Human RPL7 and DDX21 function synergistically with HTLV-1 Gag to overcome genomic RNA structural barriers that block tRNAPro primer annealing.

Human T-cell leukemia virus type 1 (HTLV-1), an oncogenic retrovirus, uses human tRNAPro to prime reverse transcription. How tRNAPro is annealed to the primer-binding site (PBS), which is embedded in a stable hairpin structure in the genomic RNA, remains unclear. In contrast to human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein, which robustly chaperones transfer RNA (tRNA) annealing to the HIV-1 PBS, HTLV-1 NC protein displays very weak chaperone function. Recombinantly-purified HTLV-1 Gag was only slightly more effective at chaperoning the annealing of tRNAPro to the PBS than NC protein. To identify potential HTLV-1 Gag interacting co-chaperones in cells, we performed affinity tagging/purification-mass spectrometry. Two significant hits, ribosomal protein L7 (RPL7) and DDX21, were validated by reciprocal co-IP studies in cells. Domain mapping revealed that HTLV-1 Gag interacts with RPL7 and DDX21 through the zinc fingers of NC protein in an RNA-independent fashion. Both RPL7 and DDX21 are packaged into virions, and each protein alone was more effective than HTLV-1 Gag at annealing tRNAPro to the PBS. Further synergistic effects were observed for the Gag/RPL7/DDX21 combination in overcoming structural constraints at the PBS to promote tRNAPro annealing. The mechanistic insights gained from these studies may be exploited for the development of new therapeutic strategies aimed at targeting HTLV-1 RT.