Cellular requirements for the budding pathways of avian sarcoma virus and human immunodeficiency virus, type 1

The retroviral Gag polyprotein, the main structural component of virus particles, encodes all the elements necessary for assembly and release of VLPs from cells. Among these is the late assembly (L) domain, which functions as binding sites to recruit cellular proteins required for membrane scission and budding. Three classes of L domains, defined by conserved core motifs, exist within retroviruses and other enveloped virus families. The P(T/S)AP and YPxL motifs (both encoded by HIV-1 Gag) bind to components of the ESCRT pathway, Tsg101 and AIP1/Alix, respectively. The PPxY motif (encoded by ASV Gag) binds to Nedd4-like E3 ubiquitin ligases. Nedd4 ubiquitinates ASV Gag and links it to the ESCRT machinery for budding.;In the following study, we show that HIV-1 and ASV Gag utilize different components of the ESCRT pathway for budding. Covalently linking ESCRT proteins to the C-terminus of ASV Gag/Deltap2b and HIV-1 Gag/P7L rescues the budding defect caused by the L domain mutations. Though ESCRT-I proteins, Tsg101 and Vps37C, efficiently rescued ASV Gag/Deltap2b and HIV-1 Gag/P7L budding, siRNA-mediated depletion of these proteins in mammalian or avian cells did not affect ASV Gag release. The ESCRT-II protein, Eap20, partially rescues ASV Gag/Deltap2b, but did not rescue HIV-1 Gag/P7L. Consequently, siRNA knockdown of Eap20 inhibits ASV Gag release but not HIV-1 Gag. The ESCRT-III protein, Chmp6, rescued both ASV Gag/Deltap2b and HIV-1 Gag/P7L. Overexpression of a dominant-negative fragment of Chmp6 inhibited ASV and HIV VLP release indicating that both budding complexes required the ESCRT-III proteins for budding.We also observe that HIV and ASV VLPs bud from or pass through different cellular membranes during egress with ASV Gag associating the endosomal lipid analog, N-Rh-PE. ASV Gag derived particles, but not HIV-1 Gag particles, purified from N-Rh-PE-treated COS cells are labeled with the tracer. ASV Gag associates with N-Rh-PE in an L domain-dependent manner. The ASV Gag/Deltap2b-Eap20 chimera rescues budding through N-Rh-PE-positive membranes.;Finally, we show that the ubiquitin-like protein, ISG15, inhibits ASV and HIV-1 Gag budding. ISG15 prevents the interaction between the Gag budding complexes and Vps4E228Q. We correlate the inability to recruit Vps4 to the ISGylation of Chmp5.