ALIX Is Recruited Temporarily into HIV-1 Budding Sites at the End of Gag Assembly

Ku, Pei I.; Bendjennat, Mourad; Ballew, Jeff; Landesman, Michael B.; Saffarian, Saveez

doi:10.1371/journal.pone.0096950

Cited by 31 publications

(48 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…During the early stage of viral budding, the Gag protein in its ubiquitinated form recruits AIP1 and Tsg101/ESCRT-I to initiate ESCRT-mediated assembly (385). Thereafter, the downstream ESCRT-III and VPS4 factors are recruited to complete viral budding (386). Experimental evidence suggests that HIV-1 NC Gag physically interacts with AIP1 (378) and Tsg101 (387).…”

Section: Gag -Tsg101/aip1-p6 Gag Associationmentioning

confidence: 99%

See 1 more Smart Citation

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

Clercq

2016

Microbiol Mol Biol Rev

View full text Add to dashboard Cite

SUMMARYThe HIV genome encodes a small number of viral proteins (i.e., 16), invariably establishing cooperative associations among HIV proteins and between HIV and host proteins, to invade host cells and hijack their internal machineries. As a known example, the HIV envelope glycoprotein GP120 is closely associated with GP41 for viral entry. From a genome-wide perspective, a hypothesis can be worked out to determine whether 16 HIV proteins could develop 120 possible pairwise associations either by physical interactions or by functional associations mediated via HIV or host molecules. Here, we present the first systematic review of experimental evidence on HIV genome-wide protein associations using a large body of publications accumulated over the past 3 decades. Of 120 possible pairwise associations between 16 HIV proteins, at least 34 physical interactions and 17 functional associations have been identified. To achieve efficient viral replication and infection, HIV protein associations play essential roles (e.g., cleavage, inhibition, and activation) during the HIV life cycle. In either a dispensable or an indispensable manner, each HIV protein collaborates with another viral protein to accomplish specific activities that precisely take place at the proper stages of the HIV life cycle. In addition, HIV genome-wide protein associations have an impact on anti-HIV inhibitors due to the extensive cross talk between drug-inhibited proteins and other HIV proteins. Overall, this study presents for the first time a comprehensive overview of HIV genome-wide protein associations, highlighting meticulous collaborations between all viral proteins during the HIV life cycle.

show abstract

Section: Gag -Tsg101/aip1-p6 Gag Associationmentioning

confidence: 99%

“…Gag interacts with AIP1 (386,388,389) and Tsg101 (390). During viral budding, AIP1 is packaged into viral particles through the interaction between the Bro1 domain of AIP1 and the zinc finger domains of NC Gag (378).…”

Section: Gag -Tsg101/aip1-p6 Gag Associationmentioning

confidence: 99%

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

Clercq

2016

Microbiol Mol Biol Rev

View full text Add to dashboard Cite

show abstract

“…Two late domains within the p6 domain of Gag, PTAP and YPXL, interact with the ESCRT-I protein TSG101 and ESCRT-associated protein ALIX, respectively, which then recruit ESCRT-III for the final scission to occur (3). Our understanding of the recruitment of the ESCRTs during Gag accumulation at the membrane has benefitted from biochemical analyses of budding events (6)(7)(8)(9), as well as from advanced fluorescence microscopy techniques which can examine the temporal dynamics of these events (10)(11)(12)(13). Interestingly, TSG101 and ALIX show different dynamics once engaged with Gag; TSG101 co-occurs with Gag as Gag multimerises (14), whereas ALIX is only recruited when Gag puncta reach maximum intensity (13).…”

Section: Introductionmentioning

confidence: 99%

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

Meng

Ramirez

Scherer

et al. 2020

Preprint

View full text Add to dashboard Cite

The scission of lipid membranes is a common biological process, often mediated by ESCRT complexes in concert with VPS4 assembling around the separation point. The functions of the ESCRT-I and ESCRT-III complexes are well established in certain of these cellular processes; however, the role of ESCRT-II remains contentious. Here, we devised a SNAP-tag fluorescent labelling strategy to understand the domain requirements of EAP45, the main component of ESCRT-II, in HIV egress, late endosome recruitment, and cytokinesis. We used TIRF microscopy to measure the spatial co-occurrence of the HIV structural polyprotein Gag with full length EAP45 in both fixed and live cells. Gag colocalises with the full length EAP45 comparably to ALIX, but this is lost on deletion of the EAP45 N terminus. Our findings reveal the H0 domain of the EAP45 protein is essential for linking to ESCRT-I during HIV budding and in anchoring at the late endosomal membrane, however in cytokinesis it is the Glue domain that is critical. ESCRT-II | EAP45 | HIV | Gag | cytokinesis| TIRF | SPT | colocalisationCorrespondence: cfk23@cam.ac.uk / amll1@medschl.cam.ac.uk AUTHOR

