A Large Extension to HIV-1 Gag, Like Pol, Has Negative Impacts on Virion Assembly

Abstract

The GagPol protein of HIV-1 harbors viral enzymes, such as protease (PR), reverse transcriptase, and integrase, that are all crucial for virion infectivity. Previous studies have suggested that expression of GagPol alone does not produce viral particles and that the budding defect is caused by the presence of the Pol region. However, it has remained unknown why GagPol fails to produce viral particles. We show here that HIV-1 GagPol is incapable of membrane binding and subsequent particle assembly. Our confocal data indicated that, despite full N-myristoylation, GagPol protein failed to target plasma membrane with diffuse distribution in the cytoplasm. Membrane flotation analysis confirmed these findings. Progressive C-terminal truncation of GagPol to give GagPR allowed for plasma membrane targeting but still not for particle production. Conversely, the C-terminal addition of a noncognate protein, such as ß-galactosidase or 4 tandem GFP, to Gag impaired the membrane affinity, indicating that the Pol region, a large extension to Gag, inhibits membrane binding in the context of GagPol. The addition of the 10 N-terminal amino acids of Fyn kinase [Fyn(10)], a tight membrane-binding signal, conferred plasma membrane targeting on GagPol, but the Fyn(10)GagPol did not produce viral particles. The defect in particle budding was not rescued by the introduction of the PTAP motif, which is responsible for a late stage of viral particle budding. Rather, electron microscopy suggested that the budding defect of GagPR occurred at an early stage of particle morphogenesis. Our data, which were consistent with previous observations, demonstrate the defects of GagPol in membrane binding and particle assembly.

FRET analysis of HIV‐1 Gag and GagPol interactions

“…All derivatives used in this study were from the HIV‐1 molecular clone pNL4‐3 and contained inactive PR (D25N mutation). The derivative expressing Gag with a FLAG peptide at the C terminus without GagPol and that expressing GagPol with a HA peptide at the C terminus in which the gag and pol genes were placed in‐frame by deleting the frameshifting signal (frameshift mutation) were described previously .…”

“…In many cell types, the expression of HIV‐1 Gag alone produces a Gag virus‐like particle, similar to immature HIV‐1 . In contrast, the GagPol protein is incapable of binding to the membrane and is only incorporated into an HIV‐1 virion by interactions, termed ‘coassembly’ with Gag . As GagPol is a precursor protein that is processed to produce the virus‐specific enzymes, PR, RT, and IN, the incorporation of GagPol into HIV‐1 particles is absolutely required for the virion infectivity.…”

The Preserved HTH-Docking Cleft of HIV-1 Integrase Is Functionally Critical

“…Unlike Gag, which itself can form viral like particles (VLP), Gag-Pol alone is incapable of plasma membrane targeting or particle production and can only be incorporated into viral particles by co-assembling with Gag (reviewed in (Haraguchi et al, 2012)). Although the mechanism by which the ScFv inhibits HIV-1 particle production is not established, the high-resolution structure of IN CCD in complex with Fab2 clearly indicates that Fab interferes with IN interfaces essential for CTD and HTH-docking and functional multimerization.…”

“…Increasing the size of Gag by C-terminal fusion of large protein extensions mimicking the size of Pol domain has indeed been shown to impair membrane affinity of Gag (Haraguchi et al, 2012). Nevertheless, specific targeting of IN has previously been shown to inhibit virus particle maturation or production.…”