Session 4Oral Abstract Presentations HIV Replication: Entry and Assembly Session Day and Time: Tuesday 10 am - 12:30 pm Presentation Time: 11:45 Room: Ballroom A
24 The Role of ESCRT-I in Retroviral Budding Juan Martin-Serrano*, Trinity Zang, Paul D. Bieniasz Aaron Diamond AIDS Res Ctr, Rockefeller Univ, New York, NY
Background: Retroviral late-budding or "L"-domains are required for the efficient release of nascent virions. The 3 known types of L-domain designated according to essential tetrapeptide motifs (PTAP, PPXY, or YPDL) each bind distinct cellular cofactors. We and others have demonstrated that recruitment of an ESCRT-I subunit, Tsg101, a component of the class E vacuolar protein sorting (VPS) machinery, is required for the budding of viruses such as HIV-1 and Ebola that encode a PTAP-type L-domain, but subsequent events remain undefined.
Methods: The ability of Tsg101 to multimerize and to bind vps28 was determined by yeast 2-hybrid assay and by co-precipitation analysis. Viral particle formation was determined by infectivity assay and by ultracentrifugation of culture supernatants from transfected cells followed by western blotting. Viral protein induced re-localization of Tsg101 and vps28 was visualized by deconvolution microscopy.
Results: We demonstrate that VPS28, a second component of ESCRT-I, binds to a sequence close to the Tsg101 C-terminus and is, therefore, recruited to the plasma membrane by HIV-1 Gag. In addition, we show that Tsg101 exhibits a multimerization activity. Using a complementation assay in which Tsg101 is artificially recruited to sites of HIV-1 assembly, we demonstrate that the integrity of the VPS28-binding and multimerization sites within Tsg101 is required for particle budding, as is a putative leucine zipper. A minimal multimerizing Tsg101 domain is a dominant negative inhibitor of PTAP-mediated HIV-1 budding, but does not inhibit YPDL-type or PPXY-type L-domain function. Nevertheless, YDPL-type L-domain activity is inhibited by expression of a catalytically inactive mutant of the class E VPS ATPase, VPS4.
Conclusions: These results indicate that all 3 classes of retroviral L-domains require a functioning class E VPS pathway in order to effect budding. However, the PTAP-type L-domain appears unique in its requirement for an intact, or nearly intact, ESCRT-I complex.
