Imaging the interaction of HIV-1 genomes and Gag during assembly of individual viral particles

Abstract

The incorporation of viral genomes into particles has never previously been imaged in live infected cells. Thus, for many viruses it is unknown how the recruitment and packaging of genomes into virions is temporally and spatially related to particle assembly. Here, we devised approaches to simultaneously image HIV-1 genomes, as well as the major HIV-1 structural protein, Gag, to reveal their dynamics and functional interactions during the assembly of individual viral particles. In the absence of Gag, HIV-1 RNA was highly dynamic, moving in and out of the proximity of the plasma membrane. Conversely, in the presence of Gag, RNA molecules docked at the membrane where their lateral movement slowed and then ceased as Gag assembled around them and they became irreversibly anchored. Viral genomes were not retained at the membrane when their packaging signals were mutated, nor when expressed with a Gag mutant that was not myristoylated. In the presence of a Gag mutant that retained membrane-and RNAbinding activities but could not assemble into particles, the viral RNA docked at the membrane but continued to drift laterally and then often dissociated from the membrane. These results, which provide visualization of the recruitment and packaging of genomes into individual virus particles, demonstrate that a small number of Gag molecules recruit viral genomes to the plasma membrane where they nucleate the assembly of complete virions.

Feline Immunodeficiency Virus Gag Is a Nuclear Shuttling Protein

“…A central question is whether Gag and the viral genomic RNA (gRNA) associate at the plasma membrane or earlier during assembly and, if so, whether this occurs in the cytosol; in association with organelles, e.g., cotranslationally; or even in the nucleus, as was described previously for an alpharetrovirus (17). Human immunodeficiency virus type 1 (HIV-1) gRNAs become anchored at the plasma membrane before particle assembly is detectable, but it is not clear whether the Gag-gRNA complexes of this and other lentiviruses first form in the cytoplasm and then transit to the plasma membrane or the gRNA traffics there independently (26,27). Biochemical experiments supported the former scenario, with a monomer or low-order multimers forming on HIV-1 gRNAs and higher-order multimer formation depending on subsequent plasma membrane interactions (33).…”

T-body formation precedes virus-like particle maturation inS. cerevisiae

“…23 Thus, the assembly of immature Ty1 VLPs resembles the behavior of HIV-1, where Gag and HIV-1 RNA accumulation in the plasma membrane occurs prior to virion assembly. 24,25 The fact that T-bodies do not seem to be required for VLP maturation and cDNA synthesis "per se", along with the Gag self-assembly properties to form VLPs in an heterologous prokaryote host, raises the question of the functionality of T-bodies in S. cerevisiae. Although this is still an open question, we favor the idea that the kinetics of VLP formation is stimulated by locally concentrating Ty1 elements.…”

Properties and functions of the nucleocapsid protein in virus assembly

“…[51][52][53] However, NC-mediated fraying of the NA structure depends on the overall stability of the NA and requires natural mismatches as for wild-type TAR in order to take place. 46,54,57 This NC-mediated fraying of the NA structure largely relies on the hydrophobic plateau of the ZnF domain. 58 The second NC chaperoning component consists in NC-directed annealing of two NAs with complementary sequences when present in the same complex, namely TAR and cTAR sequences.…”