Scaffolding viral protein NC nucleates phase separation of the HIV-1 biomolecular condensate

Influence of HIV-1 genomic RNA on the formation of Gag biomolecular condensates

“…Therefore, we expected that the Zn-chelating Cys-His boxes in the NC domain of Gag were primarily responsible for condensate formation. This notion was supported by our most recent observation that in cells, the HIV-1 NC protein serves as a scaffold onto which other viral proteins condense to form HIV-1 BMCs [45]. We therefore performed experiments to generate phase diagrams for each recombinant Gag protein by assessing the effect of crowding buffer containing additional NaCl with increasing protein concentrations on condensate formation (ranging from 0 to 40 µM protein; 0 to 300 mM NaCl).…”

“…Interestingly, disorder was higher in both the RNA-binding MA highly basic region (MA-HBR) (i.e., 70.00%) [51] and RNA-binding NC ZnFs (48.57%). Although the NC domain of Gag contributes to phase separation and can itself produce large cytoplasmic aggregates within stress granules [44,45,60,61], the MA domain has yet to be shown to contribute to phase separation of Gag. However, a number of studies have shown that MA forms trimers [62][63][64], and that MA alone can produce VLPs [65,66], suggesting that, similar to NC, the mature MA protein participates in protein-protein interactions.…”

“…As previously reported, WT Gag optimally phase separated into spherical condensates between concentrations of 10 - 20 µM at 150 mM NaCl (Figure 2B, D), with sizes ranging from 0.5 to 2 μm in diameter [44, 72] (Figure 2B, E). At higher viral protein concentrations [45] and increasing salt concentrations, Gag formed a gel (Figure 2B, C). At lower protein concentrations and higher salt concentrations, Gag formed rod-like structures (Figure 2B).…”

“…Formation of in vitro phase separated condensates was monitored by differential interference contrast (DIC) and fluorescence microscopy as previously described [13,44,45]. For examination of fluorescent condensates, purified proteins were labeled using the Condensates less spherical at optimum protein concentrations; rod-like at high protein concentration; Gag-gRNA BMCs less spherical at low protein concentration and little impact from gRNA on BMC morphology and decreased gelling at higher protein concentrations.…”

“…Our earlier work demonstrated that the disordered HIV-1 NC protein undergoes zinc-dependent phase separation, regulating gRNA positioning and trafficking [44]. More recently, we demonstrated that HIV-1 NC, CA, RT, and IN proteins phase separate in proportion to their degrees of intrinsic disorder, and furthermore, that virus maturation by HIV-1 PR generates NC-initiated condensates driving maintenance of functional viral core BMCs [45]. We are also intrigued by the observation that the HIV-1 MA protein also possesses high levels of predicted intrinsic disorder [46].…”