Human RPL7 and DDX21 interact with HTLV-1 Gag and enhance tRNAPro primer annealing to genomic RNA

Human T-cell leukemia virus type 1 (HTLV-1), an oncogenic retrovirus, uses human tRNA Pro to prime reverse transcription (RT). However, how tRNA Pro is annealed to the primer binding site (PBS), which is embedded in a highly structured hairpin in the genomic RNA (gRNA), remains unclear. We hypothesize that HTLV-1 Gag may have more robust chaperone activity than mature HTLV-1 nucleocapsid (NC), which in contrast to HIV-1 NC, displays relatively weak chaperone function, and that a cellular co-factor may be required to facilitate primer tRNA annealing. Recombinant HTLV-1 Gag was successfully purified for the first time and used to perform primer-annealing assays. Relative to mature NC and matrix (MA) domains, HTLV-1 Gag is only slightly more effective at chaperoning the annealing of tRNA Pro to the PBS. To identify potential HTLV-1 Gag interacting co-chaperones of tRNA annealing in cells, we performed affinity tagging/purification-mass spectrometry (AP-MS). Two significant AP-MS hits, RPL7 and DDX21, were further validated by reciprocal co-IP studies in both HEK293T and chronically HTLV-1-infected MT-2 cells. Domain mapping studies revealed that HTLV-1 Gag interacts with RPL7 and DDX21 through the zinc fingers in the NC domain independent of the presence of RNA. In addition, we showed that both RPL7 and DDX21 are packaged into virions. RPL7 or DDX21 alone was more effective than HTLV-1 Gag at annealing tRNA Pro to the PBS. Synergistic effects of the Gag/RPL7/DDX21 combination in facilitating tRNA Pro annealing to the PBS were found. Taken together, the mechanistic insights gained from these studies could be exploited for the development of new therapeutic strategies aimed at targeting HTLV-1 RT. Highlights Mechanism for HTLV-1 reverse transcription primer annealing to the primer binding site in viral RNA is proposed. Recombinant HTLV-1 Gag was purified successfully from E. coli for the first time. HTLV-1 Gag interacts directly with human RPL7 and DDX21 in cells. RPL7 and DDX21 enhance HTLV-1 Gag’s tRNA Pro primer annealing activity. tRNA Pro annealing activity: DDX21 + RPL7 + Gag > DDX21 + RPL7 > DDX21 > RPL7 > Gag.