Single-Molecule Studies of HIV-1 Gag Assembly

Abstract

Gag is the key structural protein that mediates assembly of nascent HIV-1 virions around viral genomic RNA. Previous studies have established that Gag can spontaneously assemble around a variety of nucleic acid substrates into immature virus-like particles in vitro. Additionally, substrates which include the Psi signal, a small structural element found in the 5’ UTR of the HIV-1 genome, are selectively packaged over other substrates. In fact, the Psi signal is required for selective packaging of the genomic RNA over the large background of other RNAs in the cellular milieu. However, the mechanism of this assembly process and interactions with the Psi signal that lead to specificity are poorly understood. Here we have developed a single-molecule total internal reflection fluorescence microscopy assay to monitor the earliest steps of particle assembly under controlled conditions. Individual fluorescently-labeled Gag monomers are resolved as they spontaneously bind to and dissociate from immobilized RNA substrates derived from the genomic 5’ UTR. Statistical analysis of multiple single-molecule time trajectories allowed for the determination of the relative populations of various assembly intermediates along with rate constants for individual binding and dissociation steps. Experiments were performed under a variety of conditions including varied protein concentrations, mutations to Gag and the RNA substrates, and with proposed small-molecule cofactors present. Taken together, the results from these experiments elucidate details of molecular interactions involved in the initiation of particle assembly that may be opaque to other methodologies.