PPPYEPTAP Motif Is the Late Domain of Human T-Cell Leukemia Virus Type 1 Gag and Mediates Its Functional Interaction with Cellular Proteins Nedd4 and Tsg101

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Abstract
The human T-cell leukemia virus type 1 (HTLV-1) Gag polyprotein contains two adjacent proline-rich motifs (sequence PPPYEPTAP) in the C terminus of the matrix domain. Proline-to-alanine mutations were introduced into either or both motifs of HTLV-1 to determine the effect on the release of HTLV-1 virus-like particles from 293T cells. The release of both single mutants was significantly reduced, whereas a double mutation in both motifs abolished the release of the HTLV-1 particles. Two-hybrid and in vitro binding assays showed that the HTLV-1 Gag polyprotein binds both Tsg101 and Nedd4 proteins. The interaction with HTLV-1 Gag required the central WW domain of Nedd4 and the ubiquitin enzyme variant (UEV) domain of Tsg101. We expressed various fragments of Nedd4 and Tsg101 proteins in 293T cells and tested for their ability to interfere with virion release mediated by the HTLV-1 Gag-Pro polyprotein. Fragments consisting of the N-terminal UEV domain of Tsg101 and the central WW and C-terminal Hect domains of Nedd4 protein all caused transdominant inhibition of HTLV-1 particle release. Similarly, inhibition of the proteasome significantly decreased HTLV-1 particle release. Furthermore, the WW domain overexpression caused an early arrest of HTLV-1 particle morphogenesis before the membrane is deformed into the typical half-shell structure. This result suggests that Nedd4 is involved early in budding of HTLV-1.