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.
Highlights
55 Endosomal sorting complexes required for transport (ESCRTs) are a family of cellular56 proteins comprising ESCRT-0, -I, -II and -III complexes and have been reviewed extensively57 (Olmos and Carlton, 2016; Schöneberg et al, 2016; Scourfield and Martin-Serrano, 2017).58 Each is composed of two or more proteinsESCRT-0 (HRS and STAM complexes), ESCRT-I (TSG101, VPS28, VPS37, and MVB12/UBAP1), ESCRT-II (EAP20, EAP30, and EAP45), and60 ESCRT-III (CHMP1–7) being the major recognised components
We utilised a CRISPR-Cas[9] EAP45 knockout cell 38 line to assess the involvement of ESCRT-II in HIV replication
We demonstrated that the 20 absence of ESCRT-II impairs HIV budding
Summary
55 Endosomal sorting complexes required for transport (ESCRTs) are a family of cellular56 proteins comprising ESCRT-0, -I, -II and -III complexes and have been reviewed extensively57 (Olmos and Carlton, 2016; Schöneberg et al, 2016; Scourfield and Martin-Serrano, 2017).58 Each is composed of two or more proteinsESCRT-0 (HRS and STAM complexes), ESCRT-I (TSG101, VPS28, VPS37, and MVB12/UBAP1), ESCRT-II (EAP20, EAP30, and EAP45), and60 ESCRT-III (CHMP1–7) being the major recognised components. 55 Endosomal sorting complexes required for transport (ESCRTs) are a family of cellular. 56 proteins comprising ESCRT-0, -I, -II and -III complexes and have been reviewed extensively. 57 (Olmos and Carlton, 2016; Schöneberg et al, 2016; Scourfield and Martin-Serrano, 2017). 23 involved in multivesicular body (MVB) formation (Katzmann et al, 2001) and sorting of 62 ubiquitinated cargos into vesicles involving sequential recruitment of ESCRT-0, -I, -II, and -III. 63 In addition to MVB formation, ESCRT complexes are involved in a variety of cellular. functions including cytokinesis (Carlton and Martin-Serrano, 2007), the biogenesis of microvesicles and endosomes (Nabhan et al, 2012), nuclear envelope resealing 66 al., 2015; Vietri et al, 2015), membrane wound repair (Jimenez et al, 2014), neuron pruning. 67 (Zhang et al, 2014) and endolysosomal repair (Skowyra et al, 2018).
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