Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function

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Abstract
Efficient budding of HIV-1 from the plasma membrane of infected cells requires the function of a 6-kDa protein known as p6. A highly conserved Pro-Thr-Ala-Pro (PTAP) motif (the “late” or “L” domain), is critical for the virus-budding activity of p6. Recently, it was demonstrated that the product of tumor susceptibility gene 101 (TSG101), which contains at its N terminus a domain highly related to ubiquitin-conjugating (E2) enzymes, binds HIV-1 Gag in a p6-dependent fashion. We examined the impact of overexpressing the N-terminal region of TSG101 on HIV-1 particle assembly and release. We observed that this domain (referred to as TSG-5′) potently inhibits virus production. Examination of cells coexpressing HIV-1 Gag and TSG-5′ by electron microscopy reveals a defect in virus budding reminiscent of that observed with p6 L domain mutants. In addition, the effect of TSG-5′ depends on an intact p6 L domain; the assembly and release of virus-like particles produced by Gag mutants lacking a functional p6 PTAP motif is not significantly affected by TSG-5′. Furthermore, assembly and release of murine leukemia virus and Mason–Pfizer monkey virus are insensitive to TSG-5′. TSG-5′ is incorporated into virions, confirming the Gag/TSG101 interaction in virus-producing cells. Mutations that inactivate the p6 L domain block TSG-5′ incorporation. These data demonstrate a link between the E2-like domain of TSG101 and HIV-1 L domain function, and indicate that TSG101 derivatives can act as potent and specific inhibitors of HIV-1 replication by blocking virus budding.