Abstract
The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase Cδ (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding.
Highlights
The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl group, but contains structural domains similar to those of HIV-1 Gag
We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase C␦ (PH-PLC), two proteins that bind PI[4,5]P2, affects ASV Gag trafficking to the plasma membrane (PM) and budding
Ala and Glu substitutions were introduced at the Lys6, Lys13, Lys23, and Lys24 sites, and the resulting ASV Gag mutants were transfected into the DF-1 chicken fibroblast cell line
Summary
The matrix domain of the Gag protein from avian sarcoma virus contains a PI[4,5]P2-binding site that targets Gag to the cell periphery. The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; it is unclear whether the phospholipid PI[4,5]P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. the role of PI[4,5]P2 in ASV Gag localization and budding has been controversial. In another study employing a similar enzyme-mediated depletion approach, 5ptaseIV, phosphoinositide 5-phosphatase IV; PS, phosphatidylserine; VLP, virus-like particle; HA, hemagglutinin
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