show abstract

“…We visualized the recruitment of ALIX during assembly of individual HIV Gag virus like particles (VLPs) on the basal membrane of HeLa cells stably expressing ALIX-h30-eGFP. ALIX-h30-eGFP links ALIX with eGFP through a stiff 30 amino acid super helical linker and is functional with the same efficiency as WT in rescue of PTAP - HIV virion release 14 . To generate fluorescent VLPs for fluorescent microscopy, cells were transfected with plasmids encoding HIV Gag-mCherry under a CMV promoter.…”

mentioning

confidence: 99%

“…Live imaging of HIV as well as MVB budding has been previously used for visualizing recruitment of ESCRTs during membrane session events 14,17,20,26,44-47 . Our study shows how disturbing previously characterized biochemical interactions can result in surprising recruitment profiles of ESCRTs observed in live cells and therefore underscores the usefulness of the imaging methods for further characterizing these interactions in vivo.…”

mentioning

confidence: 99%

Abrogating ALIX interactions results in stuttering of the ESCRT machinery

Gupta

Bendjennat

Saffarian

2019

Preprint

Self Cite

View full text Add to dashboard Cite

ESCRTs are cellular proteins that catalyze the fission of membranes and play an important role 6 in biology of disease including cancer and infectious virus release 1 . ESCRT associated protein 7ALIX plays an essential role in HIV budding 2-4 , exosome release 5,6 , down regulation of G-8 protein coupled receptors 7 , cytokinesis 8,9 and multi vesicular budding 1 . The consensus view is 9that ALIX plays a role by binding to the viral late domains 2-4 /Syntenin late domain 5 /Cepp55 8,9 10 and helps recruit downstream protein CHMP4 2,8,10,11 which along with VPS4 catalyzes the 11 fission of the membrane 12,13 . Using live imaging we have visualized the recruitment of ALIX, 12CHMP4 and VPS4 during budding of HIV with abrogated Gag-ALIX interactions. Based on the 13 canonical view, we were expecting to find reduced recruitment of ALIX under these conditions. 14 Instead we report observing multiple rounds of transient recruitment of ALIX, CHMP4 and VPS4 15prior to virion release. We further show that during each, transient recruitment, stoichiometry of 16all ESCRT components remained the same regardless of mutations abrogating ALIX and Gag 17interaction. In addition, mutations abrogating interactions between Gag and TSG101 result in 18 recruitment of ESCRTs with a substantial delay while maintaining similar stoichiometry. Our 19 results demonstrate that recruitment of ESCRTs is driven by a robust network of interactions 20 resulting in an "On/Off" switch behavior and ALIX's interactions with late domains of HIV Gag 21 play a crucial role during final catalysis of membrane fission after assembly of the full ESCRT 22 machinery. 23We visualized the recruitment of ALIX during assembly of individual HIV Gag virus like particles 24 (VLPs) on the basal membrane of HeLa cells stably expressing ALIX-h30-eGFP. ALIX-h30-25 eGFP links ALIX with eGFP through a stiff 30 amino acid super helical linker and is functional 26with the same efficiency as WT in rescue of PTAP -HIV virion release 14 . To generate 27fluorescent VLPs for fluorescent microscopy, cells were transfected with plasmids encoding HIV 28Gag-mCherry under a CMV promoter. Once VLP assembly commenced at the basal 29 membrane, the membrane was imaged with a TIRF penetration depth of 150 nm using 30 consecutive 488 nm and 561 nm illuminations every 15 seconds for 1.5 hours (methods). 31Individual VLPs were identified and analyzed from their initiation until full assembly which 32 corresponds to a stable fluorescence signal from HIV Gag-mCherry as shown in Figure 1. From 33 40 VLPs analyzed we detected 75% single transient recruitment of ALIX at the end of assembly 34 and 25% showing an average of two transient recruitment events. 35 ALIX interacts directly with HIV Gag through the YPXL late domain motif on p6 Gag 2,7,15 . In 36Gag(YP -) we abrogated this interaction by incorporating ( 36 SR 37 ) in place of ( 36 YP 37 ) as 37 previously characterized 3 . We visualized the recruitment of ALIX during assembly of individual 38HIV Gag(YP -)-mCherry VLPs on the plasma membra...

show abstract

ALIX Is Recruited Temporarily into HIV-1 Budding Sites at the End of Gag Assembly

Cited by 31 publications

References 68 publications

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

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

Abrogating ALIX interactions results in stuttering of the ESCRT machinery

Contact Info

Product

Resources

About