HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

Abstract

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal-complex-required-for-transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma-membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag plasma-membrane-binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma-membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding-site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared to p6 in GST-pull-down experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

Updating on Roles of HIV Intrinsic Factors: A Review of Their Antiviral Mechanisms and Emerging Functions

“…The results suggest a transient ESCRT-matrix interaction that is replaced by Gag plasma membrane binding. These results further indicate that matrix interactions may block the ESCRT function [157].…”

The Role of Exosome and the ESCRT Pathway on Enveloped Virus Infection

“…The retrovirus budding, especially human immunodeficiency virus type 1 (HIV-1), is particularly similar to the biogenic origin of exosomes [82]. As reported, the TSG101 and ALIX proteins of ESCRT are critical for the budding and release of HIV-1 [83]. Exosome surface marker molecules such as CD63 and CD81 are also involved in HIV-1 budding and infection [84].…”

Exosomes in the life cycle of viruses and the pathogenesis of viral infections

“…В процессе выхода из клетки участвует белок Gag ВИЧ-1, который накапливается и заякоревается на плазматической мембране. Изучение ключевого компонента ESCRT -TSG101 с использованием системы Таким образом, взаимодействие TSG101 с Gag ВИЧ может служить потенциальной мишенью для противовирусной терапии [28][29][30]. Сборку вирусных частиц и секрецию экзосом также регулируют белки семейства Rab [31].…”