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Viral Replication: Post-Integration Events
Monday, 1:30 - 3:30 pm
Background: The retrovirus polyprotein Gag is the only viral protein required for formation, budding, and release of virus-like particles (VLP) from the plasma membrane. Some current models support the idea that single Gag molecules bind to and subsequently multimerize at the inner face of the plasma membrane. However some studies suggest that Gag-Gag interaction is independent of membrane binding. The aim of this study is to gain a better understanding of HIV-1 assembly.
Methods: Gag precursor and different Gag mutants were fused at the C-terminus to GFP, CFP or YFP. These constructs were transfected in 293T cells and analyzed by deconvolution microscopy (Delta Vision), membrane flotation assays and fluorescence resonance energy transfer (FRET).
Results: The Gag-GFP expressed at low levels localizes at the cytoplasm, but overexpresion of unlabeled Gag directs Gag-GFP to the plasma membrane. A mutant lacking the globular head of matrix (dGH-GFP) localizes at the plasma membrane independently of the overexpression of unlabeled Gag. A mutant in the myristoylation site of Gag (G2A-GFP) changed from cytoplasmic to membrane localization with high levels of unlabeled Gag. The same result was observed with the double mutant G2AdGH-GFP. Interestingly, the fusion of aminoacids 1 through 9 of Gag precursor containing the myristoylation site to GFP localized this protein at the plasma membrane. Finally, the cytoplasmic distribution of MACA-GFP was not altered by the expression of more unlabeled Gag. All these data were corroborated by membrane flotation assays. On the other hand, FRET analysis shows that all these constructs except MACA-GFP were able to multimerize.
Conclusions: Altogether, these data suggest that the first event in HIV-1 assembly is a multimerization of Gag in the cytoplasm, probably through NC, that could produce conformational changes in matrix and expose the myristoylation site to anchor the Gag oligomer to the inner face of the plasma membrane. Thus, the function of matrix in assembly could be to avoid incorporation of single Gag molecules to the plasma membrane, masking the myristoylation site until the multimerization of Gag is achieved.
Keywords: Gag multimerization; HIV-1; FRET