ESCRT‐II functions by linking to ESCRT‐I in human immunodeficiency virus‐1 budding

Abstract

Human immunodeficiency virus (HIV) uses the ESCRT (endosomal sorting complexes required for transport) protein pathway to bud from infected cells. Despite the roles of ESCRT‐I and ‐III in HIV budding being firmly established, participation of ESCRT‐II in this process has been controversial. EAP45 is a critical component of ESCRT‐II. Previously, we utilised a CRISPR‐Cas9 EAP45 knockout cell line to assess the involvement of ESCRT‐II in HIV replication. We demonstrated that the absence of ESCRT‐II impairs HIV budding. Here, we show that virus spread is also defective in physiologically relevant CRISPR/Cas9 EAP45 knockout T cells. We further show reappearance of efficient budding by re‐introduction of EAP45 expression into EAP45 knockout cells. Using expression of selected mutants of EAP45, we dissect the domain requirement responsible for this function. Our data show at the steady state that rescue of budding is only observed in the context of a Gag/Pol, but not a Gag expressor, indicating that the size of cargo determines the usage of ESCRT‐II. EAP45 acts through the YPXL‐ALIX pathway as partial rescue is achieved in a PTAP but not a YPXL mutant virus. Our study clarifies the role of ESCRT‐II in the late stages of HIV replication and reinforces the notion that ESCRT‐II plays an integral part during this process as it does in sorting ubiquitinated cargos and in cytokinesis.

Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

“…The discrepancy between these two studies may have arisen from minor amounts of residual ESCRT-II in the siRNA KD study still being sufficient for functional budding. Notably in these studies, cell proliferation was largely unperturbed by the depletion of EAP45 and yet there was up to a 10 fold decrease in virus spreading, suggesting that EAP45 is redundant for cell growth but is critical for viral multiplication and spread (26). This, together with the apparent requirement for early ESCRT proteins to ensure the specific recruitment of the Gag-pol polyprotein (26), underlines the importance of understanding the mechanistic roles of ESCRT-II in HIV budding.…”

“…Notably in these studies, cell proliferation was largely unperturbed by the depletion of EAP45 and yet there was up to a 10 fold decrease in virus spreading, suggesting that EAP45 is redundant for cell growth but is critical for viral multiplication and spread (26). This, together with the apparent requirement for early ESCRT proteins to ensure the specific recruitment of the Gag-pol polyprotein (26), underlines the importance of understanding the mechanistic roles of ESCRT-II in HIV budding. Further insights into these protein-protein interactions could help in developing a new class of antivirals to treat HIV.…”

“…Although ESCRT-II's role as the canonical bridge in linking to ESCRT-I in MVB formation (22,23) and cytokinesis is established (24), the same role has been controversial in HIV budding. An earlier siRNA knockdown study of EAP20 suggested ESCRT-II is dispensable for HIV budding in 293T cells (19), however, our recent study using CRISPR/EAP45 knockout (KO) HAP1 and T cells showed that EAP45 is required for efficient HIV budding and spreading (25,26). The specificity of this effect was demonstrated by the ability of EAP45 in trans to rescue the EAP45 KO cell-mediated virus budding and by its dependency on the H0 linker region for interacting with ESCRT-I (26).…”

“…An earlier siRNA knockdown study of EAP20 suggested ESCRT-II is dispensable for HIV budding in 293T cells (19), however, our recent study using CRISPR/EAP45 knockout (KO) HAP1 and T cells showed that EAP45 is required for efficient HIV budding and spreading (25,26). The specificity of this effect was demonstrated by the ability of EAP45 in trans to rescue the EAP45 KO cell-mediated virus budding and by its dependency on the H0 linker region for interacting with ESCRT-I (26). The discrepancy between these two studies may have arisen from minor amounts of residual ESCRT-II in the siRNA KD study still being sufficient for functional budding.…”

Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine

“…However, PCR require multiple steps to run the assay, including an upfront nucleic acid extraction and amplification steps, and require dedicated instrumentation [30][31][32]. By contrast, CRISPR-Cas-based assays can be run directly on primary clinical samples as a single reaction and performed using minimal equipment [33][34][35][36]. This is especially relevant for detection of SARS-CoV-2, where testing has been limited in many places due to a lack of testing materials and personal protective equipment [37][38][39].…”