disease-resistant strategies targeting viral entry receptors only work for specific subgroups. This study identifies that the host protein eEF1A associates with ALV reverse transcriptase via conserved key residues to promote viral reverse transcription and replication, and this regulatory mechanism is conserved across multiple ALV subgroups. These findings provide a novel target for developing broad-spectrum ALV-resistant poultry breeds through genome editing, with important implications for poultry disease control.