Residues E124, K129, and R134 in eEF1A Domain I Promote ALV‑J Replication via direct interaction 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 a direct interaction 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 eEF1A-RT interaction. Alanine substitution at these EKR residues disrupted eEF1A-RT binding, 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 interaction and viral replication. Notably, this eEF1A-RT interaction and its functional significance are conserved across ALV subgroups A, B, K, and J. Together, our work elucidate that three residues E124, K129, and R134 within domain I of eEF1A mediate the direct interaction 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, broaden the understanding of how retroviruses co‑opt host factors to facilitate this critical process, and provide a rational basis for genome editing of eEF1A to generate broad‑spectrum ALV‑resistant poultry lines.