Abstract
Members of the Nedd4 family of E3 ubiquitin ligases bind the L domain in avian sarcoma virus (ASV) Gag and facilitate viral particle release. Translational fusion of ASV Gag with an L domain deletion (Deltap2b) to proteins that comprise ESCRT-I, -II, and -III (the endocytic sorting complexes required for transport) rescued both Gag ubiquitination and particle release from cells. The ESCRT-I factors Vps37C or Tsg101 were more effective in rescue of Gag/Deltap2b budding than the ESCRT-II factor Eap20 or the ESCRT-III component CHMP6. Thus ESCRT components can substitute for Nedd4 family members in ASV Gag release. Unlike wild type, ASV Gag/Deltap2b -ESCRT chimeras failed to co-immunoprecipitate with co-expressed hemagglutinin-tagged Nedd4, indicating that Nedd4 was not stably associated with these Gag fusions. Release of the Gag-ESCRT-I or -II fusions was inhibited by a dominant negative mutant of Vps4 ATPase similar to wild type ASV Gag. In contrast to ASV Gag, HIV-1 Gag containing an L domain inactivating mutation (P7L) was efficiently rescued by fusion to a component of ESCRT-III (Chmp6) but not ESCRT-II (Eap20). Depletion of the endogenous pool of Eap20 (ESCRT-II) had little effect on HIV-1 Gag release but blocked ASV Gag release. In contrast, depletion of the endogenous pool of Vps37C (ESCRT-I) had little effect on ASV but blocked HIV-1 Gag release. Furthermore, an N-terminal fragment of Chmp6 inhibited both HIV-1 and ASV Gag release in a dominant negative manner. Taken together, these results indicate that ASV and HIV-1 Gag utilize different combinations of ESCRT proteins to facilitate the budding process, although they share some common elements.
Highlights
Retroviruses, and many other enveloped viruses, evolved mechanisms to exploit components of the endocytic sorting pathway to bud from cells efficiently
ESCRT Proteins Complement an L domain Deletion when Covalently Linked to the C Terminus of avian sarcoma virus (ASV) Gag/⌬p2b—Previous studies established that covalently linking Hrs [24], or ESCRT-I proteins Tsg101 [25], Vps37B [26], or Vps37C [21] to the C-terminal end of HIV-1 Gag/⌬p6 restored the release of virus-like particles (VLPs), supporting the view that the PTAP motif functions to recruit ESCRT-I activity to the site of viral particle assembly
The most efficient rescue of VLP release occurred by translationally linking Gag/ ⌬p2b to the ESCRT-I complex using Tsg101 or Vps37C
Summary
Retroviruses, and many other enveloped viruses, evolved mechanisms to exploit components of the endocytic sorting pathway to bud from cells efficiently. Distinct classes of L domain sequences exist among retroviruses (with core motifs of PTAP, PPXY, and YPXL), each functions as a binding site to recruit different components of the vacuolar protein sorting (Vps) machinery. The specific role of ubiquitin in retrovirus budding remains unclear, it may potentially function as a sorting signal to facilitate the interaction between Gag and ubiquitin-binding domains in some of the factors of the ESCRT machinery. Tethering the ESCRT-II protein Eap to the C terminus of Gag complements the L domain mutation of ASV Gag/⌬p2b but not HIV Gag/P7L This suggests that, unlike HIV-1 Gag, ASV Gag requires a functional ESCRT-II complex for release. We observe that siRNA-mediated depletion of Eap in 293/E cells significantly inhibits the release of ASV but not HIV-1 Gag. Tethering the ESCRT-I protein, Vps37C, to ASV Gag/⌬p2b rescues budding and ubiquitination. The biochemical analysis presented in this manuscript, together with our previous findings, indicate that ASV and HIV-1 Gag use parallel pathways dependent on different components of the ESCRT machinery to bud from cells
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