Real-Time Imaging of HIV-1 Protease Activation and Substrate Cleavage in Single Virions Assembling on the Plasma Membrane.

The timing of HIV-1 protease (PR) activation and the determinants of orderly Gag and Gag-Pol polyprotein cleavage, leading to structural maturation of virions, are not well-understood. Here, we employed total internal reflection microscopy to visualize PR activation and cleavage of a FRET-based fluorescent substrate in single HIV-1 particles assembled on the cell's ventral membrane. PR activity manifested as a reduction in FRET signal in a relatively small fraction of nascent viral particles. By contrast, most virions released from cells contained a processed FRET substrate, suggesting that PR activation may be delayed or suppressed in most virions assembled at the ventral membrane. A combination of single-particle tracking and FRET measurements detected PR activity, on average, within ~20 min after the onset of particle assembly, while cleavage of a FRET substrate in single virions was completed within a few minutes. Importantly, substrates containing distinct PR cleavage sequences derived from Gag and Gag-Pol polyproteins were processed with different rates and efficiency. Our findings validate the use of surrogate FRET substrates to assess the timing of PR activation in single virions and elucidate the determinants of the tightly orchestrated order of Gag and Gag-Pol cleavage required for the formation of infectious virions.