Single-molecule imaging of HIV-1 RNA translation reveals rapid kinetics and nucleocapsid-dependent efficiency

The HIV-1 full-length RNA (FL-RNA) serves both as the viral genome and as the messenger RNA encoding the Gag and Gag-Pol polyproteins. Regulation of FL-RNA translation is therefore central to HIV-1 gene expression and replication. The nucleocapsid domain of Gag (NC) is a multifunctional RNA-binding protein best known for its role in genomic RNA packaging, yet its potential contribution to viral RNA translation remains poorly understood. Here, we used single-molecule imaging to visualize translation of HIV-1 FL-RNA in living cells. We engineered an HIV-1 molecular clone containing SunTag repeats in Gag and MS2 stem-loops in the viral RNA and analyzed mutants of the NC domain impaired in RNA binding. We found that only a subset of cytoplasmic FL-RNA molecules is actively translated under our experimental conditions, and that NC deletion reduced this fraction. Live-cell imaging revealed rapid elongation kinetics (~24 amino acids per second) and heterogeneous polysomes containing multiple ribosomes per viral RNA. Deletion of the NC zinc-fingers or of the entire NC domain markedly reduced twice the ribosome loading onto individual RNAs without affecting elongation speed but decreasing initiation time. Together, these results provide a quantitative view of HIV-1 FL-RNA translation dynamics in living cells and identify the nucleocapsid domain as a key determinant of efficient ribosome loading on the viral genome.