Dataset Information

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 with p6 in GST-pulldown 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.

SUBMITTER: Meusser B

PROVIDER: S-EPMC7939435 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundHIV-1 relies on the host ESCRTs for release from cells. HIV-1 Gag engages ESCRTs by directly binding TSG101 or Alix. ESCRTs also sort ubiquitinated membrane proteins through endosomes to facilitate their lysosomal degradation. The ability of ESCRTs to recognize and process ubiquitinated proteins suggests that ESCRT-dependent viral release may also be controlled by ubiquitination. Although both Gag and ESCRTs undergo some level of ubiquitination, definitive demonstration that ubiquitin is required for viral release is lacking. Here we suppress ubiquitination at viral budding sites by fusing the catalytic domain of the Herpes Simplex UL36 deubiquitinating enzyme (DUb) onto TSG101, Alix, or Gag.ResultsExpressing DUb-TSG101 suppressed Alix-independent HIV-1 release and viral particles remained tethered to the cell surface. DUb-TSG101 had no effect on budding of MoMLV or EIAV, two retroviruses that rely on the ESCRT machinery for exit. Alix-dependent virus release such as EIAV's, and HIV-1 lacking access to TSG101, was instead dramatically blocked by co-expressing DUb-Alix. Finally, Gag-DUb was unable to support virus release and dominantly interfered with release of wild type HIV-1. Fusion of UL36 did not effect interactions with Alix, TSG101, or Gag and all of the inhibitory effects of UL36 fusion were abolished when its catalytic activity was ablated. Accordingly, Alix, TSG101 and Gag fused to inactive UL36 functionally replaced their unfused counterparts. Interestingly, coexpression of the Nedd4-2s ubiquitin ligase suppressed the ability of DUb-TSG101 to inhibit HIV-1 release while also restoring detectable Gag ubiquitination at the membrane. Similarly, incorporation of Gag-Ub fusion proteins into virions lifted DUb-ESCRT inhibitory effect. In contrast, Nedd4-2s did not suppress the inhibition mediated by Gag-DUb despite restoring robust ubiquitination of TSG101/ESCRT-I at virus budding sites.ConclusionsThese studies demonstrate a necessary and natural role for ubiquitin in ESCRT-dependent viral release and indicate a critical role for ubiquitination of Gag rather than ubiquitination of ESCRTs themselves.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets